90 results on '"Ann-Bin Shyu"'
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2. Tob2 phosphorylation regulates global mRNA turnover to reshape transcriptome and impact cell proliferation
- Author
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Kai Lieh Huang, Krista Strouz, Ann-Bin Shyu, and Chyi-Ying A. Chen
- Subjects
RNA Stability ,Mutant ,Cell Cycle Proteins ,Biology ,Polyadenylation ,Poly(A)-Binding Proteins ,Article ,Cell Line ,Transcriptome ,03 medical and health sciences ,CCR4-NOT complex ,Humans ,RNA, Messenger ,Phosphorylation ,Molecular Biology ,030304 developmental biology ,Cell Proliferation ,0303 health sciences ,Messenger RNA ,Cell growth ,Kinase ,030302 biochemistry & molecular biology ,Cell cycle ,Cell biology ,HEK293 Cells ,Poly A - Abstract
Tob2, an anti-proliferative protein, promotes deadenylation through recruiting Caf1 deadenylase to the mRNA poly(A) tail by simultaneously interacting with both Caf1 and poly(A)-binding protein (PABP). Previously, we found that changes in Tob2 phosphorylation can alter its PABP-binding ability and deadenylation-promoting function. However, it remained unknown regarding the relevant kinase(s). Moreover, it was unclear whether Tob2 phosphorylation modulates the transcriptome and whether the phosphorylation is linked to Tob2's anti-proliferative function. In this study, we found that c-Jun amino-terminal kinase (JNK) increases phosphorylation of Tob2 at many Ser/Thr sites in the intrinsically disordered region (IDR) that contains two separate PABP-interacting PAM2 motifs. JNK-induced phosphorylation or phosphomimetic mutations at these sites weaken the Tob2–PABP interaction. In contrast, JNK-independent phosphorylation of Tob2 at serine 254 (S254) greatly enhances Tob2 interaction with PABP and its ability to promote deadenylation. We discovered that both PAM2 motifs are required for Tob2 to display these features. Combining mass spectrometry analysis, poly(A) size-distribution profiling, transcriptome-wide mRNA turnover analyses, and cell proliferation assays, we found that the phosphomimetic mutation at S254 (S254D) enhances Tob2's association with PABP, leading to accelerated deadenylation and decay of mRNAs globally. Moreover, the Tob2–S254D mutant accelerates the decay of many transcripts coding for cell cycle related proteins and enhances anti-proliferation function. Our findings reveal a novel mechanism by which Ccr4–Not complex is recruited by Tob2 to the mRNA 3′ poly(A)-PABP complex in a phosphorylation dependent manner to promote rapid deadenylation and decay across the transcriptome, eliciting transcriptome reprogramming and suppressed cell proliferation.
- Published
- 2020
3. Corrigendum: Antagonistic actions of two human Pan3 isoforms on global mRNA turnover
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Yueqiang Zhang, Yu Xiang, Chyi-Ying A. Chen, Ann-Bin Shyu, and Leng Han
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0301 basic medicine ,Gene isoform ,03 medical and health sciences ,Messenger RNA ,030104 developmental biology ,Text mining ,business.industry ,Biology ,business ,Molecular Biology ,Article ,Cell biology - Abstract
Deadenylation is a fundamental process that regulates eukaryotic gene expression. Mammalian deadenylation exhibits biphasic kinetics, with the Pan2–Pan3 and Ccr4–Caf1 deadenylase complexes mediating the first and second phase, respectively; however, the significance of the biphasic nature of deadenylation in mRNA turnover remains unclear. In this study, we discovered that two distinct isoforms of human Pan3 display opposing properties necessary for coordinating the two phases of deadenylation. The shorter isoform (Pan3S) interacts more strongly with PABP than the longer isoform (Pan3L) does. Pan2 deadenylase activity is enhanced by Pan3S but suppressed by Pan3L. Knocking down individual Pan3 isoforms has opposing effects on the global poly(A) tail length profile, P-body formation, and different mRNA decay pathways. Transcriptome-wide analysis of Pan3 knockdown effects on mRNA turnover shows that depleting either Pan3 isoform causes profound and extensive changes in mRNA stability globally. These results reveal a new fundamental step governing mammalian mRNA metabolism. We propose that the first phase of deadenylation, coordinated through the interplay among the two Pan3 isoforms, Pan2, and PABP, represents a cytoplasmic mRNA maturation step important for proper mRNA turnover.
- Published
- 2018
- Full Text
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4. Comprehensive Characterization of Alternative Polyadenylation in Human Cancer
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Yang Xia, Michael R. Blackburn, Zheng Chen, Ning Yuan Chen, Youqiong Ye, Lixia Diao, Tingting Mills, Ann-Bin Shyu, Yang Yang, Zhao Zhang, Fatma Muge Ozguc, Harry Karmouty-Quintana, Yanyan Lou, Chunyan Cai, Seung Hee Yoo, Leng Han, Rodney E. Kellems, Gordon B. Mills, Yu Xiang, and Yoonjin Kim
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0301 basic medicine ,Cancer Research ,Polyadenylation ,education ,Computational biology ,Biology ,Poly(A)-Binding Protein I ,Genome ,03 medical and health sciences ,Neoplasms ,mental disorders ,Biomarkers, Tumor ,Tumor Cells, Cultured ,Humans ,RNA, Messenger ,3' Untranslated Regions ,Gene ,Regulation of gene expression ,Genome, Human ,Three prime untranslated region ,Proportional hazards model ,High-Throughput Nucleotide Sequencing ,Articles ,Prognosis ,Chromatin ,Gene Expression Regulation, Neoplastic ,030104 developmental biology ,Oncology ,psychological phenomena and processes ,Human cancer ,Follow-Up Studies - Abstract
Background Alternative polyadenylation (APA) is emerging as a major post-transcriptional mechanism for gene regulation, and dysregulation of APA contributes to several human diseases. However, the functional consequences of APA in human cancer are not fully understood. Particularly, there is no large-scale analysis in cancer cell lines. Methods We characterized the global APA profiles of 6398 patient samples across 17 cancer types from The Cancer Genome Atlas and 739 cancer cell lines from the Cancer Cell Line Encyclopedia. We built a linear regression model to explore the correlation between APA factors and APA events across different cancer types. We used Spearman correlation to assess the effects of APA events on drug sensitivity and the Wilcoxon rank-sum test or Cox proportional hazards model to identify clinically relevant APA events. Results We revealed a striking global 3'UTR shortening in cancer cell lines compared with tumor samples. Our analysis further suggested PABPN1 as the master regulator in regulating APA profile across different cancer types. Furthermore, we showed that APA events could affect drug sensitivity, especially of drugs targeting chromatin modifiers. Finally, we identified 1971 clinically relevant APA events, as well as alterations of APA in clinically actionable genes, suggesting that analysis of the complexity of APA profiles could have clinical utility. Conclusions Our study highlights important roles for APA in human cancer, including reshaping cellular pathways and regulating specific gene expression, exemplifying the complex interplay between APA and other biological processes and yielding new insights into the action mechanism of cancer drugs.
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- 2017
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5. Emerging Themes in Regulation of Global mRNA Turnover in cis
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Ann-Bin Shyu and Chyi-Ying A. Chen
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0301 basic medicine ,Genetics ,Messenger RNA ,Polyadenylation ,RNA Stability ,Nonsense-mediated decay ,Eukaryota ,RNA ,Translation (biology) ,Biology ,Biochemistry ,Article ,Cell biology ,Transcriptome ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,P-bodies ,Humans ,RNA, Messenger ,Molecular Biology ,030217 neurology & neurosurgery ,Function (biology) - Abstract
Messenger RNA (mRNA) is the molecule that conveys genetic information from DNA to the translation apparatus. mRNAs in all organisms display a wide range of stability, and mechanisms have evolved to selectively and differentially regulate individual mRNA stability in response to intra-cellular and extra-cellular cues. In recent years, three seemingly distinct aspects of RNA biology—mRNA N6-methyladenosine (m6A) modification, alternative 3’ end processing and polyadenylation (APA), and mRNA codon usage—have been linked to mRNA turnover, and all three aspects function to regulate global mRNA stability in cis. Here, we discuss the discovery and molecular dissection of these mechanisms in relation to how they impact the intrinsic decay rate of mRNA in eukaryotes, leading to transcriptome reprogramming.
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- 2017
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6. MiR-26 down-regulates TNF-α/NF-κB signalling and IL-6 expression by silencing HMGA1 and MALT1
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Ann-Bin Shyu, Yi Fang Ho, Chyi-Ying A. Chen, and Jeffrey T. Chang
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0301 basic medicine ,Lung Neoplasms ,medicine.medical_treatment ,Down-Regulation ,Adenocarcinoma ,Cell Line ,03 medical and health sciences ,RNA interference ,microRNA ,Genetics ,medicine ,Humans ,Gene silencing ,Gene Silencing ,HMGA1a Protein ,Molecular Biology ,3' Untranslated Regions ,biology ,Interleukin-6 ,Tumor Necrosis Factor-alpha ,NF-kappa B ,NFKB1 ,HMGA1 ,Hedgehog signaling pathway ,Neoplasm Proteins ,MicroRNAs ,030104 developmental biology ,Cytokine ,A549 Cells ,Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein ,Caspases ,biology.protein ,Cancer research ,Signal transduction ,Transcriptome ,Signal Transduction - Abstract
MiR-26 has emerged as a key tumour suppressor in various cancers. Accumulating evidence supports that miR-26 regulates inflammation and tumourigenicity largely through down-regulating IL-6 production, but the underlying mechanism remains obscure. Here, combining a transcriptome-wide approach with manipulation of cellular miR-26 levels, we showed that instead of directly targeting IL-6 mRNA for gene silencing, miR-26 diminishes IL-6 transcription activated by TNF-α through silencing NF-κB signalling related factors HMGA1 and MALT1. We demonstrated that miR-26 extensively dampens the induction of many inflammation-related cytokine, chemokine and tissue-remodelling genes that are activated via NF-κB signalling pathway. Knocking down both HMGA1 and MALT1 by RNAi had a silencing effect on NF-κB-responsive genes similar to that caused by miR-26. Moreover, we discovered that poor patient prognosis in human lung adenocarcinoma is associated with low miR-26 and high HMGA1 or MALT1 levels and not with levels of any of them individually. These new findings not only unravel a novel mechanism by which miR-26 dampens IL-6 production transcriptionally but also demonstrate a direct role of miR-26 in down-regulating NF-κB signalling pathway, thereby revealing a more critical and broader role of miR-26 in inflammation and cancer than previously realized.
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- 2016
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7. ROCK inhibition enhances microRNA function by promoting deadenylation of targeted mRNAs via increasing PAIP2 expression
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Jiahuai Han, Young Jun Kang, Chikako Shibata, Felicia Han, Takeshi Yoshikawa, Takahiro Kishikawa, Motoyuki Otsuka, Kazuhiko Koike, Motoko Ohno, Ann-Bin Shyu, Jianfeng Wu, Akemi Takata, and Chyi-Ying A. Chen
- Subjects
Pyridines ,RNA-binding protein ,Biology ,Cell Line ,03 medical and health sciences ,Mice ,0302 clinical medicine ,microRNA ,Genetics ,Animals ,Humans ,ROCK1 ,RNA, Messenger ,Enzyme Inhibitors ,Protein kinase A ,Transcription factor ,030304 developmental biology ,Regulation of gene expression ,0303 health sciences ,rho-Associated Kinases ,RNA-Binding Proteins ,Molecular biology ,Amides ,Cell biology ,Repressor Proteins ,MicroRNAs ,HEK293 Cells ,Hepatocyte nuclear factor 4 ,Gene Expression Regulation ,Hepatocyte Nuclear Factor 4 ,Hepatocyte nuclear factor 4 alpha ,030220 oncology & carcinogenesis ,RNA ,Caco-2 Cells - Abstract
The reduced expression levels and functional impairment of global miRNAs are related to various human diseases, including cancers. However, relatively little is known about how global miRNA function may be upregulated. Here, we report that global miRNA function can be enhanced by Rho-associated, coiled-coil-containing protein kinase (ROCK) inhibitors. The regulation of miRNA function by ROCK inhibitors is mediated, at least in part, by poly(A)-binding protein-interacting protein 2 (PAIP2), which enhances poly(A)-shortening of miRNA-targeted mRNAs and leads to global upregulation of miRNA function. In the presence of a ROCK inhibitor, PAIP2 expression is enhanced by the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) through increased ROCK1 nuclear localization and enhanced ROCK1 association with HNF4A. Our data reveal an unexpected role of ROCK1 as a cofactor of HNF4A in enhancing PAIP2 transcription. ROCK inhibitors may be useful for the various pathologies associated with the impairment of global miRNA function.
- Published
- 2015
8. Correction for Ezzeddine et al., 'Evidence Providing New Insights into TOB-Promoted Deadenylation and Supporting a Link between TOB's Deadenylation-Enhancing and Antiproliferative Activities'
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Chyi-Ying A. Chen, Ann-Bin Shyu, and Nader Ezzeddine
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Tumor Suppressor Proteins ,Intracellular Signaling Peptides and Proteins ,Proteins ,Cell Cycle Proteins ,Cell Biology ,Computational biology ,Biology ,Poly(A)-Binding Proteins ,Cell Line ,Repressor Proteins ,Mice ,Ribonucleases ,Gene Expression Regulation ,Exoribonucleases ,NIH 3T3 Cells ,Animals ,Humans ,RNA, Messenger ,Author Correction ,Molecular Biology ,Cell Proliferation ,Protein Binding - Abstract
The mammalian TOB1 and TOB2 proteins have emerged as key players in repressing cell proliferation. Accumulating evidence indicates that TOBs regulate mRNA deadenylation. A recruitment model was proposed in which TOBs promote deadenylation by recruiting CAF1-CCR4 deadenylase complex to the 3' end of mRNAs by simultaneously binding CAF1 and PABP. However, the exact molecular mechanism underlying TOB-promoted deadenylation remains unclear. It is also unclear whether TOBs' antiproliferative and deadenylation-promoting activities are connected. Here, we combine biochemical analyses with a functional assay directly monitoring deadenylation and mRNA decay to characterize the effects of tethering TOBs or their mutant derivatives to mRNAs. The results provide direct evidence supporting the recruitment model and reveal a link between TOBs' antiproliferative and deadenylation-promoting activities. We also find that TOBs' actions in deadenylation are independent of the phosphorylation state of three serines known to regulate antiproliferative actions, suggesting that TOBs arrest cell growth through at least two different mechanisms. TOB1 and TOB2 were interchangeable in the properties tested here, indicating considerable functional redundancy between the two proteins. We propose that their multiple modes of modulating mRNA turnover and arresting cell growth permit the TOB proteins to coordinate their diverse roles in controlling cell growth and differentiation.
- Published
- 2017
9. Antagonistic actions of two human Pan3 isoforms on global mRNA turnover
- Author
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Ann-Bin Shyu, Jeffrey T. Chang, Leng Han, Chyi-Ying A. Chen, Yu Xiang, and Yueqiang Zhang
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0301 basic medicine ,Gene isoform ,Polyadenylation ,RNA Stability ,Plasma protein binding ,Biology ,Cell Line ,03 medical and health sciences ,Mice ,Gene expression ,microRNA ,Animals ,Humans ,Protein Isoforms ,Protein Interaction Domains and Motifs ,RNA, Messenger ,Molecular Biology ,Cell Proliferation ,Regulation of gene expression ,Gene knockdown ,Messenger RNA ,Molecular biology ,Cell biology ,MicroRNAs ,030104 developmental biology ,Gene Expression Regulation ,Gene Knockdown Techniques ,Exoribonucleases ,Mutation ,Carrier Proteins ,Poly A ,Transcriptome ,Corrigendum ,Protein Binding - Abstract
Deadenylation is a fundamental process that regulates eukaryotic gene expression. Mammalian deadenylation exhibits biphasic kinetics, with the Pan2–Pan3 and Ccr4–Caf1 deadenylase complexes mediating the first and second phase, respectively; however, the significance of the biphasic nature of deadenylation in mRNA turnover remains unclear. In this study, we discovered that two distinct isoforms of human Pan3 display opposing properties necessary for coordinating the two phases of deadenylation. The shorter isoform (Pan3S) interacts more strongly with PABP than the longer isoform (Pan3L) does. Pan2 deadenylase activity is enhanced by Pan3S but suppressed by Pan3L. Knocking down individual Pan3 isoforms has opposing effects on the global poly(A) tail length profile, P-body formation, and different mRNA decay pathways. Transcriptome-wide analysis of Pan3 knockdown effects on mRNA turnover shows that depleting either Pan3 isoform causes profound and extensive changes in mRNA stability globally. These results reveal a new fundamental step governing mammalian mRNA metabolism. We propose that the first phase of deadenylation, coordinated through the interplay among the two Pan3 isoforms, Pan2, and PABP, represents a cytoplasmic mRNA maturation step important for proper mRNA turnover.
- Published
- 2017
10. Phosphorylation at intrinsically disordered regions of PAM2 motif-containing proteins modulates their interactions with PABPC1 and influences mRNA fate
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Amanda B. Chadee, Yueqiang Zhang, Chyi-Ying A. Chen, Kai Lieh Huang, and Ann-Bin Shyu
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Threonine ,Cytoplasm ,Amino Acid Motifs ,Biology ,Transfection ,Intrinsically disordered proteins ,Poly(A)-Binding Protein I ,Mice ,PABPC1 ,Report ,Serine ,Animals ,Humans ,Gene silencing ,Protein phosphorylation ,Amino Acid Sequence ,RNA, Messenger ,Phosphorylation ,Molecular Biology ,Peptide sequence ,Cells, Cultured ,Binding Sites ,Cell biology ,Biochemistry ,NIH 3T3 Cells ,Poly A - Abstract
Cytoplasmic poly(A)-binding protein (PABP) C1 recruits different interacting partners to regulate mRNA fate. The majority of PABP-interacting proteins contain a PAM2 motif to mediate their interactions with PABPC1. However, little is known about the regulation of these interactions or the corresponding functional consequences. Through in silico analysis, we found that PAM2 motifs are generally embedded within an extended intrinsic disorder region (IDR) and are located next to cluster(s) of potential serine (Ser) or threonine (Thr) phosphorylation sites within the IDR. We hypothesized that phosphorylation at these Ser/Thr sites regulates the interactions between PAM2-containing proteins and PABPC1. In the present study, we have tested this hypothesis using complementary approaches to increase or decrease phosphorylation. The results indicate that changing the extent of phosphorylation of three PAM2-containing proteins (Tob2, Pan3, and Tnrc6c) alters their ability to interact with PABPC1. Results from experiments using phospho-blocking or phosphomimetic mutants in PAM2-containing proteins further support our hypothesis. Moreover, the phosphomimetic mutations appreciably affected the functions of these proteins in mRNA turnover and gene silencing. Taken together, these results provide a new framework for understanding the roles of intrinsically disordered proteins in the dynamic and signal-dependent control of cytoplasmic mRNA functions.
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- 2013
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11. 3' UTR shortening represses tumor-suppressor genes in trans by disrupting ceRNA crosstalk
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Hyun Jung Park, Ann-Bin Shyu, Soyeon Kim, Ping Ji, Kaifu Chen, Lei Li, Wei Li, Chioniso P. Masamha, Benjamin Rodriguez, Eric J. Wagner, Jianzhong Su, David Baillat, Zheng Xia, Camila R. Fontes-Garfias, and Joel R. Neilson
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0301 basic medicine ,Polyadenylation ,Biology ,Proto-Oncogene Mas ,Article ,03 medical and health sciences ,Cell Line, Tumor ,Neoplasms ,Proto-Oncogenes ,Genetics ,Humans ,Genes, Tumor Suppressor ,RNA, Messenger ,Genes, Suppressor ,Psychological repression ,3' Untranslated Regions ,Regulation of gene expression ,Gene knockdown ,Competing endogenous RNA ,Three prime untranslated region ,Cell biology ,Gene Expression Regulation, Neoplastic ,Crosstalk (biology) ,MicroRNAs ,030104 developmental biology ,NFIA ,MCF-7 Cells ,RNA ,HeLa Cells - Abstract
Widespread mRNA 3′ UTR shortening through alternative polyadenylation 1 promotes tumor growth in vivo 2 . A prevailing hypothesis is that it induces proto-oncogene expression in cis through escaping microRNA-mediated repression. Here we report a surprising enrichment of 3′UTR shortening among transcripts that are predicted to act as competing-endogenous RNAs (ceRNAs) for tumor-suppressor genes. Our model-based analysis of the trans effect of 3′ UTR shortening (MAT3UTR) reveals a significant role in altering ceRNA expression. MAT3UTR predicts many trans-targets of 3′ UTR shortening, including PTEN, a crucial tumor-suppressor gene 3 involved in ceRNA crosstalk 4 with nine 3′UTR-shortening genes, including EPS15 and NFIA. Knockdown of NUDT21, a master 3′ UTR-shortening regulator 2 , represses tumor-suppressor genes such as PHF6 and LARP1 in trans in a miRNA-dependent manner. Together, the results of our analysis suggest a major role of 3′ UTR shortening in repressing tumor-suppressor genes in trans by disrupting ceRNA crosstalk, rather than inducing proto-oncogenes in cis. Shortening of mRNA 3′ UTRs is often observed in cancer. A combination of model-based analysis and experiments suggests that 3′ UTR shortening disrupts competing endogenous RNA crosstalk, thus influencing tumor-suppressor expression in trans.
- Published
- 2016
12. CFIm25 regulates glutaminase alternative terminal exon definition to modulate miR-23 function
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Zheng Xia, Natoya Peart, Scott D. Collum, Chioniso P. Masamha, Wei Li, Ann-Bin Shyu, and Eric J. Wagner
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0301 basic medicine ,Gene isoform ,Polyadenylation ,Biology ,03 medical and health sciences ,Exon ,Glutaminase ,Report ,microRNA ,Humans ,Molecular Biology ,Psychological repression ,3' Untranslated Regions ,Alternative splicing ,Exons ,Isoenzymes ,Alternative Splicing ,MicroRNAs ,030104 developmental biology ,HEK293 Cells ,Biochemistry ,RNA splicing ,Poly A ,HeLa Cells - Abstract
Alternative polyadenylation (APA) and alternative splicing (AS) provide mRNAs with the means to avoid microRNA repression through selective shortening or differential usage of 3′UTRs. The two glutaminase (GLS) mRNA isoforms, termed KGA and GAC, contain distinct 3′UTRs with the KGA isoform subject to repression by miR-23. We show that depletion of the APA regulator CFIm25 causes a strong shift to the usage of a proximal poly(A) site within the KGA 3′UTR and also alters splicing to favor exclusion of the GAC 3′UTR. Surprisingly, we observe that while miR-23 is capable of down-regulating the shortened KGA 3′UTR, it has only minor impact on the full-length KGA 3′UTR, demonstrating that additional potent negative regulation of GLS expression exists beyond this single microRNA targeting site. Finally, we show that the apoptosis induced upon down-regulation of the GAC isoform can be alleviated through concurrent reduction in CFIm25 expression, revealing the sensitivity of glutaminase expression to the levels of RNA processing factors. These results exemplify the complex interplay between RNA processing and microRNA repression in controlling glutamine metabolism in cancer cells.
- Published
- 2016
13. Evidence Providing New Insights into TOB-Promoted Deadenylation and Supporting a Link between TOB's Deadenylation-Enhancing and Antiproliferative Activities
- Author
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Nader Ezzeddine, Ann-Bin Shyu, and Chyi-Ying A. Chen
- Subjects
Regulation of gene expression ,Messenger RNA ,biology ,Cell growth ,Mutant ,Articles ,Cell Biology ,Plasma protein binding ,Cell biology ,Poly(A)-binding protein ,biology.protein ,Phosphorylation ,Cell Cycle Protein ,Molecular Biology - Abstract
The mammalian TOB1 and TOB2 proteins have emerged as key players in repressing cell proliferation. Accumulating evidence indicates that TOBs regulate mRNA deadenylation. A recruitment model was proposed in which TOBs promote deadenylation by recruiting CAF1-CCR4 deadenylase complex to the 3′ end of mRNAs by simultaneously binding CAF1 and PABP. However, the exact molecular mechanism underlying TOB-promoted deadenylation remains unclear. It is also unclear whether TOBs' antiproliferative and deadenylation-promoting activities are connected. Here, we combine biochemical analyses with a functional assay directly monitoring deadenylation and mRNA decay to characterize the effects of tethering TOBs or their mutant derivatives to mRNAs. The results provide direct evidence supporting the recruitment model and reveal a link between TOBs' antiproliferative and deadenylation-promoting activities. We also find that TOBs' actions in deadenylation are independent of the phosphorylation state of three serines known to regulate antiproliferative actions, suggesting that TOBs arrest cell growth through at least two different mechanisms. TOB1 and TOB2 were interchangeable in the properties tested here, indicating considerable functional redundancy between the two proteins. We propose that their multiple modes of modulating mRNA turnover and arresting cell growth permit the TOB proteins to coordinate their diverse roles in controlling cell growth and differentiation.
- Published
- 2012
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14. RNA Foci, CUGBP1, and ZNF9 Are the Primary Targets of the Mutant CUG and CCUG Repeats Expanded in Myotonic Dystrophies Type 1 and Type 2
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Benedikt Schoser, Nikolai A. Timchenko, Giovanni Meola, Partha S. Sarkar, Claudia Huichalaf, Lubov Timchenko, Bingwen Jin, Karlie Jones, Ann-Bin Shyu, Christiane Schneider-Gold, and Polina Iakova
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congenital, hereditary, and neonatal diseases and abnormalities ,Transcription, Genetic ,Mutant ,RNA-binding protein ,Biology ,Myotonic dystrophy ,CELF1 Protein ,Cell Line ,Pathology and Forensic Medicine ,chemistry.chemical_compound ,Transcription (biology) ,medicine ,Humans ,Myotonic Dystrophy ,MBNL1 ,RNA-Binding Proteins ,RNA ,Regular Article ,medicine.disease ,Molecular biology ,chemistry ,Cytoplasm ,Doxycycline ,Mutation ,Myotonic Disorders - Abstract
Expansions of noncoding CUG and CCUG repeats in myotonic dystrophies type 1 (DM1) and DM2 cause complex molecular pathology, the features of which include accumulation of RNA aggregates and misregulation of the RNA-binding proteins muscleblind-like 1 (MBNL1) and CUG-binding protein 1 (CUGBP1). CCUG repeats also decrease amounts of the nucleic acid binding protein ZNF9. Using tetracycline (Tet)-regulated monoclonal cell models that express CUG and CCUG repeats, we found that low levels of long CUG and CCUG repeats result in nuclear and cytoplasmic RNA aggregation with a simultaneous increase of CUGBP1 and a reduction of ZNF9. Elevation of CUGBP1 and reduction of ZNF9 were also observed before strong aggregation of the mutant CUG/CCUG repeats. Degradation of CUG and CCUG repeats normalizes ZNF9 and CUGBP1 levels. Comparison of short and long CUG and CCUG RNAs showed that great expression of short repeats form foci and alter CUGBP1 and ZNF9; however, long CUG/CCUG repeats misregulate CUGBP1 and ZNF9 much faster than high levels of the short repeats. These data suggest that correction of DM1 and DM2 might be achieved by complete and efficient degradation of CUG and CCUG repeats or by a simultaneous disruption of CUG/CCUG foci and correction of CUGBP1 and ZNF9.
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- 2011
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15. Hu antigen R and tristetraprolin: Counter-regulators of rat apical sodium-dependent bile acid transporter by way of effects on messenger RNA stability
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Frank Chen, Benjamin L. Shneider, and Ann-Bin Shyu
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medicine.medical_specialty ,Hepatology ,Bile acid ,Organic anion transporter 1 ,Reabsorption ,medicine.drug_class ,MRNA stabilization ,Biology ,Apical membrane ,digestive system ,Intestinal absorption ,Endocrinology ,Internal medicine ,Symporter ,medicine ,biology.protein ,Enterohepatic circulation - Abstract
The apical sodium dependent bile acid transporter (ASBT) is the major carrier protein involved in the ileal reabsorption of bile acids (1, 2). ASBT also transports bile acids across the apical membrane of renal proximal convoluted tubule cells and cholangiocytes. Ileal transport plays a critical role in the enterohepatic circulation of bile salts. Bile acids are essential for normal liver function, in particular for maintenance of bile flow. In addition, they are essential for intestinal absorption of fat and fat-soluble vitamins. ASBT mediated ileal bile acid transport leads to physiologically relevant signaling to the gallbladder and liver via ileal secretion of fibroblast growth factor-19 (3, 4). Both deficiency and surplus of bile acids can lead to liver-based pathologic processes. As such, a number of mechanisms exist permitting tight regulation of bile acid homeostasis, thereby preventing disease (5). The regulation of ASBT expression is complex and has been the subject of many recent investigations. Mechanisms of transcriptional control of ASBT expression have been elucidated over the past 10 years (1). More recent studies have implicated post-transcriptional processes in regulating ASBT expression (6-8). Normal ontogeny of ileal ASBT expression in the rat is biphasic, with fetal expression, postnatal repression and induction at weaning (9). Postnatal repression of ASBT expression may provide a critical signal to enhance hepatic synthesis of bile acids thereby expanding the bile acid pool size. Descriptive analyses of the ontogeny of ASBT in rat ileum and kidney suggest that ASBT expression may be controlled in part by regulated changes in mRNA stability (10, 11). During normal development in the rat ileum there is a greater than 100-fold increase in steady-state ASBT mRNA levels, while there is only a 10-fold difference in ASBT transcription as assessed by nuclear run-on assays (10, 11). In preweaning kidney steady-state ASBT mRNA levels are 10-fold higher than in the ileum, yet transcription rates are similar. These findings suggest that mRNA stabilization contributes to the steady-state accumulation of ASBT mRNA in the adult ileum. Moreover, they also support that differential stabilization of ASBT mRNA plays a critical role in controlling ASBT expression in a tissue-specific manner. Currently, there are no data that describe a molecular mechanism for the regulation of ASBT expression via changes in mRNA stability and thus the following investigations were undertaken.
- Published
- 2011
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16. Unraveling regulation and new components of human P-bodies through a protein interaction framework and experimental validation
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Chyi-Ying A. Chen, Dinghai Zheng, and Ann-Bin Shyu
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RNA Stability ,In silico ,Blotting, Western ,Transfection ,F-box protein ,Heterogeneous-Nuclear Ribonucleoproteins ,Protein–protein interaction ,Mice ,P-bodies ,Animals ,Humans ,Protein phosphorylation ,RNA, Messenger ,Phosphorylation ,RNA Processing, Post-Transcriptional ,RNA, Small Interfering ,Molecular Biology ,Regulation of gene expression ,biology ,Computational Biology ,Translation (biology) ,Protein ubiquitination ,Cell biology ,14-3-3 Proteins ,Gene Expression Regulation ,Gene Knockdown Techniques ,Perspective ,NIH 3T3 Cells ,biology.protein ,Cytoplasmic Structures ,RNA Interference ,Protein Processing, Post-Translational - Abstract
The cellular factors involved in mRNA degradation and translation repression can aggregate into cytoplasmic domains known as GW bodies or mRNA processing bodies (P-bodies). However, current understanding of P-bodies, especially the regulatory aspect, remains relatively fragmentary. To provide a framework for studying the mechanisms and regulation of P-body formation, maintenance, and disassembly, we compiled a list of P-body proteins found in various species and further grouped both reported and predicted human P-body proteins according to their functions. By analyzing protein–protein interactions of human P-body components, we found that many P-body proteins form complex interaction networks with each other and with other cellular proteins that are not recognized as P-body components. The observation suggests that these other cellular proteins may play important roles in regulating P-body dynamics and functions. We further used siRNA-mediated gene knockdown and immunofluorescence microscopy to demonstrate the validity of our in silico analyses. Our combined approach identifies new P-body components and suggests that protein ubiquitination and protein phosphorylation involving 14-3-3 proteins may play critical roles for post-translational modifications of P-body components in regulating P-body dynamics. Our analyses provide not only a global view of human P-body components and their physical interactions but also a wealth of hypotheses to help guide future research on the regulation and function of human P-bodies.
- Published
- 2011
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17. Mechanisms of deadenylation-dependent decay
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Chyi-Ying A. Chen and Ann-Bin Shyu
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Untranslated region ,Messenger RNA ,Nonsense-mediated decay ,microRNA ,P-bodies ,Gene silencing ,RNA ,Biology ,Molecular Biology ,Biochemistry ,Molecular biology ,mRNA surveillance ,Cell biology - Abstract
Degradation of messenger RNAs (mRNAs) plays an essential role in modulation of gene expression and in quality control of mRNA biogenesis. Nearly all major mRNA decay pathways characterized thus far in eukaryotes are initiated by deadenylation, i.e., shortening of the mRNA 3(') poly(A) tail. Deadenylation is often a rate-limiting step for mRNA degradation and translational silencing, making it an important control point for both processes. In this review, we discuss the fundamental principles that govern mRNA deadenylation in eukaryotes. We use several major mRNA decay pathways in mammalian cells to illustrate mechanisms and regulation of deadenylation-dependent mRNA decay, including decay directed by adenine/uridine-rich elements (AREs) in the 3(') -untranslated region (UTR), the rapid decay mediated by destabilizing elements in protein-coding regions, the surveillance mechanism that detects and degrades nonsense-containing mRNA [i.e., nonsense-mediated decay (NMD)], the decay directed by miRNAs, and the default decay pathway for stable messages. Mammalian mRNA deadenylation involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. Decapping takes place after deadenylation and may serve as a backup mechanism to trigger mRNA decay if initial deadenylation is compromised. In addition, we discuss how deadenylation impacts the dynamics of RNA processing bodies (P-bodies), where nontranslatable mRNAs can be degraded or stored. Possible models for mechanisms of various deadenylation-dependent mRNA decay pathways are also discussed.
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- 2010
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18. BTG/TOB factors impact deadenylases
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Fabienne Mauxion, Ann-Bin Shyu, Bertrand Séraphin, and Chyi-Ying A. Chen
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Genetics ,Regulation of gene expression ,BTG2 ,Protein Conformation ,Tumor Suppressor Proteins ,Protein subunit ,Cell Cycle Proteins ,Biology ,Models, Biological ,Biochemistry ,Article ,Cell biology ,Protein structure ,Apoptosis ,Transcriptional regulation ,Animals ,Humans ,Cell Cycle Protein ,Molecular Biology ,Function (biology) - Abstract
BTG/TOB factors are a family of antiproliferative proteins whose expression is altered in numerous cancers. They have been implicated in cell differentiation, development and apoptosis. Although proposed to affect transcriptional regulation, these factors interact with CAF1, a subunit of the main eukaryotic deadenylase, and with poly(A)-binding-proteins, strongly suggesting a role in post-transcriptional regulation of gene expression. The recent determination of the structures of BTG2, TOB1 N-terminal domain (TOB1N138) and TOB1N138–Caf1 complexes support a role for BTG/TOB proteins in mRNA deadenylation, a function corroborated by recently published functional characterizations. We highlight possible molecular mechanisms by which BTG/TOB proteins influence deadenylation and discuss the need for a better understanding of BTG/TOB physiological functions.
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- 2009
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19. Ago–TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps
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Chyi-Ying A. Chen, Ann-Bin Shyu, Zhenfang Xia, and Dinghai Zheng
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RNA Stability ,Blotting, Western ,Eukaryotic Initiation Factor-2 ,Nonsense-mediated decay ,Mutant ,Biology ,Autoantigens ,Models, Biological ,mRNA turnover ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Structural Biology ,P-bodies ,microRNA ,Animals ,Humans ,Immunoprecipitation ,RNA-Induced Silencing Complex ,Gene silencing ,RNA, Small Interfering ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Gene knockdown ,Messenger RNA ,Caf1 ,RNA-Binding Proteins ,Pan2 ,deadenylation ,Argonaute ,Blotting, Northern ,Molecular biology ,Argonaute protein ,Cell biology ,TNRC6 ,MicroRNAs ,Gene Knockdown Techniques ,Argonaute Proteins ,NIH 3T3 Cells ,030217 neurology & neurosurgery ,Protein Binding - Abstract
MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain largely unclear. Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.
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- 2009
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20. Coordinated Changes in mRNA Turnover, Translation, and RNA Processing Bodies in Bronchial Epithelial Cells following Inflammatory Stimulation
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Ling Song, Ann-Bin Shyu, Yuxin Zhai, Michael R. Blackburn, Chyi-Ying A. Chen, Zhenping Zhong, and Zhenfang Xia
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Chemokine ,RNA Stability ,Bronchi ,Inflammation ,Cell Line ,Proinflammatory cytokine ,Mice ,Gene expression ,P-bodies ,medicine ,Animals ,Humans ,RNA, Messenger ,Interleukin 8 ,Molecular Biology ,Chemokine CCL2 ,Regulation of gene expression ,biology ,Tumor Necrosis Factor-alpha ,Interleukin-8 ,Epithelial Cells ,Articles ,Cell Biology ,Molecular biology ,Cell biology ,MicroRNAs ,Gene Expression Regulation ,RNA, Ribosomal ,Protein Biosynthesis ,biology.protein ,Tumor necrosis factor alpha ,Interleukin-4 ,medicine.symptom - Abstract
Bronchial epithelial cells play a pivotal role in airway inflammation, but little is known about posttranscriptional regulation of mediator gene expression during the inflammatory response in these cells. Here, we show that activation of human bronchial epithelial BEAS-2B cells by proinflammatory cytokines interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) leads to an increase in the mRNA stability of the key chemokines monocyte chemotactic protein 1 and IL-8, an elevation of the global translation rate, an increase in the levels of several proteins critical for translation, and a reduction of microRNA-mediated translational repression. Moreover, using the BEAS-2B cell system and a mouse model, we found that RNA processing bodies (P bodies), cytoplasmic domains linked to storage and/or degradation of translationally silenced mRNAs, are significantly reduced in activated bronchial epithelial cells, suggesting a physiological role for P bodies in airway inflammation. Our study reveals an orchestrated change among posttranscriptional mechanisms, which help sustain high levels of inflammatory mediator production in bronchial epithelium during the pathogenesis of inflammatory airway diseases.
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- 2008
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21. Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells
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Wenmiao Zhu, Ann-Bin Shyu, Chyi-Ying A. Chen, Xiangwei He, Dinghai Zheng, and Nader Ezzeddine
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Receptors, CCR4 ,Polyadenylation ,RNA Stability ,Nonsense-mediated decay ,Biology ,Models, Biological ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Ribonucleases ,Animals ,Humans ,RNA, Messenger ,Research Articles ,030304 developmental biology ,Ribonucleoprotein ,0303 health sciences ,Gene knockdown ,Messenger RNA ,030302 biochemistry & molecular biology ,RNA ,Proteins ,Cell Biology ,Molecular biology ,Transport protein ,Cell biology ,Repressor Proteins ,Protein Transport ,chemistry ,Puromycin ,Multiprotein Complexes ,Exoribonucleases ,NIH 3T3 Cells ,Cytoplasmic Structures ,Poly A ,Protein Binding - Abstract
Deadenylation is the major step triggering mammalian mRNA decay. One consequence of deadenylation is the formation of nontranslatable messenger RNA (mRNA) protein complexes (messenger ribonucleoproteins [mRNPs]). Nontranslatable mRNPs may accumulate in P-bodies, which contain factors involved in translation repression, decapping, and 5′-to-3′ degradation. We demonstrate that deadenylation is required for mammalian P-body formation and mRNA decay. We identify Pan2, Pan3, and Caf1 deadenylases as new P-body components and show that Pan3 helps recruit Pan2, Ccr4, and Caf1 to P-bodies. Pan3 knockdown causes a reduction of P-bodies and has differential effects on mRNA decay. Knocking down Caf1 or overexpressing a Caf1 catalytically inactive mutant impairs deadenylation and mRNA decay. P-bodies are not detected when deadenylation is blocked and are restored when the blockage is released. When deadenylation is impaired, P-body formation is not restorable, even when mRNAs exit the translating pool. These results support a dynamic interplay among deadenylation, mRNP remodeling, and P-body formation in selective decay of mammalian mRNA.
- Published
- 2008
22. Human TOB, an Antiproliferative Transcription Factor, Is a Poly(A)-Binding Protein-Dependent Positive Regulator of Cytoplasmic mRNA Deadenylation
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Dinghai Zheng, Nader Ezzeddine, Chyi-Ying A. Chen, Wenmiao Zhu, Yukiko M. Yamashita, Ann-Bin Shyu, Tsung Cheng Chang, Akio Yamashita, and Zhenping Zhong
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Polyadenylation ,Immunoprecipitation ,Phenylalanine ,RNA Stability ,Recombinant Fusion Proteins ,Poly(A)-Binding Protein I ,Mice ,PABPC1 ,Chlorocebus aethiops ,Poly(A)-binding protein ,CCR4-NOT complex ,Animals ,Humans ,RNA, Messenger ,RNA, Small Interfering ,Molecular Biology ,Transcription factor ,Messenger RNA ,biology ,Tumor Suppressor Proteins ,Intracellular Signaling Peptides and Proteins ,Articles ,Cell Biology ,Molecular biology ,Protein Structure, Tertiary ,Multiprotein Complexes ,COS Cells ,Exoribonucleases ,NIH 3T3 Cells ,biology.protein ,Transcription Factors - Abstract
In mammalian cells, mRNA decay begins with deadenylation, which involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. The regulation of the critical deadenylation step and its relationship with RNA-processing bodies (P-bodies), which are thought to be a site where poly(A)-shortened mRNAs get degraded, are poorly understood. Using the Tet-Off transcriptional pulsing approach to investigate mRNA decay in mouse NIH 3T3 fibroblasts, we found that TOB, an antiproliferative transcription factor, enhances mRNA deadenylation in vivo. Results from glutathione S-transferase pull-down and coimmunoprecipitation experiments indicate that TOB can simultaneously interact with the poly(A) nuclease complex CCR4-CAF1 and the cytoplasmic poly(A)-binding protein, PABPC1. Combining these findings with those from mutagenesis studies, we further identified the protein motifs on TOB and PABPC1 that are necessary for their interaction and found that interaction with PABPC1 is necessary for TOB's deadenylation-enhancing effect. Moreover, our immunofluorescence microscopy results revealed that TOB colocalizes with P-bodies, suggesting a role of TOB in linking deadenylation to the P-bodies. Our findings reveal a new mechanism by which the fate of mammalian mRNA is modulated at the deadenylation step by a protein that recruits poly(A) nuclease(s) to the 3′ poly(A) tail-PABP complex.
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- 2007
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23. Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells
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Yukiko M. Yamashita, Zhenping Zhong, Wenmiao Zhu, Akio Yamashita, Ann-Bin Shyu, Chyi-Ying A. Chen, and Tsung Cheng Chang
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Cell type ,Gene knockdown ,Messenger RNA ,Transcription, Genetic ,RNA Stability ,Cell Cycle ,Method ,Transfection ,Biology ,Cell cycle ,Molecular biology ,Cell Line ,Cell biology ,Kinetics ,Cell culture ,Cell Line, Tumor ,Gene expression ,Humans ,RNA, Messenger ,RNA, Small Interfering ,Promoter Regions, Genetic ,Proto-Oncogene Proteins c-fos ,Molecular Biology ,K562 cells - Abstract
Development of transcriptional pulsing approaches using the c-fos and Tet-off promoter systems greatly facilitated studies of mRNA turnover in mammalian cells. However, optimal protocols for these approaches vary for different cell types and/or physiological conditions, limiting their widespread application. In this study, we have further optimized transcriptional pulsing systems for different cell lines and developed new protocols to facilitate investigation of various aspects of mRNA turnover. We apply the Tet-off transcriptional pulsing strategy to investigate ARE-mediated mRNA decay in human erythroleukemic K562 cells arrested at various phases of the cell cycle by pharmacological inhibitors. This application facilitates studies of the role of mRNA stability in control of cell-cycle dependent gene expression. To advance the investigation of factors involved in mRNA turnover and its regulation, we have also incorporated recently developed transfection and siRNA reagents into the transcriptional pulsing approach. Using these protocols, siRNA and DNA plasmids can be effectively cotransfected into mouse NIH3T3 cells to obtain high knockdown efficiency. Moreover, we have established a tTA-harboring stable line using human bronchial epithelial BEAS-2B cells and applied the transcriptional pulsing approach to monitor mRNA deadenylation and decay kinetics in this cell system. This broadens the application of the transcriptional pulsing system to investigate the regulation of mRNA turnover related to allergic inflammation. Critical factors that need to be considered when employing these approaches are characterized and discussed.
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- 2007
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24. Poly(A) Nuclease Interacts with the C-terminal Domain of Polyadenylate-binding Protein Domain from Poly(A)-binding Protein
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Nadeem Siddiqui, Jeanne Marie Palermino, Kalle Gehring, Tsung Cheng Chang, Ann-Bin Shyu, and David A. Mangus
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Magnetic Resonance Spectroscopy ,Saccharomyces cerevisiae Proteins ,Protein subunit ,Amino Acid Motifs ,Molecular Sequence Data ,Protein domain ,Poly(A)-Binding Proteins ,Biochemistry ,Evolution, Molecular ,Fungal Proteins ,Ribonucleases ,Protein structure ,Two-Hybrid System Techniques ,Poly(A)-binding protein ,Humans ,Amino Acid Sequence ,RNA, Messenger ,Molecular Biology ,Messenger RNA ,Sequence Homology, Amino Acid ,biology ,Cell Biology ,Messenger ribonucleoprotein complex ,Molecular biology ,Protein Structure, Tertiary ,Cell biology ,Exoribonucleases ,biology.protein ,Sequence motif ,Sterile alpha motif ,Protein Binding - Abstract
The poly(A)-binding protein (PABP) is an essential protein found in all eukaryotes and is involved in an extensive range of cellular functions, including translation, mRNA metabolism, and mRNA export. Its C-terminal region contains a peptide-interacting PABC domain that recruits proteins containing a highly specific PAM-2 sequence motif to the messenger ribonucleoprotein complex. In humans, these proteins, including Paip1, Paip2, eRF3 (eukaryotic release factor 3), Ataxin-2, and Tob2, are all found to regulate translation through varying mechanisms. The following reports poly(A) nuclease (PAN) as a PABC-interacting partner in both yeast and humans. Their interaction is mediated by a PAM-2 motif identified within the PAN3 subunit. This site was identified in various fungal and animal species suggesting that the interaction is conserved throughout evolution. Our results indicate that PABP is directly involved in recruiting a deadenylase to the messenger ribonucleoprotein complex. This demonstrates a novel role for the PABC domain in mRNA metabolic processes and gives further insight into the function of PABP in mRNA maturation, export, and turnover.
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- 2007
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25. Comparative Peptide Binding Studies of the PABC Domains from the Ubiquitin-protein Isopeptide Ligase HYD and Poly(A)-binding Protein
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Guennadi Kozlov, Nadia S Lim, Laurent Volpon, Ann-Bin Shyu, Olivia Groover, Gregory De Crescenzo, Kalle Gehring, Tsung Cheng Chang, and Nadeem Siddiqui
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chemistry.chemical_classification ,DNA ligase ,biology ,Immunoprecipitation ,Isothermal titration calorimetry ,Peptide ,Peptide binding ,Cell Biology ,Protein degradation ,Biochemistry ,chemistry ,Ubiquitin ,Poly(A)-binding protein ,biology.protein ,Molecular Biology - Abstract
The PABC domain is a peptide-binding domain that is specifically found in poly(A)-binding protein (PABP) and a HECT ubiquitin-protein isopeptide ligase (E3) known as HYD (hyperplastic discs), EDD (E3 isolated by differential display), or Rat100. The PABC domain of PABP recruits various regulatory proteins and translation factors to poly(A) mRNAs through binding of a conserved 12-amino acid peptide motif, PAM2 (PABP-interacting motif 2). In contrast, little is known about the specificity or function of the domain from HYD. Here, we used isothermal calorimetry and surface plasmon resonance titrations to show that the PABC domain of HYD binds PAM2 peptides with micromolar affinity. NMR chemical shift perturbations were used to map the peptide-binding site in the PABC domain of HYD. The structural features of binding are very similar to those of the interactions with the domain of PABP, which explains the overlapping peptide specificity and binding affinity. We identified the anti-proliferative Tob proteins as potential binding partners of HYD. This was confirmed by glutathione S-transferase pulldown and immunoprecipitation experiments demonstrating the interaction with full-length Tob2. Altogether, our results point to a role of the PABC domain as a protein-protein interaction domain that brings together the processes of translation, ubiquitin-mediated protein degradation, and cell cycle control.
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- 2006
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26. UNR, a new partner of poly(A)-binding protein, plays a key role in translationally coupled mRNA turnover mediated by the c-fos major coding-region determinant
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Akio Yamashita, Avak Kahvejian, Wenmiao Zhu, Ann-Bin Shyu, Tsung Cheng Chang, Simon Durdan, Chyi-Ying A. Chen, Nahum Sonenberg, and Yukiko M. Yamashita
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Receptors, CCR4 ,Molecular Sequence Data ,Biology ,Poly(A)-Binding Proteins ,Ribosome ,Mice ,Eukaryotic translation ,Sequence Homology, Nucleic Acid ,Poly(A)-binding protein ,Genetics ,Protein biosynthesis ,Animals ,Coding region ,RNA, Messenger ,Binding site ,Messenger RNA ,Binding Sites ,Base Sequence ,Genes, fos ,Translation (biology) ,Research Papers ,Molecular biology ,Cell biology ,Protein Biosynthesis ,NIH 3T3 Cells ,biology.protein ,Receptors, Chemokine ,Developmental Biology - Abstract
Messenger RNA decay mediated by the c-fos major protein coding-region determinant of instability (mCRD) is a useful system for studying translationally coupled mRNA turnover. Among the five mCRD-associated proteins identified previously, UNR was found to be an mCRD-binding protein and also a PABP-interacting protein. Interaction between UNR and PABP is necessary for the full destabilization function of the mCRD. By testing different classes of mammalian poly(A) nucleases, we identified CCR4 as a poly(A) nuclease involved in the mCRD-mediated rapid deadenylation in vivo and also associated with UNR. Blocking either translation initiation or elongation greatly impeded poly(A) shortening and mRNA decay mediated by the mCRD, demonstrating that the deadenylation step is coupled to ongoing translation of the message. These findings suggest a model in which the mCRD/UNR complex serves as a “landing/assembly” platform for formation of a deadenylation/decay mRNA-protein complex on an mCRD-containing transcript. The complex is dormant prior to translation. Accelerated deadenylation and decay of the transcript follows ribosome transit through the mCRD. This study provides new insights into a mechanism by which interplay between mRNA turnover and translation determines the lifespan of an mCRD-containing mRNA in the cytoplasm.
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- 2004
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27. Rapid Deadenylation Triggered by a Nonsense Codon Precedes Decay of the RNA Body in a Mammalian Cytoplasmic Nonsense-Mediated Decay Pathway
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Chyi-Ying A. Chen and Ann-Bin Shyu
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Exonuclease ,Cytoplasm ,RNA Stability ,Nonsense mutation ,Nonsense-mediated decay ,Gene Expression ,RNA-binding protein ,Heterogeneous-Nuclear Ribonucleoproteins ,Mice ,Eukaryotic translation ,Protein biosynthesis ,Animals ,RNA, Messenger ,RNA Processing, Post-Transcriptional ,Molecular Biology ,Genes, Dominant ,Mammals ,Messenger RNA ,biology ,RNA-Binding Proteins ,RNA ,3T3 Cells ,Cell Biology ,Molecular biology ,Adenosine Monophosphate ,Globins ,DNA-Binding Proteins ,Gene Expression Regulation ,Codon, Nonsense ,Protein Biosynthesis ,Mutation ,Trans-Activators ,biology.protein ,RNA Helicases ,Transcription Factors - Abstract
Nonsense-mediated mRNA decay (NMD) is an RNA surveillance pathway that detects and destroys aberrant mRNAs containing nonsense or premature termination codons (PTCs) in a translation-dependent manner in eukaryotes. In yeast, the NMD pathway bypasses the deadenylation step and directly targets PTC-containing messages for decapping, followed by 5′-to-3′ exonuclease digestion of the RNA body. In mammals, most PTC-containing mRNAs are subject to active nucleus-associated NMD. Here, using two distinct transcription-pulsing approaches to monitor mRNA deadenylation and decay kinetics, we demonstrate the existence of an active cytoplasmic NMD pathway in mammalian cells. In this pathway, a nonsense codon triggers accelerated deadenylation that precedes decay of the PTC-containing mRNA body. Transcript is stabilized when accelerated deadenylation is impeded by blocking translation initiation; by ectopically expressing two RNA-binding proteins, UNR and NSAP1; or by ectopically expressing a UPF1 dominant-negative mutant. These results are consistent with the notion that the nonsense codon can function in the cytoplasm by promoting rapid removal of the poly(A) tail as a necessary first step in the decay process.
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- 2003
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28. Highly Selective Actions of HuR in Antagonizing AU-Rich Element-Mediated mRNA Destabilization
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Chyi-Ying A. Chen, Ann-Bin Shyu, and Nianhua Xu
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MRNA destabilization ,RNA Stability ,Molecular Sequence Data ,ELAV-Like Protein 1 ,Gene Expression ,Biology ,Response Elements ,Heterogeneous-Nuclear Ribonucleoproteins ,3T3 cells ,Mice ,Downregulation and upregulation ,In vivo ,medicine ,Animals ,Humans ,Heterogeneous Nuclear Ribonucleoprotein D0 ,RNA, Messenger ,Heterogeneous-Nuclear Ribonucleoprotein D ,Binding site ,Molecular Biology ,Cell Nucleus ,AU-rich element ,Binding Sites ,Base Sequence ,RNA-Binding Proteins ,RNA ,3T3 Cells ,Cell Biology ,Molecular biology ,Cell biology ,medicine.anatomical_structure ,ELAV Proteins ,Ribonucleoproteins ,Antigens, Surface ,Proto-Oncogene Proteins c-fos - Abstract
Human RNA-binding protein HuR, a nucleocytoplasmic shuttling protein, is a ubiquitously expressed member of the family of Hu proteins, which consist of two N-terminal RNA recognition motifs (RRM1 and RRM2), a hinge region, and a C-terminal RRM (RRM3). Although in vitro experiments showed indiscriminate binding of Hu proteins synthesized in bacterial systems to many different AU-rich elements (AREs), in vivo studies have pointed to a cytoplasmic role for HuR protein in antagonizing the rapid decay of some specific ARE-containing mRNAs, depending on physiological situations. By ectopically overexpressing HuR and its mutant derivatives in NIH 3T3 cells to mimic HuR upregulation of specific ARE-containing mRNAs in other systems, we have examined the in vivo ARE-binding specificity of HuR and dissected its functionally critical domains. We show that in NIH 3T3 cells, HuR stabilizes reporter messages containing only the c-fos ARE and not other AREs. Two distinct binding sites were identified within the c-fos ARE, the 5' AUUUA-containing domain and the 3' U-stretch-containing domain. These actions of HuR are markedly different from those of another ARE-binding protein, hnRNP D (also termed AUF1), which in vivo recognizes AUUUA repeats found in cytokine AREs and can exert both stabilizing and destabilizing effects. Further experiments showed that any combination of two of the three RRM domains of HuR is sufficient for strong binding to the c-fos ARE in vitro and to exert an RNA stabilization effect in vivo comparable to that of intact HuR and that the hinge region containing nucleocytoplasmic shuttling signals is dispensable for the stabilization effect of HuR. Our data suggest that the ARE-binding specificity of HuR in vivo is modulated to interact only with and thus regulate specific AREs in a cell type- and physiological state-dependent manner.
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- 2002
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29. RNA surveillance by nuclear scanning?
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Miles F. Wilkinson and Ann-Bin Shyu
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Mammals ,Nuclear Proteins ,RNA ,Translation (biology) ,Cell Biology ,RNA surveillance ,Biology ,Gene mutation ,Cell biology ,medicine.anatomical_structure ,Gene Expression Regulation ,Codon, Nonsense ,Cytoplasm ,Gene expression ,RNA Precursors ,medicine ,Animals ,Nucleus - Abstract
There are many quality-control mechanisms that ensure high fidelity of gene expression. One of these is the nonsense-mediated decay (NMD) pathway, which destroys aberrant mRNAs that contain premature termination codons generated as a result of biosynthetic errors or random and programmed gene mutations. Two complexes that initially bind to RNA in the nucleus have been suggested to be involved in NMD in the cytoplasm. Here we propose an alternative model that involves nuclear scanning, on the basis of recent evidence for nuclear translation.
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- 2002
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30. Emerging mechanisms of mRNP remodeling regulation
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Ann-Bin Shyu and Chyi-Ying A. Chen
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Cell Nucleus ,Messenger RNA ,Extramural ,fungi ,Ubiquitination ,RNA ,Biology ,Bioinformatics ,Biochemistry ,Poly(A)-Binding Proteins ,Article ,Cell biology ,Messenger RNP ,Intrinsically Disordered Proteins ,Ribonucleoproteins ,Protein Biosynthesis ,Protein biosynthesis ,Humans ,RNA, Messenger ,Phosphorylation ,Molecular Biology ,Function (biology) - Abstract
The assembly and remodeling of the components of messenger ribonucleoprotein particles (mRNPs) are important in determining the fate of a messenger RNA (mRNA). A combination of biochemical and cell biology research, recently complemented by genome-wide high-throughput approaches, has led to significant progress on understanding the formation, dynamics, and function of mRNPs. These studies also advanced the challenging process of identifying the evolving constituents of individual mRNPs at various stages during an mRNA's lifetime. While research on mRNP remodeling in general has been gaining momentum, there has been relatively little attention paid to the regulatory aspect of mRNP remodeling. Here, we discuss the results of some new studies and potential mechanisms for regulation of mRNP remodeling.
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- 2014
31. A Mechanism for Translationally Coupled mRNA Turnover
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Christophe Grosset, Ann-Bin Shyu, Nianhua Xu, Hélène Jacquemin-Sablon, Chyi-Ying A. Chen, and Nahum Sonenberg
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Messenger RNA ,Polyadenylation ,Biochemistry ,Biochemistry, Genetics and Molecular Biology(all) ,Binding protein ,RNA ,RNA-binding protein ,Translation (biology) ,Biology ,Heterogeneous ribonucleoprotein particle ,Ribosome ,General Biochemistry, Genetics and Molecular Biology ,Cell biology - Abstract
mRNA turnover mediated by the major protein-coding-region determinant of instability (mCRD) of the c-fos proto-oncogene transcript illustrates a functional interplay between mRNA turnover and translation. We show that the function of mCRD depends on its distance from the poly(A) tail. Five mCRD-associated proteins were identified: Unr, a purine-rich RNA binding protein; PABP, a poly(A) binding protein; PAIP-1, a poly(A) binding protein interacting protein; hnRNP D, an AU-rich element binding protein; and NSAP1, an hnRNP R-like protein. These proteins form a multiprotein complex. Overexpression of these proteins stabilized mCRD-containing mRNA by impeding deadenylation. We propose that a bridging complex forms between the poly(A) tail and the mCRD and ribosome transit disrupts or reorganizes the complex, leading to rapid RNA deadenylation and decay.
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- 2000
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32. The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism
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Helmut Holtmann, Klaus Resch, Arno Wilhelm, Ann-Bin Shyu, Michael Kracht, Matthias Gaestel, Monika Müller, Reinhard Winzen, Birgit Ritter, and Chyi-Ying A. Chen
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RNA Stability ,Molecular Sequence Data ,MAPK7 ,Gene Expression ,Protein Serine-Threonine Kinases ,Biology ,Mitogen-activated protein kinase kinase ,Transfection ,p38 Mitogen-Activated Protein Kinases ,General Biochemistry, Genetics and Molecular Biology ,MAP2K7 ,Genes, Reporter ,Humans ,ASK1 ,RNA, Messenger ,c-Raf ,3' Untranslated Regions ,Molecular Biology ,DNA Primers ,Base Sequence ,General Immunology and Microbiology ,MAP kinase kinase kinase ,Interleukin-6 ,General Neuroscience ,MAPKAPK2 ,Interleukin-8 ,Intracellular Signaling Peptides and Proteins ,Molecular biology ,Cytokines ,Cyclin-dependent kinase 9 ,Mitogen-Activated Protein Kinases ,HeLa Cells ,Interleukin-1 ,Signal Transduction ,Research Article - Abstract
Stabilization of mRNAs contributes to the strong and rapid induction of genes in the inflammatory response. The signaling mechanisms involved were investigated using a tetracycline-controlled expression system to determine the half-lives of interleukin (IL)-6 and IL-8 mRNAs. Transcript stability was low in untreated HeLa cells, but increased in cells expressing a constitutively active form of the MAP kinase kinase kinase MEKK1. Destabilization and signal-induced stabilization was transferred to the stable beta-globin mRNA by a 161-nucleotide fragment of IL-8 mRNA which contains an AU-rich region, as well as by defined AU-rich elements (AREs) of the c-fos and GM-CSF mRNAs. Of the different MEKK1-activated signaling pathways, no significant effects on mRNA degradation were observed for the SAPK/JNK, extracellular regulated kinase and NF-kappaB pathways. Selective activation of the p38 MAP kinase (=SAPK2) pathway by MAP kinase kinase 6 induced mRNA stabilization. A dominant-negative mutant of p38 MAP kinase interfered with MEKK1 and also IL-1-induced stabilization. Furthermore, an active form of the p38 MAP kinase-activated protein kinase (MAPKAP K2 or MK2) induced mRNA stabilization, whereas a negative interfering MK2 mutant interfered with MAP kinase kinase 6-induced stabilization. These findings indicate that the p38 MAP kinase pathway contributes to cytokine/stress-induced gene expression by stabilizing mRNAs through an MK2-dependent, ARE-targeted mechanism.
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- 1999
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33. Study of mRNA turnover never decays
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Ann-Bin Shyu
- Subjects
Genetics ,Gene knockdown ,Mature messenger RNA ,RISC complex ,Hydrolysis ,Nonsense-mediated decay ,Translation (biology) ,Biology ,Cell biology ,Open Reading Frames ,microRNA ,RNA splicing ,P-bodies ,RNA, Messenger ,Molecular Biology ,Personal Reflections - Abstract
As I reflect on what the field of mRNA turnover has achieved over the past two decades, the first things that pop up in my mind are the significant recognition the field has attained and how far the field has advanced. I recall that in the early 1990s, mRNA degradation was viewed by many researchers as an uncontrolled scavenger process, much like a cellular trash can where cells dump old mRNAs that are no longer in use. Of course, without transcription there will be no gene expression in the first place. It is thus not surprising that, when dealing with problems of regulation of eukaryotic gene expression, researchers at the time intuitively placed regulation mainly at the level of transcription. This idea was prevalent even though degradation of mRNA had been shown to be one of the principal means by which prokaryotic genes are regulated. It was not until the mid 1990s that researchers started to appreciate that modulation of mRNA stability plays a critical role in regulating gene expression, allowing cells to change the levels of gene expression rapidly in response to a dynamic cellular environment. In the early and mid 1990s, use of budding yeast and a transcriptional pulsing approach permitted investigators to chase a newly synthesized mRNA into its decay products in a genetically manipulated background. This work helped elucidate the mechanistic steps and basic constituents of mRNA turnover machinery. The development of siRNA-based gene knockdown technologies in the early 2000s greatly facilitated deciphering the mRNA decay pathways in mammalian cells. Cumulative evidence establishes that deadenylation, i.e., shortening of the 3′ poly(A) tail, is the major means of triggering eukaryotic mRNA turnover. Unexpectedly, instead of being followed by 3′ to 5′ exonucleolytic digestion of the mRNA body, deadenylation primarily leads to removal of the 5′ cap of mRNA followed by rapid 5′ to 3′ digestion of the mRNA body. At first glance, this major route of mammalian mRNA decay may seem indirect and inefficient. However, it is clear now that this sequence of steps in mRNA decay serves important purposes. Decapping prevents additional loading of ribosomes at the same time allowing ribosomes already on the targeted mRNAs to finish translation without generating potentially cytotoxic truncated polypeptide chains. This mRNA degradation sequence also adds another layer of regulation of mRNA function. Depending on their environment, cells can either reuse or rescue the poly(A) shortened mRNA for later translation or degrade that mRNA permanently. It is therefore not entirely surprising to find that microRNAs, after forming the RISC complex on target mRNAs, shunt those mRNAs to this major route of mRNA degradation and accelerate their deadenylation. The discovery of GW/P-bodies in 2002–2003 brought much excitement to the mRNA turnover field and to the entire RNA biology field, and it drove much biological research in the next decade. GW/P-bodies are cytoplasmic foci of messenger ribonucleoprotein particle (mRNP) aggregates containing a plethora of translation repressors and mRNA decay factors. The information about mRNA decay pathways and participating factors that accumulated through the 1990s helped pave the way to understanding GW/P-bodies. Few could have imagined at the beginning that investigation of these cytoplasmic foci would eventually occupy hundreds of biologists. Although it has been suggested that GW/P-bodies function in translational control and mRNA degradation, their exact role(s) in regulating gene expression, and how their assembly and disassembly is controlled, remain challenging and critical issues in the RNA field. Another important development in the mRNA turnover field that caught the attention of many biologists was the elucidation of how rapid decay of an mRNA is triggered by a nonsense or premature termination codon (PTC) in the open-reading-frame (ORF). This intriguing yet elusive problem of how eukaryotic cells discern whether the ORF of a mature mRNA is prematurely terminated and thus needs to be promptly degraded drew the efforts of many investigators starting in the late 1990s. These efforts led to a fairly thorough understanding of the mechanism of nonsense-mediated mRNA decay (NMD) and its manifestation in human genetic diseases. Several important principles regarding mRNA processing in the nucleus and its metabolism in the cytoplasm emerged. One principle is that the experience of an mRNA in the nucleus influences that mRNA's fate in the cytoplasm. As it turns out, after pre-mRNA splicing, a group of proteins, termed the exon-junction-complex (EJC), associate with mRNAs undergoing maturation in the nucleus. The EJC proteins help guide the pre-mRNA through subsequent processing steps and/or nuclear export. Some EJC constituents accompany mRNA to the cytoplasm, where they influence the translation, stability and localization of the mRNA. A second principle is that mRNAs fulfill their functions in the form of mRNPs. One prominent feature of mRNP complexes is that they are highly dynamic, with rapid exchanges of mRNP protein constituents dictating the mRNA functions and fate at each step during their metabolism. These alterations in mRNP protein composition are collectively termed mRNP remodeling. Any failure to appropriately remodel an mRNP complex has the potential to disrupt downstream events that determine the mRNA's fate, as exemplified by the NMD process. Thus, mRNPs represent highly dynamic, functional forms of mRNAs. A third principle is that many mRNP constituent proteins shuttle between the nucleus and cytoplasm to fulfill their functions in mRNA metabolism. These shuttling proteins usually live a double-life in eukaryotic cells, with one role in the nucleus and another in the cytoplasm, and disrupting the proteins’ shuttling leads to formation of dysfunctional mRNPs. The molecular constituents of individual mRNPs and their remodeling during the lifetime of mRNAs are exciting and relatively underexplored areas of research in RNA biology. With over 800 putative human RNA-binding proteins (RNA-BPs) and the many additional proteins that interact with the RNA-BPs, identifying the makeup of individual mRNPs and defining specific roles for individual RNP proteins are challenging but important tasks. Equally challenging is elucidation of the post-translational modification mechanisms that regulate mRNP remodeling, as these modifications could impact the fate of mRNAs in a transcript-specific or transcriptome-wide manner. At this point, few signaling pathways associated with mRNP remodeling have been identified. A combination of RNA-affinity chromatography and quantitative proteomics may prove useful for characterizing modifications of the RNA-binding proteome. The physiological importance of mRNA-BPs and mRNP complexes is underscored by their linkage to many human diseases. One recent example is a malfunction of hnRNPA1 that results in mRNP aggregation and is linked to neuronal degenerative diseases. There is also the instance of mutations in a protein segregase, p97, that have been genetically linked to inclusion body myopathy, Paget's disease, frontotemporal dementia, and amyotrophic lateral sclerosis. The molecular pathogenesis of these diseases remains poorly understood. It has been proposed that some severe neurodegenerative diseases associated with p97 mutations involve a defect in mRNP remodeling that results in mRNP aggregation and aberrant mRNA translation. It is clear from recent studies that mammalian p97 is critical for autophagic clearance of stress granule-related and pathogenic RNP granules, which accumulate in p97-linked degenerative diseases. Studies of the regulation of mRNP remodeling may suggest ways to therapeutically manipulate a subset of mRNPs under particular physiological or pathological conditions. It has recently become evident just how rapidly new techniques, such as proteome- and transcriptome-based technologies, can catch fire and spread to labs that don't specialize in methods development but nevertheless are able to rapidly adopt—and improve upon—these new approaches. In the next decade, we should expect to see a more comprehensive grasp of the makeup of individual mRNPs, of mRNP dynamics, and of the regulation of mRNP remodeling and its connections to mRNP-related human diseases.
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- 2015
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34. RNA stabilization by the AU-rich element binding protein, HuR, an ELAV protein
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Sheila S.Y. Peng, Ann-Bin Shyu, Chyi-Ying A. Chen, and Nianhua Xu
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Cytoplasm ,ELAV-Like Protein 1 ,RNA-binding protein ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Mice ,Genes, jun ,medicine ,Animals ,Humans ,RNA, Messenger ,RNA Processing, Post-Transcriptional ,Molecular Biology ,Transcription factor ,Cell Nucleus ,AU-rich element ,Messenger RNA ,General Immunology and Microbiology ,General Neuroscience ,Genes, fos ,RNA-Binding Proteins ,RNA ,3T3 Cells ,Molecular biology ,Cell nucleus ,medicine.anatomical_structure ,ELAV Proteins ,Ribonucleoproteins ,Antigens, Surface ,Research Article - Abstract
An important paradigm for post‐transcriptional regulation is the control of cytoplasmic mRNA stability mediated by AU‐rich elements (AREs) in the 3′ untranslated region of transcripts encoding oncoproteins, cytokines and transcription factors. While many RNA‐binding proteins have been shown to bind to AREs in vitro , neither the functional consequences nor the physiological significance of their interactions are known. Here we demonstrate a role for the embryonic lethal abnormal visual (ELAV) RNA‐binding protein HuR in mRNA turnover in vivo . The ELAV family of RNA‐binding proteins is highly conserved in vertebrates. In humans, there are four members; HuR is expressed in all proliferating cells, whereas Hel‐N1, HuC and HuD are expressed in terminally differentiated neurons. We show that elevation of cytoplasmic HuR levels inhibits c‐ fos ARE‐mediated RNA decay but has little effect on rapid decay directed by c‐ jun ARE. It appears that HuR has little effect on deadenylation but delays onset of decay of the RNA body and slows down its subsequent decay. We also show that HuR can be induced to redistribute from the nucleus to the cytoplasm and that this redistribution is associated with an altered function. Modulation of the ARE‐mediated decay pathway through controlling distribution of the ELAV proteins between nucleus and cytoplasm may be a mechanism by which cell growth and differentiation is regulated.
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- 1998
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35. A broader role for AU-rich element-mediated mRNA turnover revealed by a new transcriptional pulse strategy
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Ann-Bin Shyu, Paul Loflin, Chyi-Ying A. Chen, and Nianhua Xu
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Adenosine ,Transcription, Genetic ,Nonsense-mediated decay ,Population ,Repressor ,Regulatory Sequences, Nucleic Acid ,Biology ,Transfection ,Mice ,Gene expression ,P-bodies ,Tumor Cells, Cultured ,Genetics ,Animals ,Humans ,RNA, Messenger ,Promoter Regions, Genetic ,education ,Uridine ,Transcription factor ,AU-rich element ,education.field_of_study ,Base Sequence ,3T3 Cells ,Tetracycline ,Molecular biology ,Globins ,Cell biology ,Repressor Proteins ,Mutagenesis, Site-Directed ,Leukemia, Erythroblastic, Acute ,Research Article ,Plasmids - Abstract
The widespread occurrence of AU-rich elements (AREs) in mRNAs encoding proteins with diversified functions and synthesized under a vast variety of physiological conditions suggests that AREs are involved in finely tuned and stringent control of gene expression. Thus it is important to investigate the regulation of ARE-mediated mRNA decay in a variety of mammalian cells in different physiological states. The tetracycline (Tet)-regulatory promoter system appears appropriate for these investigations. However, we found that efficient degradation of mRNAs bearing different AREs cannot be observed simply by blocking constitutive transcription from the Tet-regulated promoter with Tet, possibly due to saturation of the cellular decay machinery. In addition, deadenylation kinetics and their relationship to mRNA decay cannot be adequately measured under these conditions. To overcome these obstacles we have developed a new strategy that employs the Tet-regulated promoter system to achieve a transient burst of transcription that results in synthesis of a population of cytoplasmic mRNAs fairly homogeneous in size. Using this new system we show that ARE-destabilizing function, necessary for down-regulating mRNAs for cytokines, growth factors and transcription factors, is maintained in quiescent or growth-arrested cells as well as in saturation density-arrested NIH 3T3 cells. We also demonstrate that the ARE-mediated decay pathway is conserved between NIH 3T3 fibroblasts and K562 erythroblasts. These in vivo observations support a broader role for AREs in the control of cell growth and differentiation. In addition, we observed that there is a significant difference in deadenylation and decay rates for beta-globin mRNA expressed in these two cell lines. Deadenylation and decay of beta-globin mRNA in K562 cells is extraordinarily slow compared with NIH 3T3 cells, suggesting that the increased stability gained by beta-globin mRNA in K562 cells is mainly controlled at the deadenylation step. Our strategy for studying mammalian mRNA turnover now permits a more general application to different cell lines harboring the Tet-regulated system under various physiological conditions.
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- 1998
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36. Protein segregase meddles in remodeling of mRNA–protein complexes
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Ann-Bin Shyu and Chyi-Ying A. Chen
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Cyclin-Dependent Kinase Inhibitor p21 ,RNA Stability ,Mice ,Ubiquitin ,Stress, Physiological ,Genetics ,Animals ,Humans ,RNA, Messenger ,Gene ,Adenosine Triphosphatases ,Messenger RNA ,biology ,Mechanism (biology) ,Ubiquitination ,A protein ,Membrane Proteins ,Nuclear Proteins ,Metabolism ,Blood Proteins ,Cell biology ,Biochemistry ,ELAV Proteins ,Ribonucleoproteins ,Perspective ,biology.protein ,Biogenesis ,Developmental Biology ,HeLa Cells ,Protein Binding - Abstract
The assembly and disassembly of ribonucleoproteins (RNPs) are dynamic processes that control every step of RNA metabolism, including mRNA stability. However, our knowledge of how RNP remodeling is achieved is largely limited to RNA helicase functions. Here, we report a previously unknown mechanism that implicates the ATPase p97, a protein-remodeling machine, in the dynamic regulation of mRNP disassembly. We found that p97 and its cofactor, UBXD8, destabilize p21, MKP-1, and SIRT1, three established mRNA targets of the RNA-binding protein HuR, by promoting release of HuR from mRNA. Importantly, ubiquitination of HuR with a short K29 chain serves as the signal for release. When cells are subjected to stress conditions, the steady-state levels of HuR ubiquitination change, suggesting a new mechanism through which HuR mediates the stress response. Our studies reveal a new paradigm in RNA biology: nondegradative ubiquitin signaling-dependent disassembly of mRNP promoted by the p97-UBXD8 complex to control mRNA stability.
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- 2013
37. CFIm25 links alternative polyadenylation to glioblastoma tumour suppression
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Zheng Xia, Todd R. Albrecht, Chioniso P. Masamha, Eric J. Wagner, Min Li, Jingxuan Yang, Wei Li, and Ann-Bin Shyu
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Male ,Polyadenylation ,Carcinogenesis ,Repressor ,Biology ,medicine.disease_cause ,Article ,Cell Line ,Mice ,Downregulation and upregulation ,Cell Line, Tumor ,microRNA ,medicine ,Animals ,Humans ,RNA, Messenger ,3' Untranslated Regions ,Cell Proliferation ,Regulation of gene expression ,mRNA Cleavage and Polyadenylation Factors ,Gene knockdown ,Multidisciplinary ,Three prime untranslated region ,Gene Expression Profiling ,Molecular biology ,Cell biology ,Gene Expression Regulation, Neoplastic ,Gene Knockdown Techniques ,Heterografts ,Regression Analysis ,Glioblastoma ,HeLa Cells - Abstract
CFIm25 is identified as a factor that prevents messenger RNAs being shortened due to altered 3′ polyadenylation, which typically occurs when cells undergo high proliferation and correlates with increased tumorigenic activity in glioblastoma tumours. Cells undergoing high proliferation display mRNAs that are shortened as a result of decreased 3′ polyadenylation. Eric Wagner and colleagues have identified CFIm25 (a 25-kilodalton component of the cleavage factor Im complex involved in pre-mRNA 3′-processing) as a factor that prevents polyadenylation shortening. In its absence, poly(A) tails are shorter and proliferation is enhanced in about 11% of expressed mRNAs in HeLa cells. This polyadenylation shortening correlates with the upregulation of several oncogenes, and in glioblastoma cells with higher tumorigenic activity. The global shortening of messenger RNAs through alternative polyadenylation (APA) that occurs during enhanced cellular proliferation represents an important, yet poorly understood mechanism of regulated gene expression1,2. The 3′ untranslated region (UTR) truncation of growth-promoting mRNA transcripts that relieves intrinsic microRNA- and AU-rich-element-mediated repression has been observed to correlate with cellular transformation3; however, the importance to tumorigenicity of RNA 3′-end-processing factors that potentially govern APA is unknown. Here we identify CFIm25 as a broad repressor of proximal poly(A) site usage that, when depleted, increases cell proliferation. Applying a regression model on standard RNA-sequencing data for novel APA events, we identified at least 1,450 genes with shortened 3′ UTRs after CFIm25 knockdown, representing 11% of significantly expressed mRNAs in human cells. Marked increases in the expression of several known oncogenes, including cyclin D1, are observed as a consequence of CFIm25 depletion. Importantly, we identified a subset of CFIm25-regulated APA genes with shortened 3′ UTRs in glioblastoma tumours that have reduced CFIm25 expression. Downregulation of CFIm25 expression in glioblastoma cells enhances their tumorigenic properties and increases tumour size, whereas CFIm25 overexpression reduces these properties and inhibits tumour growth. These findings identify a pivotal role of CFIm25 in governing APA and reveal a previously unknown connection between CFIm25 and glioblastoma tumorigenicity.
- Published
- 2013
38. Expression of NGFI-B mRNA in a rat focal cerebral ischemia-reperfusion model
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Johnny T. Cheng, Chung Y. Hsu, Teng-Nan Lin, Shu Ing Chi, Ann-Bin Shyu, Janice J. Chen, Grace Y. Sun, and Shr Jie Wang
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Male ,endocrine system ,medicine.medical_specialty ,Ischemia ,Hippocampus ,Biology ,Brain Ischemia ,Cellular and Molecular Neuroscience ,Internal medicine ,Gene expression ,medicine ,Animals ,Nerve Growth Factors ,RNA, Messenger ,Northern blot ,Molecular Biology ,In Situ Hybridization ,Analysis of Variance ,Dentate gyrus ,medicine.disease ,Rats ,Disease Models, Animal ,medicine.anatomical_structure ,Nerve growth factor ,Endocrinology ,nervous system ,Cerebral cortex ,Cerebrovascular Circulation ,Neuroscience ,Immediate early gene ,hormones, hormone substitutes, and hormone antagonists - Abstract
Cerebral ischemia is known to induce the expression of several immediate early genes (IEGs), including c-fos and c-jun, which subsequently regulate a number of late effector genes. In this study, we examined the expression of NGFI-B (or nur77) mRNA in a rat focal cerebral ischemia-reperfusion model. NGFI-B is a member of the IEGs which encodes for a nuclear receptor and is rapidly induced by nerve growth factor (NGF). Northern blot analysis showed a rapid but transient enhancement of NGFI-B mRNA, a peak level for which was observed at 30 min of reperfusion following 60 min ischemic insult. At the peak level, quantitative analysis of the blot indicated a 12-fold and 4-fold increase of NGFI-B mRNA in the ischemic cortex and ipsilateral hippocampus, respectively, as compared to the sham-operated control. No apparent changes in mRNA levels were observed within contralateral sites of the cortex. Results from in situ hybridization showed that severe ischemia (60 min) resulted in a marked increase of NGFI-B mRNA throughout the entire ischemic cerebral cortex. The increase was particularly notable in the frontal, occipital, perirhinal and piriform cortical regions and in the dentate gyrus and CA1–3 regions of the ipsilateral hippocampus. A marked induction was also noted in the ipsilateral caudate putamen. Unlike the induction profile of NGFI-B mRNA, severe ischemia resulted in bilateral increases of its family gene, NGFI-A mRNA. The spatial induction profile is similar to that of NGFI-B mRNA in both hemispheres, except within the region of the contralateral dentate gyrus which showed low levels of NGFI-A mRNA. The expression pattern of NGF and BDNF mRNA, upstream genes of NGFI-B, were also examined. Interestingly, the temporal and spatial expression patterns of BDNF mRNA were very similar to that of NGFI-A mRNA under the same conditions, whereas increased NGF and NGFI-B mRNA were observed only in the ipsilateral hemisphere. It is likely that multiple and/or overlapping pathways are activated subsequent to ischemic challenge which in turn are crucial for cell survival and/or functional recovery following focal cerebral ischemia.
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- 1996
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39. Functional Characterization of a Non-AUUUA AU-Rich Element from the c-jun Proto-Oncogene mRNA: Evidence for a Novel Class of AU-Rich Elements
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Chyi-Ying A. Chen, Sheila S.Y. Peng, and Ann-Bin Shyu
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Untranslated region ,Proto-Oncogene Proteins c-jun ,Heterogeneous Nuclear Ribonucleoprotein A1 ,Molecular Sequence Data ,Biology ,Transfection ,DNA-binding protein ,Ribosome ,Mice ,Viral Proteins ,Genes, jun ,Transcription (biology) ,Polysome ,Heterogeneous-Nuclear Ribonucleoprotein Group A-B ,Animals ,RNA, Messenger ,Molecular Biology ,Ribonucleoprotein ,Genetics ,AU-rich element ,Messenger RNA ,Base Sequence ,DNA Helicases ,3T3 Cells ,Cell Biology ,Cell biology ,DNA-Binding Proteins ,Thymus Hormones ,Ribonucleoproteins ,Poly A ,Research Article - Abstract
AU-rich RNA-destabilizing elements (AREs) found in the 3' untranslated regions of many labile mRNAs encoding proto-oncoproteins and cytokines generally contain (i) one or more copies of the AUUUA pentanucleotide and (ii) a high content of uridylate and sometimes also adenylate residues. Recently, we have identified a potent ARE from the 3' untranslated region of c-jun proto-oncogene mRNA that does not contain the AUUUA motif. In an attempt to further our understanding of the general principles underlying mechanisms by which AREs direct rapid and selective mRNA degradation, in this study we have characterized the functionally important structural features and properties of this non-AUUUA ARE. Like AUUUA-containing AREs, this non-AUUUA ARE directs rapid shortening of the poly(A) tail as a necessary first step for mRNA degradation. It can be further dissected into three structurally and functionally distinct regions, designated domains I, II, and III. None of three domains alone is able to significantly destabilize the stable beta-globin mRNA. The two unlinked domains, I and III, together are necessary and sufficient for specifying the full destabilizing function of this non-AUUUA ARE. Domain II appears functionally dispensable but can partially substitute for domain I. Domain swaps made between the c-jun non-AUUUA and the c-fos AUUUA-containing AREs reveal that the two AREs, while sharing no sequence homology, appear to contain sequence domains that are structurally distinct but functionally overlapping and exchangeable. These data support the idea that the ultimate destabilizing function of an individual ARE is determined by its own unique combination of structurally distinct and functionally interdependent domains. Our polysome profile studies show tha the destabilizing function of the c-jun non-AUUUA ARE does not require any active transit by ribosomes of the mRNA bearing it, further corroborating that the destabilizing function of AREs is not tightly coupled to ongoing translation by ribosomes. Moreover, unlike AUUUA-containing AREs, the c-jun ARE is insensitive to blockage of its effects by addition of transcription inhibitors. Thus, our data provide further evidence for the existence of a novel class of ARE with unique properties.
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- 1996
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40. AU-rich elements: characterization and importance in mRNA degradation
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Chyi-Ying A. Chen and Ann-Bin Shyu
- Subjects
AU-rich element ,Untranslated region ,Regulation of gene expression ,Base Composition ,Transcription, Genetic ,MRNA destabilization ,Three prime untranslated region ,Adenine ,ELAV-Like Protein 1 ,RNA-Binding Proteins ,MRNA stabilization ,Biology ,Biochemistry ,Molecular biology ,Cell biology ,Gene Expression Regulation ,Protein Biosynthesis ,Animals ,RNA, Messenger ,Uracil ,Molecular Biology ,Transcription factor ,Conserved Sequence ,Signal Transduction - Abstract
Adenylate/uridylate-rich elements (AREs) are found in the 3' untranslated region (UTR) of many messenger RNAs (mRNAs) that code for proto-oncogenes, nuclear transcription factors and cytokines. They represent the most common determinant of RNA stability in mammalian cells. Moreover, ARE-directed mRNA degradation is influenced by many exogenous factors, including phorbol esters, calcium ionophores, cytokines and transcription inhibitors. These observations suggest that AREs play a critical role in the regulation of gene expression during cell growth and differentiation, and in the immune response.
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- 1995
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41. Deadenylation and P-bodies
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Chyi-Ying A. Chen and Ann-Bin Shyu
- Subjects
Saccharomyces cerevisiae Proteins ,RNA Stability ,Saccharomyces cerevisiae ,Biology ,Microbodies ,Article ,Ribonucleases ,RNA interference ,microRNA ,P-bodies ,Protein biosynthesis ,Animals ,Drosophila Proteins ,Humans ,RNA, Messenger ,Ribonucleoprotein ,Genetics ,Regulation of gene expression ,Messenger RNA ,RNA ,Cell biology ,MicroRNAs ,Gene Expression Regulation ,Ribonucleoproteins ,Protein Biosynthesis ,Exoribonucleases ,RNA Interference ,Carrier Proteins - Abstract
Deadenylation is the major step in triggering mRNA decay and results in mRNA translation inhibition in eukaryotic cells. Therefore, it is plausible that deadenylation also induces the mRNP remodeling required for formation of GW bodies or RNA processing bodies (P-bodies), which harbor translationally silenced mRNPs. In this chapter, we discuss several examples to illustrate the roles of deadenylation in regulating gene expression. We highlight several lines of evidence indicating that even though non-translatable mRNPs may be prepared and/or assembled into P-bodies in different ways, deadenylation is always a necessary, and perhaps the earliest, step in mRNA decay pathways that enable mRNP remodeling required for P-body formation. Thus, deadenylation and the participating deadenylases are not simply required for preparing mRNA substrates; they play an indispensable role both structurally and functionally in P-body formation and regulation.
- Published
- 2012
42. Analysis of interferon-beta mRNA stability control after poly(I:C) stimulation using RNA metabolic labeling by ethynyluridine
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Shigeo Ohno, Kaito Abe, Satoshi Umemura, Akio Yamashita, Ann-Bin Shyu, and Tomoaki Ishigami
- Subjects
Messenger RNA ,RNA Stability ,Transcription, Genetic ,medicine.medical_treatment ,Biophysics ,RNA ,Stimulation ,Cell Biology ,Interferon-beta ,Biology ,Biochemistry ,Molecular biology ,Article ,Cell Line ,De novo synthesis ,Immune system ,Cytokine ,Terminator (genetics) ,Poly I-C ,medicine ,Humans ,RNA, Messenger ,Molecular Biology ,Uridine - Abstract
Interferon-beta (IFN-β) is a critical antiviral cytokine and is essential for innate and acquired immune responses to pathogens. Treatment with polyinosinic:polycytidylic acid (poly(I:C)) induces transient accumulation of IFN-β mRNA, which involves an increase and a decrease of IFN-β mRNA. This phenomenon has been extensively analyzed as a model for understanding the mechanisms of transient gene induction in response to external stimuli. Using a new RNA metabolic labeling method with ethynyluridine to directly measure de novo RNA synthesis and RNA stability, we reassessed both de novo synthesis and degradation of IFN-β mRNA. We found that transcriptional activity is maintained after the maximum accumulation of IFN-β mRNA following poly(I:C) treatment on immortalized human bronchial epithelial cells. We also observed an unexpected change in the stability of IFN-β mRNA before and after the maximum accumulation. The results indicate that this method of RNA metabolic labeling provides a general approach for the simultaneous analysis of transcriptional activity and mRNA stability coupled with transcriptional timing.
- Published
- 2012
43. Multiple elements in the c-fos protein-coding region facilitate mRNA deadenylation and decay by a mechanism coupled to translation
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Joel G. Belasco, Cheryl L. Wellington, Michael E. Greenberg, Susan C. Schiavi, Ann-Bin Shyu, and Chyi-Ying A Chen
- Subjects
Genetics ,Untranslated region ,Messenger RNA ,Polyadenylation ,Nonsense-mediated decay ,Translation (biology) ,Cell Biology ,Biology ,Biochemistry ,Ribosome ,Cell biology ,Regulatory sequence ,Coding region ,Molecular Biology - Abstract
The c-fos proto-oncogene transcript is one of the most labile mammalian mRNAs known. Rapid degradation of c-fos mRNA is mediated by both the c-fos protein-coding region and an AU-rich element in the 3'-untranslated region. Here we present evidence that the c-fos coding region contains multiple destabilizing elements that can function independently to facilitate both deadenylation and decay of mRNA. The ability of these coding region destabilizing elements to direct deadenylation and decay requires the assembly of ribosomes at the 5' end of this domain and, most likely, translation of the message.
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- 1994
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44. Mechanisms of deadenylation-dependent decay
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Chyi-Ying A, Chen and Ann-Bin, Shyu
- Subjects
Kinetics ,RNA Stability ,Animals ,Humans ,RNA, Messenger ,RNA Processing, Post-Transcriptional ,Poly A ,Polyadenylation ,Models, Biological ,Article ,Signal Transduction - Abstract
Degradation of messenger RNAs (mRNAs) plays an essential role in modulation of gene expression and in quality control of mRNA biogenesis. Nearly all major mRNA decay pathways characterized thus far in eukaryotes are initiated by deadenylation, i.e., shortening of the mRNA 3(') poly(A) tail. Deadenylation is often a rate-limiting step for mRNA degradation and translational silencing, making it an important control point for both processes. In this review, we discuss the fundamental principles that govern mRNA deadenylation in eukaryotes. We use several major mRNA decay pathways in mammalian cells to illustrate mechanisms and regulation of deadenylation-dependent mRNA decay, including decay directed by adenine/uridine-rich elements (AREs) in the 3(') -untranslated region (UTR), the rapid decay mediated by destabilizing elements in protein-coding regions, the surveillance mechanism that detects and degrades nonsense-containing mRNA [i.e., nonsense-mediated decay (NMD)], the decay directed by miRNAs, and the default decay pathway for stable messages. Mammalian mRNA deadenylation involves two consecutive phases mediated by the PAN2-PAN3 and the CCR4-CAF1 complexes, respectively. Decapping takes place after deadenylation and may serve as a backup mechanism to trigger mRNA decay if initial deadenylation is compromised. In addition, we discuss how deadenylation impacts the dynamics of RNA processing bodies (P-bodies), where nontranslatable mRNAs can be degraded or stored. Possible models for mechanisms of various deadenylation-dependent mRNA decay pathways are also discussed.
- Published
- 2011
45. Regulation of mRNA Turnover
- Author
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Ann-Bin Shyu and Chyi-Ying A. Chen
- Subjects
Untranslated region ,Messenger RNP ,Messenger RNA ,microRNA ,Nonsense-mediated decay ,P-bodies ,Gene silencing ,Biology ,Molecular biology ,mRNA surveillance ,Cell biology - Abstract
Publisher Summary This chapter discusses how mRNA decay can be triggered and highlights some recent studies on regulation of mRNA turnover in mammalian cells. mRNA turnover is a complex set of cellular processes regulated by mechanisms independent from transcription. Regulation of mRNA turnover is important for controlling the abundance of cellular transcripts and thus the levels of protein expression. mRNA degradation in the cytoplasm can be initiated by deadenylation, decapping, or endonucleolytic cleavage within the message to generate 5′ and 3′ fragments. mRNA decay pathways in eukaryotic cells involve multiple steps and are more complicated than simply creating free ends by the three theoretical ways followed by exonucleolytic digestion. The importance of deadenylation in regulation of gene expression is further emphasized by two recent developments. First, a group of abundant, evolutionarily conserved small silencing RNAs, termed microRNAs (miRNAs), are capable of promoting decay of their mRNA targets by accelerating deadenylation, thereby achieving gene silencing. Second, non-translatable mRNA-protein complexes (mRNPs) are found in RNA processing bodies, a newly discovered cytoplasmic domain related to translation repression and mRNA decay. Both translation and stability of mRNAs are affected by deadenylation. The importance of deadenylation in mRNA turnover can be observed in all major mRNA decay pathways, including decay directed by AU-rich elements (AREs) in the 3′ UTR, the rapid decay mediated by destabilizing elements in protein coding regions, the surveillance mechanism that detects and degrades nonsense containing mRNA, and the default decay pathway for stable messages such as β-globin mRNA.
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- 2010
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46. Two cellular proteins bind specifically to a purine-rich sequence necessary for the destabilization function of a c-fos protein-coding region determinant of mRNA instability
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Ann-Bin Shyu, Yun You, and Chyi-Ying A. Chen
- Subjects
Ultraviolet Rays ,Molecular Sequence Data ,Biology ,Proto-Oncogene Mas ,DNA-binding protein ,Mice ,Sequence Homology, Nucleic Acid ,Polysome ,Animals ,Humans ,Gene family ,Coding region ,RNA, Messenger ,Binding site ,Molecular Biology ,Sequence (medicine) ,Base Composition ,Messenger RNA ,Binding Sites ,Base Sequence ,3T3 Cells ,Cell Biology ,Molecular biology ,DNA-Binding Proteins ,Purines ,Cytoplasm ,Mutagenesis, Site-Directed ,Proto-Oncogene Proteins c-fos ,Research Article - Abstract
The c-fos proto-oncogene mRNA is rapidly degraded within minutes after its appearance in the cytoplasm of growth factor-stimulated mammalian fibroblasts. At least two functionally independent sequence elements are responsible for the lability of c-fos mRNA. One of these determinants is located within a 0.32-kb sequence present in the protein-coding region. We demonstrate by gel mobility shift experiments and UV cross-linking that at least two protein factors specifically interact with a 56-nucleotide purine-rich sequence located at the 5' end of the 0.32-kb coding region determinant of mRNA instability (CRDI). One protein is predominantly associated with the polysomes, while the other is detected in the post-ribosomal supernatant. Sequence comparison of members of the fos gene family revealed that the high purine content of the protein-binding region is conserved through evolution. Deletion of this region from the 0.32-kb CRDI severely impedes its function as an RNA-destabilizing element. Our results suggest that binding of the two proteins to the purine-rich sequence may participate in the rapid mRNA decay mediated by this 0.32-kb c-fos CRDI.
- Published
- 1992
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47. U-rich sequence-binding proteins (URBPs) interacting with a 20-nucleotide U-rich sequence in the 3' untranslated region of c-fos mRNA may be involved in the first step of c-fos mRNA degradation
- Author
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Yun You, Ann-Bin Shyu, and Chyi-Ying A. Chen
- Subjects
Untranslated region ,Transcription, Genetic ,Ultraviolet Rays ,Molecular Sequence Data ,RNA-binding protein ,Regulatory Sequences, Nucleic Acid ,Biology ,Mice ,Consensus Sequence ,Consensus sequence ,Animals ,Electrophoretic mobility shift assay ,RNA, Messenger ,Molecular Biology ,Protein Synthesis Inhibitors ,Messenger RNA ,Binding Sites ,Base Sequence ,Three prime untranslated region ,Binding protein ,Nucleic acid sequence ,Genes, fos ,RNA-Binding Proteins ,3T3 Cells ,Cell Biology ,Molecular biology ,Cell biology ,Cross-Linking Reagents ,Protein Biosynthesis ,Research Article - Abstract
Rapid decay of the c-fos transcript plays a critical role in controlling transforming potential of the c-fos proto-oncogene. One of the mRNA instability determinants is a 75-nucleotide AU-rich element (ARE) present in the 3' untranslated region of the c-fos transcript. It appears to control two steps in the process of c-fos mRNA degradation: removal of the poly(A) tail, which does not require the AUUUA motifs, and subsequent degradation of deadenylated mRNA, which appears to be dependent on the AUUUA motifs. In this study, we report the identification of four U-rich sequence binding proteins (URBPs) that specifically interact with a 20-nucleotide U-rich sequence within the c-fos ARE. Gel mobility shift assay and competition experiments showed that these protein factors form three specific band-shifted complexes with the c-fos ARE. Binding activity of one of the protein factors, a 37-kDa protein, is significantly affected by serum induction and by pretreatment of cells with drugs known to stabilize many of the immediate-early gene mRNAs. Combining UV cross-linking with a new approach, designated sequential RNase digestion, we were able to better determine the molecular masses of these cellular proteins. The binding sites for the four proteins were all mapped to a 20-nucleotide U-rich sequence located at the 3' half of the c-fos ARE, which contains no AUUUA pentanucleotides but stretches of uridylate residues. Single U-to-A point mutations in each of the three AUUUA motifs within the c-fos ARE have little effect on formation of the mobility-shifted complexes. Our data indicate c-fos ARE-protein interaction involves recognition of U stretches rather than recognition of the AUUUA motifs. We propose that UTBP binding may be involved in the first step, removal of the Poly(A) tail, in the c-fos ARE-mediated decay pathway.
- Published
- 1992
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48. Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay
- Author
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Ann-Bin Shyu, Joel G. Belasco, and Michael E. Greenberg
- Subjects
Untranslated region ,Polyadenylation ,Molecular Sequence Data ,Biology ,medicine.disease_cause ,Cell Line ,Mice ,Proto-Oncogene Proteins ,Proto-Oncogenes ,Genetics ,medicine ,Animals ,RNA, Messenger ,Northern blot ,AU-rich element ,Messenger RNA ,Mutation ,Base Sequence ,Transfection ,Molecular biology ,Globins ,Cell biology ,Kinetics ,Decapping complex ,Rabbits ,Poly A ,Proto-Oncogene Proteins c-fos ,Developmental Biology - Abstract
The mechanisms by which c-fos mRNA is targeted for decay have been examined. Rapid removal of the poly(A) tail occurs before the transcribed portion of the c-fos message is degraded. Identification of the determinants that mediate c-fos message deadenylation reveals that they coincide directly with previously characterized determinants of c-fos mRNA instability, one in the protein-coding region and the other an AU-rich element (ARE) in the 3'-untranslated region. Insertion of either of these c-fos instability elements into the stable beta-globin message confers the property of rapid deadenylation. Mutation of the ARE indicates that this sequence controls two steps in the process of c-fos mRNA degradation: removal of the poly(A) tail, which does not require intact AUUUA pentanucleotides within the ARE, and subsequent degradation of the transcribed portion of the message, which appears to be dependent on the AUUUA pentanucleotides. These results indicate that structurally distinct instability determinants within the transcribed portion of labile messages can function by promoting rapid removal of the poly(A) tail as a first step in the decay process.
- Published
- 1991
- Full Text
- View/download PDF
49. Messenger RNA half-life measurements in mammalian cells
- Author
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Chyi-Ying A, Chen, Nader, Ezzeddine, and Ann-Bin, Shyu
- Subjects
Mammals ,Transcription, Genetic ,RNA Stability ,Animals ,Humans ,RNA, Messenger ,Promoter Regions, Genetic ,Article ,Half-Life - Abstract
The recognition of the importance of mRNA turnover in regulating eukaryotic gene expression has mandated the development of reliable, rigorous, and “user-friendly” methods to accurately measure changes in mRNA stability in mammalian cells. Frequently, mRNA stability is studied indirectly by analyzing the steady-state level of mRNA in the cytoplasm; in this case, changes in mRNA abundance are assumed to reflect only mRNA degradation, an assumption that is not always correct. Although direct measurements of mRNA decay rate can be performed with kinetic labeling techniques and transcriptional inhibitors, these techniques often introduce significant changes in cell physiology. Furthermore, many critical mechanistic issues as to deadenylation kinetics, decay intermediates, and precursor-product relationships cannot be readily addressed by these methods. In light of these concerns, we have previously reported transcriptional pulsing methods based on the c-fos serum-inducible promoter and the tetracycline-regulated (Tet-off) promoter systems to better explain mechanisms of mRNA turnover in mammalian cells. In this chapter, we describe and discuss in detail different protocols that use these two transcriptional pulsing methods. The information described here also provides guidelines to help develop optimal protocols for studying mammalian mRNA turnover in different cell types under a wide range of physiologic conditions.
- Published
- 2008
50. Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation
- Author
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Fabiola V. Rivas, Géraldine Mathonnet, Thomas R. Sundermeier, Jennifer A. Doudna, Yuri V. Svitkin, Nahum Sonenberg, Witold Filipowicz, John R. Yates, Hansruedi Mathys, Thomas F. Duchaine, James A. Wohlschlegel, Ann-Bin Shyu, Jakob T. Zipprich, Gregory J. Hannon, Marc R. Fabian, Chyi-Ying A. Chen, and Martin Jinek
- Subjects
Receptors, CCR4 ,MRNA destabilization ,RNA-induced silencing complex ,RNA Stability ,Eukaryotic Initiation Factor-2 ,Transfection ,Autoantigens ,Poly(A)-Binding Proteins ,Article ,Carcinoma, Krebs 2 ,Mice ,Ribonucleases ,microRNA ,Poly(A)-binding protein ,CCR4-NOT complex ,Gene silencing ,Animals ,Humans ,RNA-Induced Silencing Complex ,Gene Silencing ,RNA, Messenger ,RNA Processing, Post-Transcriptional ,Molecular Biology ,Binding Sites ,biology ,Cell-Free System ,Ascites ,Proteins ,Cell Biology ,Argonaute ,Molecular biology ,Cell biology ,Protein Structure, Tertiary ,Repressor Proteins ,Kinetics ,MicroRNAs ,Protein Biosynthesis ,Argonaute Proteins ,Exoribonucleases ,biology.protein ,Eukaryotic Initiation Factor-4G ,Poly(A)-Binding Protein I ,HeLa Cells - Abstract
MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3'UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.
- Published
- 2008
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