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Rett syndrome (RTT) is a severe neurological disorder and a leading cause of intellectual disability in young females. RTT is mainly caused by mutations found in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). Despite extensive studies, the molecular mechanism underlying RTT pathogenesis is still poorly understood. Here, we report MeCP2 as a key subunit of a higher-order multiunit protein complex Rbfox/LASR. Defective MeCP2 in RTT mouse models disrupts the assembly of the MeCP2/Rbfox/LASR complex, leading to reduced binding of Rbfox proteins to target pre-mRNAs and aberrant splicing of Nrxns and Nlgn1 critical for synaptic plasticity. We further show that MeCP2 disease mutants display defective condensate properties and fail to promote phase-separated condensates with Rbfox proteins in vitro and in cultured cells. These data link an impaired function of MeCP2 with disease mutation in splicing control to its defective properties in mediating the higher-order assembly of the MeCP2/Rbfox/LASR complex., MeCP2 mutations can cause Rett syndrome, a severe childhood neurological disorder. Here the authors show that MeCP2 mediates the higher-order assembly of a large splicing complex Rbfox/LASR, which is disrupted in the mouse models of Rett syndrome.

Background Most of the microRNAs (MiRs) involved in myogenesis are transcriptional regulated. The role of MiR biogenesis in myogenesis has not been characterized yet. RNA‐binding protein Musashi 2 (Msi2) is considered to be one of the major drivers for oncogenesis and stem cell proliferation. The functions of Msi2 in myogenesis have not been explored yet. We sought to investigate Msi2‐regulated biogenesis of MiRs in myogenesis and muscle stem cell (MuSC) ageing. Methods We detected the expression of Msi2 in MuSCs and differentiated myotubes by quantitative reverse transcription PCR (RT‐qPCR) and western blot. Msi2‐binding partner human antigen R (HuR) was identified by immunoprecipitation followed by mass spectrometry analysis. The cooperative binding of Msi2 and HuR on MiR7a‐1 was analysed by RNA immunoprecipitation and electrophoresis mobility shift assays. The inhibition of the processing of pri‐MiR7a‐1 mediated by Msi2 and HuR was shown by Msi2 and HuR knockdown. Immunofluorescent staining, RT‐qPCR and immunoblotting were used to characterize the function of MiR7a‐1 in myogenesis. Msi2 and HuR up‐regulate cryptochrome circadian regulator 2 (Cry2) via MiR7a‐1 was confirmed by the luciferase assay and western blot. The post‐transcriptional regulatory cascade was further confirmed by RNAi and overexpressing of Msi2 and HuR in MuSCs, and the in vivo function was characterized by histopathological and molecular biological methods in Msi2 knockout mice. Results We identified a post‐transcription regulatory cascade governed by a pair of RNA‐binding proteins Msi2 and HuR. Msi2 is enriched in differentiated muscle cells and promotes MuSC differentiation despite its pro‐proliferation functions in other cell types. Msi2 works synergistically with another RNA‐binding protein HuR to repress the biogenesis of MiR7a‐1 in an Msi2 dose‐dependent manner to regulate the translation of the key component of the circadian core oscillator complex Cry2. Down‐regulation of Cry2 (0.6‐fold, vs. control, P

The congenital intellectual disability (ID)-causing gene mutations remain largely unclear, although many genetic variations might relate to ID. We screened gene mutations in Chinese Han children suffering from severe ID and found a single-nucleotide polymorphism (SNP) in the 5′-untranslated region (5′-UTR) of fibroblast growth factor 13 (FGF13) mRNA (NM_001139500.1:c.-32c>G) shared by three male children. In both HEK293 cells and patient-derived induced pluripotent stem cells, this SNP reduced the translation of FGF13, which stabilizes microtubules in developing neurons. Mice carrying the homologous point mutation in 5′-UTR of Fgf13 showed delayed neuronal migration during cortical development, and weakened learning and memory. Furthermore, this SNP reduced the interaction between FGF13 5′-UTR and polypyrimidine-tract-binding protein 2 (PTBP2), which was required for FGF13 translation in cortical neurons. Thus, this 5′-UTR SNP of FGF13 interferes with the translational process of FGF13 and causes deficits in brain development and cognitive functions.

The Y-box binding protein 1 (YB-1) is a member of the cold shock domain (CSD) protein family and is recognized as an oncogenic factor in several solid tumors. By binding to RNA, YB-1 participates in several steps of posttranscriptional regulation of gene expression, including mRNA splicing, stability, and translation; microRNA processing; and stress granule assembly. However, the mechanisms in YB-1–mediated regulation of RNAs are unclear. Previously, we used both systematic evolution of ligands by exponential enrichment (SELEX) and individual-nucleotide resolution UV cross-linking and immunoprecipitation coupled RNA-Seq (iCLIP-Seq) analyses, which defined the RNA-binding consensus sequence of YB-1 as CA(U/C)C. We also reported that through binding to its core motif CAUC in primary transcripts, YB-1 regulates the alternative splicing of a CD44 variable exon and the biogenesis of miR-29b-2 during both Drosha and Dicer steps. To elucidate the molecular basis of the YB-1–RNA interactions, we report high-resolution crystal structures of the YB-1 CSD in complex with different RNA oligos at 1.7 Å resolution. The structure revealed that CSD interacts with RNA mainly through π–π stacking interactions assembled by four highly conserved aromatic residues. Interestingly, YB-1 CSD forms a homodimer in solution, and we observed that two residues, Tyr-99 and Asp-105, at the dimer interface are important for YB-1 CSD dimerization. Substituting these two residues with Ala reduced CSD's RNA-binding activity and abrogated the splicing activation of YB-1 targets. The YB-1 CSD–RNA structures presented here at atomic resolution provide mechanistic insights into gene expression regulated by CSD-containing proteins.

Additional file 2: Figure S1. Distribution of RNA editing levels (Φ) across 49 analyzed tissues. Figure S2. Scatter plot of the number of edQTL sites normalized by the number of tested sites vs sample size across 49 tissues. Figure S3. Stacked bar plots for the number and proportion of shared and tissue-specific edQTL sites and ASED sites across all tissues. Figure S4. Impact of edQTL SNP on RNA secondary structure and RNA editing. Figure S5. Tissue-dependent genotype effect sizes in muscle vs non-muscle tissues. Figure S6. Allele-specific expression analysis of DHFR with respect to rs1650720. Figure S7. Comparison of miRNA quantification between qPCR and the RNA-seq based pri-miRNA proxy method. Figure S8. Simulations of edQTL and eQTL signals with miRNA targeting unedited transcripts. Figure S9. Simulations of edQTL and eQTL signals with miRNA targeting edited transcripts. Figure S10. Examples of nonsynonymous edQTL sites. Figure S11. GWAS traits associated with edQTL and eQTL signals of FADS1. Figure S12. Luciferase protein expression data for validating editing-specific targeting of miRNAs to unedited or edited 3′-UTR sequences.

Additional file 16. Review history.

Y-box-binding protein 1 (YB-1) is a multifunctional RNA binding protein involved in virtually every step of RNA metabolism. However, the functions and mechanisms of YB-1 in one of the most aggressive cancers, glioblastoma, are not well understood. In this study, we found that YB-1 protein was markedly overexpressed in glioblastoma and acted as a critical activator of both mTORC1 and mTORC2 signaling. Mechanistically, YB-1 bound the 5'UTR of CCT4 mRNA to promote the translation of CCT4, a component of the CCT chaperone complex, that in turn activated the mTOR signaling pathway by promoting mLST8 folding. In addition, YB-1 autoregulated its own translation by binding to its 5'UTR, leading to sustained activation of mTOR signaling. In patients with glioblastoma, high protein expression of YB-1 correlated with increased expression of CCT4 and mLST8 and activated mTOR signaling. Importantly, the administration of RNA decoys specifically targeting YB-1 in a mouse xenograft model resulted in slower tumor growth and better survival. Taken together, these findings uncover a disrupted proteostasis pathway involving a YB-1/CCT4/mLST8/mTOR axis in promoting glioblastoma growth, suggesting that YB-1 is a potential therapeutic target for the treatment of glioblastoma.

The congenital intellectual disability (ID)-causing gene mutations remain largely unclear, although many genetic variations might relate to ID. We screened gene mutations in Chinese Han children suffering from severe ID and found a single-nucleotide polymorphism (SNP) in the 5′-untranslated region (5′-UTR) of fibroblast growth factor 13 (FGF13) mRNA (NM_001139500.1:c.-32c>G) shared by three male children. In both HEK293 cells and patient-derived induced pluripotent stem cells, this SNP reduced the translation of FGF13, which stabilizes microtubules in developing neurons. Mice carrying the homologous point mutation in 5′-UTR of Fgf13 showed delayed neuronal migration during cortical development, and weakened learning and memory. Furthermore, this SNP reduced the interaction between FGF13 5′-UTR and polypyrimidine-tract-binding protein 2 (PTBP2), which was required for FGF13 translation in cortical neurons. Thus, this 5′-UTR SNP of FGF13 interferes with the translational process of FGF13 and causes deficits in brain development and cognitive functions.

Accumulating evidence indicates that the alternative splicing program undergoes extensive changes during cancer development and progression. The RNA-binding protein QKI-5 is frequently downregulated and exhibits anti-tumor activity in lung cancer. Howeve-r, little is known about the functional targets and regulatory mechanism of QKI-5. Here, we report that upregulation of exon 14 inclusion of cytoskeletal gene Adducin 3 (ADD3) significantly correlates with a poor prognosis in lung cancer. QKI-5 inhibits cell proliferation and migration in part through suppressing the splicing of ADD3 exon 14. Through genome-wide mapping of QKI-5 binding sites in vivo at nucleotide resolution by iCLIP-seq analysis, we found that QKI-5 regulates alternative splicing of its target mRNAs in a binding position-dependent manner. By binding to multiple sites in an upstream intron region, QKI-5 represses the splicing of ADD3 exon 14. We also identified several QKI mutations in tumors, which cause dysregulation of the splicing of QKI targets ADD3 and NUMB. Taken together, our results reveal that QKI-mediated alternative splicing of ADD3 is a key lung cancer-associated splicing event, which underlies in part the tumor suppressor function of QKI.

The histone H3K36 methyltransferase SETD2 is frequently mutated or deleted in a variety of human tumors. Nevertheless, the role of SETD2 loss in oncogenesis remains largely undefined. Here, we found that SETD2 counteracts Wnt signaling and its inactivation promotes intestinal tumorigenesis in mouse models of colorectal cancer (CRC). SETD2 was not required for intestinal homeostasis under steady state; however, upon irradiation, genetic inactivation of Setd2 in mouse intestinal epithelium facilitated the self-renewal of intestinal stem/progenitor cells as well as tissue regeneration. Furthermore, depletion of SETD2 enhanced the susceptibility to tumorigenesis in the context of dysregulated Wnt signaling. Mechanistic characterizations indicated that SETD2 downregulation affects the alternative splicing of a subset of genes implicated in tumorigenesis. Importantly, we uncovered that SETD2 ablation reduces intron retention of dishevelled segment polarity protein 2 (DVL2) pre-mRNA, which would otherwise be degraded by nonsense-mediated decay, thereby augmenting Wnt signaling. The signaling cascades mediated by SETD2 were further substantiated by a CRC patient cohort analysis. Together, our studies highlight SETD2 as an integral regulator of Wnt signaling through epigenetic regulation of RNA processing during tissue regeneration and tumorigenesis.

MicroRNA (miRNA) biogenesis is finely controlled by complex layers of post-transcriptional regulators, including RNA-binding proteins (RBPs). Here, we show that an RBP, QKI5, activates the processing of primary miR-124-1 (pri-124-1) during erythropoiesis. QKI5 recognizes a distal QKI response element and recruits Microprocessor through interaction with DGCR8. Furthermore, the recruited Microprocessor is brought to pri-124-1 stem loops by a spatial RNA-RNA interaction between two complementary sequences. Thus, mutations disrupting their base-pairing affect the strength of QKI5 activation. When erythropoiesis proceeds, the concomitant decrease of QKI5 releases Microprocessor from pri-124-1 and reduces mature miR-124 levels to facilitate erythrocyte maturation. Mechanistically, miR-124 targets TAL1 and c-MYB, two transcription factors involved in normal erythropoiesis. Importantly, this QKI5-mediated regulation also gives rise to a unique miRNA signature, which is required for erythroid differentiation. Taken together, these results demonstrate the pivotal role of QKI5 in primary miRNA processing during erythropoiesis and provide new insights into how a distal element on primary transcripts affects miRNA biogenesis.

Extracellular signals have been shown to impact on alternative pre-mRNA splicing; however, the molecular mechanisms and biological significance of signal-induced splicing regulation remain largely unknown. Here, we report that epidermal growth factor (EGF) induces splicing changes through ubiquitylation of a well-known splicing regulator, hnRNP A1. EGF signaling upregulates an E3 ubiquitin (Ub) ligase adaptor, SPRY domain-containing SOCS box protein 1 (SPSB1), which recruits Elongin B/C-Cullin complexes to conjugate lysine 29-linked polyUb chains onto hnRNP A1. Importantly, SPSB1 and ubiquitylation of hnRNP A1 have a critical role in EGF-driven cell migration. Mechanistically, EGF-induced ubiquitylation of hnRNP A1 together with the activation of SR protein kinases (SRPKs) results in the upregulation of a Rac1 splicing isoform, Rac1b, to promote cell motility. These findings unravel a novel crosstalk between protein ubiquitylation and alternative splicing in EGF/EGF receptor signaling, and identify a new EGF/SPSB1/hnRNP A1/Rac1 axis in modulating cell migration, which may have important implications for cancer treatment.

Benefited from the booming social network, reading posts from other users overinternet is becoming one of commonest ways for people to intake information. Onemay also have noticed that sometimes we tend to focus on users provide well-foundedanalysis, rather than those merely who vent their emotions. This thesis aims atfinding a simple and efficient way to recognize reliable information sources amongcountless internet users by examining the sentiments from their past posts.To achieve this goal, the research utilized a dataset of tweets about Apples stockprice retrieved from Twitter. Key features we studied include post-date, user name,number of followers of that user, and the sentiment of that tweet. Prior to makingfurther use of the dataset, tweets from users who do not have sufficient posts arefiltered out. To compare user sentiments and the derivative of Apples stock price, weuse Pearson correlation between them for to describe how well each user performs.Then we iteratively increase the weight of reliable users and lower the weight ofuntrustworthy users, the correlation between overall sentiment and the derivative ofstock price will finally converge. The final correlations for individual users are theirperformance scores. Due to the chaos of real world data, manual segmentation viadata visualization is also proposed as a denoise method to improve performance.Besides our method, other metrics can also be considered as user trust index, suchas numbers of followers of each user. Experiments are conducted to prove that ourmethod out performs others. With simple input, this method can be applied on awide range of topics including election, economy, and job market.

Alternative pre‐mRNA splicing plays important roles in regulating self‐renewal and differentiation of embryonic stem cells (ESCs). However, how specific alternative splicing programs are established in ESCs remains elusive. Here, we show that a subset of alternative splicing events in ESCs is dependent on miR‐294 expression. Remarkably, roughly 60% of these splicing events are affected by the depletion of Muscleblind‐Like Splicing Regulator 1 and 2 (Mbnl1/2). Distinct from canonical miRNA function, miR‐294 represses Mbnl1/2 through both posttranscriptional and epigenetic mechanisms. Furthermore, we uncover non‐canonical functions of MBNL proteins that bind and promote the expression of miR‐294 targets, including Cdkn1a and Tgfbr2, thereby opposing the role of miR‐294 in regulating cell proliferation, apoptosis, and epithelial–mesenchymal transition (EMT). Our study reveals extensive interactions between miRNAs and splicing factors, highlighting their roles in regulating cell type‐specific alternative splicing and defining gene expression programs during development and cellular differentiation.

Altered miRNA expression is believed to play a crucial role in a variety of human cancers; however, the mechanisms leading to the dysregulation of miRNA expression remain elusive. In this study, we report that the human Y box-binding protein (YB-1), a major mRNA packaging protein, is a novel modulator of miRNA processing in glioblastoma multiforme (GBM). Using individual nucleotide-resolution crosslinking immunoprecipitation coupled to deep sequencing (iCLIP-seq), we performed the first genome-wide analysis of the in vivo YB-1-RNA interactions and found that YB-1 preferentially recognizes a UYAUC consensus motif and binds to the majority of coding gene transcripts including pre-mRNAs and mature mRNAs. Remarkably, our data show that YB-1 also binds extensively to the terminal loop region of pri-/pre-miR-29b-2 and regulates the biogenesis of miR-29b-2 by blocking the recruitment of microprocessor and Dicer to its precursors. Furthermore, we show that down-regulation of miR-29b by YB-1, which is up-regulated in GBM, is important for cell proliferation. Together, our findings reveal a novel function of YB-1 in regulating non-coding RNA expression, which has important implications in tumorigenesis.

BS69 (aka ZMYND11) contains tandemly arranged PHD, BROMO and PWWP domains, which are chromatin recognition modalities. Here we show that BS69 selectively recognizes histone variant H3.3 lysine 36 trimethylation (H3.3K36me3) via its chromatin-binding domains. We further identify BS69 association with RNA splicing regulators including the U5 snRNP components of the spliceosome such as EFTUD2. Remarkably, RNA-seq shows that BS69 mainly regulates intron retention (IR), which is the least well-understood RNA alternative splicing event in mammalian cells. Biochemical and genetic experiments demonstrate that BS69 promotes IR by antagonizing EFTUD2 through physical interactions. We further show that regulation of IR by BS69 also depends on its binding to H3K36me3-decorated chromatin. Taken together, our study identifies an H3.3K36me3-specific reader and a regulator of IR, and reveals a novel and unexpected role of BS69 in connecting histone H3.3K36me3 to regulated RNA splicing, providing significant new insights into chromatin regulation of pre-mRNA processing.

The human Y box-binding protein-1 (YB-1) is a deoxyribonucleic acid (DNA)/ribonucleic acid (RNA)-binding protein with pleiotropic functions. Besides its roles in the regulation of transcription and translation, several recent studies indicate that YB-1 is a spliceosome-associated protein and is involved in alternative splicing, but the underlying mechanism has remained elusive. Here, we define both CAUC and CACC as high-affinity binding motifs for YB-1 by systematic evolution of ligands by exponential enrichment (SELEX) and demonstrate that these newly defined motifs function as splicing enhancers. Interestingly, on the endogenous CD44 gene, YB-1 appears to mediate a network interaction to activate exon v5 inclusion via multiple CAUC motifs in both the alternative exon and its upstream polypyrimidine tract. We provide evidence that YB-1 activates splicing by facilitating the recruitment of U2AF65 to weak polypyrimidine tracts through direct protein–protein interactions. Together, these findings suggest a vital role of YB-1 in activating a subset of weak 3′ splice sites in mammalian cells.

Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its MED23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit, MED23, but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially colocalizes with hnRNP L and the splicing machinery in the cell. Functionally, MED23 regulates a subset of hnRNP L-targeted alternative splicing (AS) and alternative cleavage and polyadenylation (APA) events, as shown by minigene reporters and exon array analysis. ChIP-seq analysis revealed that MED23 can regulate hnRNP L occupancy at their coregulated genes. Taken together, these results demonstrate a crosstalk between Mediator and the splicing machinery, providing a molecular basis for coupling mRNA processing to transcription.

Alternative pre-mRNA splicing is a key mechanism for increasing proteomic diversity and modulating gene expression. Emerging evidence indicated that the splicing program is frequently dysregulated during tumorigenesis. Cancer cells produce protein isoforms that can promote growth and survival. The RNA-binding protein QKI5 is a critical regulator of alternative splicing in expanding lists of primary human tumors and tumor cell lines. However, its biological role and regulatory mechanism are poorly defined in gastric cancer (GC) development and progression. In this study, we demonstrated that the downregulation of QKI5 was associated with pTNM stage and pM state of GC patients. Re-introduction of QKI5 could inhibit GC cell proliferation, migration, and invasion in vitro and in vivo, which might be due to the altered splicing pattern of macroH2A1 pre-mRNA, leading to the accumulation of macroH2A1.1 isoform. Furthermore, QKI5 could inhibit cyclin L1 expression via promoting macroH2A1.1 production. Thus, this study identified a novel regulatory axis involved in gastric tumorigenesis and provided a new strategy for GC therapy.

Model-based diagnosis is a new type of intelligent reasoning technology that is developed to overcome the serious defects of traditional expert system diagnosis method, which is a popular method in the 1970s. The basic point of view of model-based reasoning is that the knowledge of the internal structure and behavior of a device can be used to diagnose the device. On one hand, the actual system behavior is monitored. On the other hand, the internal model will predict system behavior. When there are non-negligible differences between the two results, a potential fault is detected by the diagnosis process [1].

We recently characterized human hnRNP L as a global regulator of alternative splicing, binding to CA-repeat and CA-rich elements. Here we report that hnRNP L autoregulates its own expression on the level of alternative splicing. Intron 6 of the human hnRNP L gene contains a short exon that, if used, introduces a premature termination codon, resulting in nonsense-mediated decay (NMD). This “poison exon” is preceded by a highly conserved CA-rich cluster extending over 800 nucleotides that binds hnRNP L and functions as an unusually extended, intronic enhancer, promoting inclusion of the poison exon. As a result, excess hnRNP L activates NMD of its own mRNA, thereby creating a negative autoregulatory feedback loop and contributing to homeostasis of hnRNP L levels. We present experimental evidence for this mechanism, based on NMD inactivation, hnRNP L binding assays, and hnRNP L-dependent alternative splicing of heterologous constructs. In addition, we demonstrate that hnRNP L cross-regulates inclusion of an analogous poison exon in the hnRNP L-like pre-mRNA, which explains the reciprocal expression of the two closely related hnRNP L proteins.

In eukaryotes, most protein-coding genes contain introns which are removed by precursor messenger RNA (pre-mRNA) splicing. Alternative splicing is a process by which multiple messenger RNAs (mRNAs) are generated from a single pre-mRNA, resulting in functionally distinct proteins. Recent genome-wide analyses of alternative splicing indicated that in higher eukaryotes alternative splicing is an important mechanism that generates proteomic complexity and regulates gene expression. Mis-regulation of splicing causes a wide range of human diseases. This review describes the current understanding of pre-mRNA splicing and the mechanisms that regulate mammalian pre-mRNA splicing. It also discusses emerging directions in the field of alternative splicing.

Alternative mRNA splicing patterns are determined by the combinatorial control of regulator proteins and their target RNA sequences. We have recently characterized human hnRNP L as a global regulator of alternative splicing, binding to diverse C/A-rich elements. To systematically identify hnRNP L target genes on a genome-wide level, we have combined splice-sensitive microarray analysis and an RNAi-knockdown approach. As a result, we describe 11 target genes of hnRNP L that were validated by RT-PCR and that represent several new modes of hnRNP L-dependent splicing regulation, involving both activator and repressor functions: first, intron retention; second, inclusion or skipping of cassette-type exons; third, suppression of multiple exons; and fourth, alternative poly(A) site selection. In sum, this approach revealed a surprising diversity of splicing-regulatory processes as well as poly(A) site selection in which hnRNP L is involved.

We have recently identified an intronic polymorphic CA-repeat region in the human endothelial nitric oxide synthase (eNOS) gene as an important determinant of the splicing efficiency, requiring specific binding of hnRNP L. Here, we analyzed the position requirements of this CA-repeat element, which revealed its potential role in alternative splicing. In addition, we defined the RNA binding specificity of hnRNP L by SELEX: not only regular CA repeats are recognized with high affinity but also certain CA-rich clusters. Therefore, we have systematically searched the human genome databases for CA-repeat and CA-rich elements associated with alternative 5' splice sites (5'ss), followed by minigene transfection assays. Surprisingly, in several specific human genes that we tested, intronic CA RNA elements could function either as splicing enhancers or silencers, depending on their proximity to the alternative 5'ss. HnRNP L was detected specifically bound to these diverse CA elements. These data demonstrated that intronic CA sequences constitute novel and widespread regulatory elements of alternative splicing.

CA dinucleotide repeat sequences are very common in the human genome. We have recently demonstrated that the polymorphic CA repeats in intron 13 of the human endothelial nitric oxide synthase (eNOS) gene function as an unusual, length-dependent splicing enhancer. The CA repeat enhancer requires for its activity specific binding of hnRNP L. Here we show that in the absence of bound hnRNP L, the pre-mRNA is cleaved directly upstream of the CA repeats. The addition of recombinant hnRNP L restores RNA stability. CA repeats are both necessary and sufficient for this specific cleavage in the 5′ adjacent RNA sequence. We conclude that—in addition to its role as a splicing activator—hnRNP L can act in vitro as a sequence-specific RNA protection factor. Based on the wide abundance of CA repetitive sequences in the human genome, this may represent a novel, generally important role of this abundant hnRNP protein.

In the human genome, dinucleotide repeats are common sequence elements of unknown functional significance. Here we demonstrate that CA repeats in intron 13 of the human endothelial nitric oxide synthase (eNOS) gene function as an unusual intronic splicing enhancer, whose activity depends on the CA repeat number. We identify the 65 kDa heterogenous nuclear ribonucleoprotein (hnRNP) L as the major factor that binds specifically and in a length-dependent manner to the CA-repeat enhancer. In addition, we show that hnRNP L functions as a specific activator of eNOS splicing, providing the first evidence for a role of hnRNP L in the regulation of mRNA splicing. We hypothesize that hnRNP L may be involved in the regulation of many other genes containing CA repeats or A/C-rich enhancers.

Lung cancer is the leading cause of cancer-related death worldwide. Aberrant splicing has been implicated in lung tumorigenesis. However, the functional links between splicing regulation and lung cancer are not well understood. Here we identify the RNA-binding protein QKI as a key regulator of alternative splicing in lung cancer. We show that QKI is frequently down-regulated in lung cancer, and its down-regulation is significantly associated with a poorer prognosis. QKI-5 inhibits the proliferation and transformation of lung cancer cells both in vitro and in vivo. Our results demonstrate that QKI-5 regulates the alternative splicing of NUMB via binding to two RNA elements in its pre-mRNA, which in turn suppresses cell proliferation and prevents the activation of the Notch signaling pathway. We further show that QKI-5 inhibits splicing by selectively competing with a core splicing factor SF1 for binding to the branchpoint sequence. Taken together, our data reveal QKI as a critical regulator of splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated regulation of the Notch signaling pathway., Author Summary Alternative pre-mRNA splicing is a key mechanism for increasing proteomic diversity and modulating gene expression. Emerging evidence indicates that splicing program is frequently deregulated during tumorigenesis, and cancer cells favor to produce protein isoforms that can promote growth and survival. Lung cancer is one of the most common cancers and the leading cause of cancer-related death worldwide. Although a number of lung cancer-related splicing events have been detected in several genome-wide analyses, much less is known about how aberrant splicing takes place in lung cancer and how it contributes to tumor development. In this study, we characterized the RNA-binding protein QKI as a new critical regulator of alternative splicing in lung cancer and as a potential marker for prognosis. Genome-wide analysis of QKI-dependent splicing by RNA-Seq identified some cancer-associated splicing changes as its targets. Our results demonstrate that QKI-5 inhibits cancer cell proliferation and prevents inappropriate activation of the Notch signaling pathway by regulating its key target, NUMB. We further showed that QKI-5 represses the inclusion of NUMB alternative exon through competing with a core splicing factor SF1. In summary, our data indicate that down-regulation of QKI causes aberrant splicing in lung cancer and suggest a novel tumor suppression mechanism involving QKI-mediated repression of Notch signaling.

Three plasmid expression vectors containing modified hepatitis B surface antigen (HBsAg) carrying preS epitopes were constructed. Transient expression afterin vitro transfection in COS-M6 cells showed that under the transcriptional control of the human cytomegalovirus (CMV) immediate early promoter, fusion genes expressed the modified HBV envelope proteins which were efficiently secreted into culture medium and presented HBsAg, preS1 and preS2 antigenicity. DNA-based immunization with these plasmids carrying preS sequences induced anti-HBs antibody in BALB/c mice. The titers of anti-HBs antibody were higher than those appeared in mice immunized with plasmid carrying S gene only. DNA injection with plasmids containing preSl sequences elicited also high titers of anti-preS1 antibody. Moreover, the antipreSl antibodies were found to appear earlier than anti-HBs antibodies.

Intramuscular injection of a plasmid encoding a modified hepatitis B surface antigen (HBsAg) induced humoral and cytotoxic responses in C57BL/6 mice. This modified HBsAg contains a preS1-derived peptide (amino acids 21 to 47), that carries the HBV receptor binding site for hepatocytes fused to the C-terminus of the small protein (at the position of amino acid 223). After a single DNA injection, the immunized mice elicited high titers of anti-HBs and anti-preS1 antibodies, and produced strong HBV specific cytotoxic T-lymphocyte (CTL) responses. The immune response induced after a single injection of this modified HBsAg gene in HBsAg-transgenic mice resulted in the clearance of circulating HBsAg and the appearance of anti-HBs and anti-preS1 antibodies. The high titers of preS1 antibody in transgenic mice were comparable to those found in non-transgenic controls and may be efficient to clear Dane particles existing in sera from chronic carriers. These data indicated that a genetic vaccine consisting of this modified HBsAg gene may have a potential use for both prophylactic and therapeutic purposes.

The loss of HBII-52 and related C/D box small nucleolar RNA (snoRNA) expression units have been implicated as a cause for the Prader–Willi syndrome (PWS). We recently found that the C/D box snoRNA HBII-52 changes the alternative splicing of the serotonin receptor 2C pre-mRNA, which is different from the traditional C/D box snoRNA function in non-mRNA methylation. Using bioinformatic predictions and experimental verification, we identified five pre-mRNAs (DPM2, TAF1, RALGPS1, PBRM1 and CRHR1) containing alternative exons that are regulated by MBII-52, the mouse homolog of HBII-52. Analysis of a single member of the MBII-52 cluster of snoRNAs by RNase protection and northern blot analysis shows that the MBII-52 expressing unit generates shorter RNAs that originate from the full-length MBII-52 snoRNA through additional processing steps. These novel RNAs associate with hnRNPs and not with proteins associated with canonical C/D box snoRNAs. Our data indicate that not a traditional C/D box snoRNA MBII-52, but a processed version lacking the snoRNA stem is the predominant MBII-52 RNA missing in PWS. This processed snoRNA functions in alternative splice-site selection. Its substitution could be a therapeutic principle for PWS.

Heterogeneous nuclear ribonucleoprotein (hnRNP) L can regulate alternative mRNA splicing in diverse ways, binding to exonic or intronic sites and acting as either an activator or repressor. To investigate the mechanistic basis of hnRNP L-regulated alternative splicing, we focus here on two specific cases of hnRNP L-dependent splice site recognition. First, in the case of TJP1 our microarray data had suggested that exon 20 inclusion is regulated by hnRNP L as a repressor. Here we demonstrate by mutational analysis that exon skipping is mediated by a short silencer sequence consisting of three hnRNP L high-score binding motifs located upstream of the 3' splice site of the regulated exon. UV crosslinking and immunoprecipitation experiments showed that hnRNP L binding interferes with 3' splice site recognition by U2AF65. Second, SLC2A2 contains a CA-repeat sequence close to the 5' splice site of the regulated exon 4. Using psoralen crosslinking, we demonstrate that hnRNP L represses splicing by preventing 5' splice site recognition of the U1 snRNP. In sum, our data provide new insights into the mechanisms of how hnRNP L-bound to intronic sites-regulates exon recognition.

Alternative splicing is one of the most important post-transcriptional processing steps that enhances genomic information by generating multiple RNA isoforms from a single gene. Recently, microarrays have been developed that can detect changes in splice site selection. Currently, the biggest challenge for the analysis of alternative splicing with microarrays is the bioinformatics analysis of array data and their low reproducibility by RT-PCR. Despite these problems, microarrays revealed an unexpected number of expressed RNAs, showed changes of alternative splicing in diseases and indicated that a splicing factor regulates a biologically meaningful set of genes.

NO, the product of endothelial NOS (eNOS), is a major regulator of vascular homeostasis and a critical factor in preventing cardiovascular diseases. We previously established a positive correlation between the number of variable CA repeats in intron 13 of human eNOS and the risk of coronary artery disease, and demonstrated that these polymorphic CA repeats function as a length-dependent splicing enhancer. By 5'-RACE polymerase chain reaction (PCR), we detected three splice variants containing novel 3' splice sites within intron 13--termed eNOS13A, eNOS13B, and eNOS13C--which share the first 13 exons of human eNOS and the same polyadenylation site at the end of the novel exon. When translated, all these splice variants would result in truncated proteins lacking eNOS activity. Coexpression of full-length eNOS with eNOS13A diminished eNOS enzyme activity in COS-7 cells by formation of heterodimers. The splice variants were expressed in endothelial cells and various human tissues. Finally, we demonstrate, using minigene transfection, that the expression of the eNOS13A splice variant is increased with high CA repeat numbers in intron 13. These data suggest a new mechanism for the regulation of eNOS activity and NO production in the cardiovascular system by truncated, dominant-negative splice variants of human eNOS.

Alternative splicing is a process by which multiple messenger RNAs (mRNAs) are generated from a single pre-mRNA, resulting in functionally distinct protein products. This is accomplished by the differential recognition of splice sites in the pre-mRNA, often regulated in a tissue- or development-specific manner. Alternative splicing constitutes not only an important mechanism in controlling gene expression in humans, but also an essential source for increasing proteome diversity. In this review we summarize the underlying mechanistic principles, focussing on the cis-acting regulatory elements. In particular, the role of short sequence repeats, which are often polymorphic, in splicing regulation is discussed.

Many studies have provided evidence that hepatitis B surface antigen (HBsAg) including preS1 and preS2 sequences could be an ideal candidate for a new hepatitis B virus (HBV) vaccine with higher efficacy. However, the large (L) protein containing the entire preS region expressed in mammalian cells is not efficiently assembled into particles and secreted. Here we report an alternative approach to include the dominant epitopes of preS1 and preS2 to the small (S) protein as fusion proteins by the recombinant DNA technology. Three fusion proteins containing preS2(120-146) and preS1(21-47) at the N-terminus and/or truncated C-terminus of S protein were expressed using the recombinant vaccinia virus system. All these fusion proteins were efficiently secreted in the particulate form, and displayed S, preS1 and/or preS2 antigenicity. Further analysis showed that these chimeric HBsAg particles elicited strong antibody responses against S, preS1 and preS2 antigens in BALB/c mice, suggesting that they could be promising candidates for a new recombinant vaccine to induce broader antibody response required for protection against hepatitis B viral infection.

U6 snRNA, as an essential component of the catalytic core of the pre-mRNA processing spliceosome, is heavily modified post-transcriptionally, with 2'-O-methylation being most common. The role of these modifications in pre-mRNA splicing as well as their physiological function in mammals have remained largely unclear. Here we report that the La-related protein LARP7 functions as a critical cofactor for 2'-O-methylation of U6 in mouse male germ cells. Mechanistically, LARP7 promotes U6 loading onto box C/D snoRNP, facilitating U6 2'-O-methylation by box C/D snoRNP. Importantly, ablation of LARP7 in the male germline causes defective U6 2'-O-methylation, massive alterations in pre-mRNA splicing, and spermatogenic failure in mice, which can be rescued by ectopic expression of wild-type LARP7 but not an U6-loading-deficient mutant LARP7. Our data uncover a novel role of LARP7 in regulating U6 2'-O-methylation and demonstrate the functional requirement of such modification for splicing fidelity and spermatogenesis in mice.