29 results on '"Medenbach J"'
Search Results
2. Integrierte Versorgung von Patienten mit Depression
- Author
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Edel, M.-A., primary, Armgart, C., primary, Echterhoff, S., primary, Wietfeld, R., primary, Mattern, W., primary, Augustin, H., primary, Bruchmann, G., primary, van Aalst, G., primary, Schulte-Florian, G., primary, Medenbach, J., primary, and Juckel, G., additional
- Published
- 2015
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3. Nobel Prize for physiology or medicine in 2023: how to dupe the cellular innate immune system using modified RNA for therapeutic treatment.
- Author
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Medenbach J and Tschochner H
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- Nobel Prize, Physiology history
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- 2024
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4. Cytosolic RGG RNA-binding proteins are temperature sensitive flowering time regulators in Arabidopsis .
- Author
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Bleckmann A, Spitzlberger N, Denninger P, Ehrnsberger HF, Wang L, Bruckmann A, Reich S, Holzinger P, Medenbach J, Grasser KD, and Dresselhaus T
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- Humans, Temperature, RNA-Binding Proteins chemistry, Cytosol metabolism, Glycine metabolism, Arabidopsis genetics, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins chemistry, Arabidopsis Proteins metabolism
- Abstract
mRNA translation is tightly regulated by various classes of RNA-binding proteins (RBPs) during development and in response to changing environmental conditions. In this study, we characterize the arginine-glycine-glycine (RGG) motif containing RBP family of Arabidopsis thaliana representing homologues of the multifunctional translation regulators and ribosomal preservation factors Stm1 from yeast (ScStm1) and human SERBP1 (HsSERBP1). The Arabidopsis genome encodes three RGG proteins named AtRGGA, AtRGGB and AtRGGC. While AtRGGA is ubiquitously expressed, AtRGGB and AtRGGC are enriched in dividing cells. All AtRGGs localize almost exclusively to the cytoplasm and bind with high affinity to ssRNA, while being capable to interact with most nucleic acids, except dsRNA. A protein-interactome study shows that AtRGGs interact with ribosomal proteins and proteins involved in RNA processing and transport. In contrast to ScStm1, AtRGGs are enriched in ribosome-free fractions in polysome profiles, suggesting additional plant-specific functions. Mutant studies show that AtRGG proteins differentially regulate flowering time, with a distinct and complex temperature dependency for each AtRGG protein. In conclusion, we suggest that AtRGGs function in fine-tuning translation efficiency to control flowering time and potentially other developmental processes in response to environmental changes., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)
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- 2023
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5. A rapid protocol for ribosome profiling of low input samples.
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Meindl A, Romberger M, Lehmann G, Eichner N, Kleemann L, Wu J, Danner J, Boesl M, Mesitov M, Meister G, König J, Leidel SA, and Medenbach J
- Subjects
- High-Throughput Nucleotide Sequencing methods, Protein Biosynthesis, Ribosomes genetics, Ribosomes metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Ribosome Profiling methods
- Abstract
Ribosome profiling provides quantitative, comprehensive, and high-resolution snapshots of cellular translation by the high-throughput sequencing of short mRNA fragments that are protected by ribosomes from nucleolytic digestion. While the overall principle is simple, the workflow of ribosome profiling experiments is complex and challenging, and typically requires large amounts of sample, limiting its broad applicability. Here, we present a new protocol for ultra-rapid ribosome profiling from low-input samples. It features a robust strategy for sequencing library preparation within one day that employs solid phase purification of reaction intermediates, allowing to reduce the input to as little as 0.1 pmol of ∼30 nt RNA fragments. Hence, it is particularly suited for the analyses of small samples or targeted ribosome profiling. Its high sensitivity and its ease of implementation will foster the generation of higher quality data from small samples, which opens new opportunities in applying ribosome profiling., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)
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- 2023
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6. Investigating the Prevalence of RNA-Binding Metabolic Enzymes in E. coli .
- Author
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Klein T, Funke F, Rossbach O, Lehmann G, Vockenhuber M, Medenbach J, Suess B, Meister G, and Babinger P
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- Prevalence, Escherichia coli genetics
- Abstract
An open research field in cellular regulation is the assumed crosstalk between RNAs, metabolic enzymes, and metabolites, also known as the REM hypothesis. High-throughput assays have produced extensive interactome data with metabolic enzymes frequently found as hits, but only a few examples have been biochemically validated, with deficits especially in prokaryotes. Therefore, we rationally selected nineteen Escherichia coli enzymes from such datasets and examined their ability to bind RNAs using two complementary methods, iCLIP and SELEX. Found interactions were validated by EMSA and other methods. For most of the candidates, we observed no RNA binding (12/19) or a rather unspecific binding (5/19). Two of the candidates, namely glutamate-5-kinase (ProB) and quinone oxidoreductase (QorA), displayed specific and previously unknown binding to distinct RNAs. We concentrated on the interaction of QorA to the mRNA of yffO , a grounded prophage gene, which could be validated by EMSA and MST. Because the physiological function of both partners is not known, the biological relevance of this interaction remains elusive. Furthermore, we found novel RNA targets for the MS2 phage coat protein that served us as control. Our results indicate that RNA binding of metabolic enzymes in procaryotes is less frequent than suggested by the results of high-throughput studies, but does occur.
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- 2023
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7. Ribosome Profiling in the Model Diatom Thalassiosira pseudonana.
- Author
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Pichler M, Meindl A, Romberger M, Eckes-Shephard A, Nyberg-Brodda CF, Buhigas C, Llaneza-Lago S, Lehmann G, Hopes A, Meister G, Medenbach J, and Mock T
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- Ribosome Profiling, Phytoplankton genetics, Diatoms genetics, Diatoms metabolism
- Abstract
Diatoms are an important group of eukaryotic microalgae, which play key roles in marine biochemical cycling and possess significant biotechnological potential. Despite the importance of diatoms, their regulatory mechanisms of protein synthesis at the translational level remain largely unexplored. Here, we describe the detailed development of a ribosome profiling protocol to study translation in the model diatom Thalassiosira pseudonana, which can easily be adopted for other diatom species. To isolate and sequence ribosome-protected mRNA, total RNA was digested, and the ribosome-protected fragments were obtained by a combination of sucrose-cushion ultracentrifugation and polyacrylamide gel electrophoresis for size selection. To minimize rRNA contamination, a subtractive hybridization step using biotinylated oligos was employed. Subsequently, fragments were converted into sequencing libraries, enabling the global quantification and analysis of changes in protein synthesis in diatoms. The development of this novel ribosome profiling protocol represents a major expansion of the molecular toolbox available for diatoms and therefore has the potential to advance our understanding of the translational regulation in this important group of phytoplankton. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Ribosome profiling in Thalassiosira pseudonana Alternate Protocol: Ribosome profiling protocol for diatoms using sucrose gradient fractionation., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)
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- 2023
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8. Targeted escape of SARS-CoV-2 in vitro from monoclonal antibody S309, the precursor of sotrovimab.
- Author
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Magnus CL, Hiergeist A, Schuster P, Rohrhofer A, Medenbach J, Gessner A, Peterhoff D, and Schmidt B
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- Amino Acids, Antibodies, Monoclonal pharmacology, Antibodies, Monoclonal, Humanized, Antibodies, Neutralizing, Caco-2 Cells, Humans, Peptidyl-Dipeptidase A metabolism, SARS-CoV-2, Spike Glycoprotein, Coronavirus metabolism, Angiotensin-Converting Enzyme 2, COVID-19
- Abstract
Class 1 and 2 monoclonal antibodies inhibit SARS-CoV-2 entry by blocking the interaction of the viral receptor-binding domain with angiotensin-converting enzyme 2 (ACE2), while class 3 antibodies target a highly conserved epitope outside the ACE2 binding site. We aimed to investigate the plasticity of the spike protein by propagating wild-type SARS-CoV-2 in the presence of class 3 antibody S309. After 12 weeks, we obtained a viral strain that was completely resistant to inhibition by S309, due to successively evolving amino acid exchanges R346S and P337L located in the paratope of S309. The antibody lost affinity to receptor-binding domains carrying P337L or both amino acid exchanges, while ACE2 binding was not affected. The resistant strain replicated efficiently in human CaCo-2 cells and was more susceptible to inhibition of fusion than the original strain. Overall, SARS-CoV-2 escaped inhibition by class 3 antibody S309 through a slow, but targeted evolution enabling immune escape and altering cell entry. This immune-driven enhancement of infectivity and pathogenicity could play an important role in the future evolution of SARS-CoV-2, which is under increasing immunological pressure from vaccination and previous infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Magnus, Hiergeist, Schuster, Rohrhofer, Medenbach, Gessner, Peterhoff and Schmidt.)
- Published
- 2022
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9. Molecular insights into RNA recognition and gene regulation by the TRIM-NHL protein Mei-P26.
- Author
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Salerno-Kochan A, Horn A, Ghosh P, Nithin C, Kościelniak A, Meindl A, Strauss D, Krutyhołowa R, Rossbach O, Bujnicki JM, Gaik M, Medenbach J, and Glatt S
- Subjects
- Animals, Drosophila genetics, Male, RNA metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Tripartite Motif Proteins genetics, Tripartite Motif Proteins metabolism, Drosophila Proteins genetics, Drosophila Proteins metabolism
- Abstract
The TRIM-NHL protein Meiotic P26 (Mei-P26) acts as a regulator of cell fate in Drosophila Its activity is critical for ovarian germline stem cell maintenance, differentiation of oocytes, and spermatogenesis. Mei-P26 functions as a post-transcriptional regulator of gene expression; however, the molecular details of how its NHL domain selectively recognizes and regulates its mRNA targets have remained elusive. Here, we present the crystal structure of the Mei-P26 NHL domain at 1.6 Å resolution and identify key amino acids that confer substrate specificity and distinguish Mei-P26 from closely related TRIM-NHL proteins. Furthermore, we identify mRNA targets of Mei-P26 in cultured Drosophila cells and show that Mei-P26 can act as either a repressor or activator of gene expression on different RNA targets. Our work reveals the molecular basis of RNA recognition by Mei-P26 and the fundamental functional differences between otherwise very similar TRIM-NHL proteins., (© 2022 Salerno-Kochan et al.)
- Published
- 2022
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10. Simple Targeted Assays for Metabolic Pathways and Signaling: A Powerful Tool for Targeted Proteomics.
- Author
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Kopczynski D, Hentschel A, Coman C, Schebb NH, Hornemann T, Mashek DG, Hartung NM, Shevchuk O, Schött HF, Lorenz K, Torta F, Burla B, Zahedi RP, Sickmann A, Kreutz MR, Ejsing CS, Medenbach J, and Ahrends R
- Subjects
- Animals, Chromatography, High Pressure Liquid, Databases, Protein, Insulin metabolism, Mice, Peptides analysis, Tandem Mass Spectrometry, Metabolic Networks and Pathways genetics, Proteomics methods, Signal Transduction genetics
- Abstract
We introduce STAMPS, a pathway-centric web service for the development of targeted proteomics assays. STAMPS guides the user by providing several intuitive interfaces for a rapid and simplified method design. Applying our curated framework to signaling and metabolic pathways, we reduced the average assay development time by a factor of ∼150 and revealed that the insulin signaling is actively controlled by protein abundance changes in insulin-sensitive and -resistance states. Although at the current state STAMPS primarily contains mouse data, it was designed for easy extension with additional organisms.
- Published
- 2020
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11. A multi-omics analysis reveals the unfolded protein response regulon and stress-induced resistance to folate-based antimetabolites.
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Reich S, Nguyen CDL, Has C, Steltgens S, Soni H, Coman C, Freyberg M, Bichler A, Seifert N, Conrad D, Knobbe-Thomsen CB, Tews B, Toedt G, Ahrends R, and Medenbach J
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- Animals, Endoplasmic Reticulum drug effects, Endoplasmic Reticulum metabolism, Humans, Methotrexate pharmacology, Pemetrexed pharmacology, Proteome drug effects, Proteome genetics, Regulon drug effects, Signal Transduction drug effects, Transcriptome drug effects, Transcriptome genetics, Antimetabolites pharmacology, Folic Acid pharmacology, Regulon genetics, Unfolded Protein Response drug effects, Unfolded Protein Response genetics
- Abstract
Stress response pathways are critical for cellular homeostasis, promoting survival through adaptive changes in gene expression and metabolism. They play key roles in numerous diseases and are implicated in cancer progression and chemoresistance. However, the underlying mechanisms are only poorly understood. We have employed a multi-omics approach to monitor changes to gene expression after induction of a stress response pathway, the unfolded protein response (UPR), probing in parallel the transcriptome, the proteome, and changes to translation. Stringent filtering reveals the induction of 267 genes, many of which have not previously been implicated in stress response pathways. We experimentally demonstrate that UPR-mediated translational control induces the expression of enzymes involved in a pathway that diverts intermediate metabolites from glycolysis to fuel mitochondrial one-carbon metabolism. Concomitantly, the cells become resistant to the folate-based antimetabolites Methotrexate and Pemetrexed, establishing a direct link between UPR-driven changes to gene expression and resistance to pharmacological treatment.
- Published
- 2020
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12. The zinc finger domains in U2AF26 and U2AF35 have diverse functionalities including a role in controlling translation.
- Author
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Herdt O, Reich S, Medenbach J, Timmermann B, Olofsson D, Preußner M, and Heyd F
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- Animals, HEK293 Cells, HeLa Cells, Humans, Mice, Protein Binding, RNA Splicing, RNA Stability, Splicing Factor U2AF chemistry, Gene Expression Regulation, Protein Biosynthesis, Splicing Factor U2AF metabolism, Zinc Fingers
- Abstract
Recent work has associated point mutations in both zinc fingers (ZnF) of the spliceosome component U2AF35 with malignant transformation. However, surprisingly little is known about the functionality of the U2AF35 ZnF domains in general. Here we have analysed key functionalities of the ZnF domains of mammalian U2AF35 and its paralog U2AF26. Both ZnFs are required for splicing regulation, whereas only ZnF2 controls protein stability and contributes to the interaction with U2AF65. These features are confirmed in a naturally occurring splice variant of U2AF26 lacking ZnF2, that is strongly induced upon activation of primary mouse T cells and localized in the cytoplasm. Using Ribo-Seq in a model T cell line we provide evidence for a role of U2AF26 in activating cytoplasmic steps in gene expression, notably translation. Consistently, an MS2 tethering assay shows that cytoplasmic U2AF26/35 increase translation when localized to the 5'UTR of a model mRNA. This regulation is partially dependent on ZnF1 thus providing a connection between a core splicing factor, the ZnF domains and the regulation of translation. Altogether, our work reveals unexpected functions of U2AF26/35 and their ZnF domains, thereby contributing to a better understanding of their role and regulation in mammalian cells.
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- 2020
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13. Auto-regulatory feedback by RNA-binding proteins.
- Author
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Müller-McNicoll M, Rossbach O, Hui J, and Medenbach J
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- Gene Expression Regulation, Humans, RNA Processing, Post-Transcriptional, RNA, Messenger genetics, RNA, Messenger metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism
- Abstract
RNA-binding proteins (RBPs) are key regulators in post-transcriptional control of gene expression. Mutations that alter their activity or abundance have been implicated in numerous diseases such as neurodegenerative disorders and various types of cancer. This highlights the importance of RBP proteostasis and the necessity to tightly control the expression levels and activities of RBPs. In many cases, RBPs engage in an auto-regulatory feedback by directly binding to and influencing the fate of their own mRNAs, exerting control over their own expression. For this feedback control, RBPs employ a variety of mechanisms operating at all levels of post-transcriptional regulation of gene expression. Here we review RBP-mediated autogenous feedback regulation that either serves to maintain protein abundance within a physiological range (by negative feedback) or generates binary, genetic on/off switches important for e.g. cell fate decisions (by positive feedback)., (© The Author(s) (2019). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS.)
- Published
- 2019
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14. RhoA regulates translation of the Nogo-A decoy SPARC in white matter-invading glioblastomas.
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Wirthschaft P, Bode J, Soni H, Dietrich F, Krüwel T, Fischer B, Knobbe-Thomsen CB, Rossetti G, Hentschel A, Mack N, Schönig K, Breckwoldt MO, Schmandke A, Pusch S, Medenbach J, Bendszus M, Schwab ME, von Deimling A, Kool M, Herold-Mende C, Reifenberger G, Ahrends R, and Tews B
- Subjects
- Animals, Binding, Competitive, Brain Neoplasms metabolism, Brain Neoplasms pathology, Glioblastoma metabolism, Glioblastoma pathology, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Inbred NOD, Neoplasm Invasiveness, Nogo Proteins genetics, Osteonectin genetics, Protein Domains, RNA Interference, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Neoplasm genetics, RNA, Neoplasm metabolism, Recombinant Proteins metabolism, Signal Transduction, Sphingosine-1-Phosphate Receptors physiology, Tumor Cells, Cultured, White Matter metabolism, Brain Neoplasms genetics, Gene Expression Regulation, Neoplastic, Glioblastoma genetics, Neoplasm Proteins physiology, Nogo Proteins biosynthesis, Osteonectin biosynthesis, Protein Biosynthesis, White Matter pathology, rhoA GTP-Binding Protein physiology
- Abstract
Glioblastomas strongly invade the brain by infiltrating into the white matter along myelinated nerve fiber tracts even though the myelin protein Nogo-A prevents cell migration by activating inhibitory RhoA signaling. The mechanisms behind this long-known phenomenon remained elusive so far, precluding a targeted therapeutic intervention. This study demonstrates that the prevalent activation of AKT in gliomas increases the ER protein-folding capacity and enables tumor cells to utilize a side effect of RhoA activation: the perturbation of the IRE1α-mediated decay of SPARC mRNA. Once translation is initiated, glioblastoma cells rapidly secrete SPARC to block Nogo-A from inhibiting migration via RhoA. By advanced ultramicroscopy for studying single-cell invasion in whole, undissected mouse brains, we show that gliomas require SPARC for invading into white matter structures. SPARC depletion reduces tumor dissemination that significantly prolongs survival and improves response to cytostatic therapy. Our finding of a novel RhoA-IRE1 axis provides a druggable target for interfering with SPARC production and underscores its therapeutic value.
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- 2019
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15. A sensitive and simple targeted proteomics approach to quantify transcription factor and membrane proteins of the unfolded protein response pathway in glioblastoma cells.
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Nguyen CDL, Malchow S, Reich S, Steltgens S, Shuvaev KV, Loroch S, Lorenz C, Sickmann A, Knobbe-Thomsen CB, Tews B, Medenbach J, and Ahrends R
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- Cell Line, Tumor, Gene Dosage, Glioblastoma chemistry, Glioblastoma pathology, Humans, Isotope Labeling, Membrane Proteins analysis, Membrane Proteins standards, Peptides standards, Proteomics standards, Transcription Factors analysis, Transcription Factors standards, Glioblastoma metabolism, Membrane Proteins metabolism, Proteomics methods, Transcription Factors metabolism, Unfolded Protein Response
- Abstract
Many cellular events are driven by changes in protein expression, measurable by mass spectrometry or antibody-based assays. However, using conventional technology, the analysis of transcription factor or membrane receptor expression is often limited by an insufficient sensitivity and specificity. To overcome this limitation, we have developed a high-resolution targeted proteomics strategy, which allows quantification down to the lower attomol range in a straightforward way without any prior enrichment or fractionation approaches. The method applies isotope-labeled peptide standards for quantification of the protein of interest. As proof of principle, we applied the improved workflow to proteins of the unfolded protein response (UPR), a signaling pathway of great clinical importance, and could for the first time detect and quantify all major UPR receptors, transducers and effectors that are not readily detectable via antibody-based-, SRM- or conventional PRM assays. As transcription and translation is central to the regulation of UPR, quantification and determination of protein copy numbers in the cell is important for our understanding of the signaling process as well as how pharmacologic modulation of these pathways impacts on the signaling. These questions can be answered using our newly established workflow as exemplified in an experiment using UPR perturbation in a glioblastoma cell lines.
- Published
- 2019
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16. Drosophila Sister-of-Sex-lethal reinforces a male-specific gene expression pattern by controlling Sex-lethal alternative splicing.
- Author
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Moschall R, Rass M, Rossbach O, Lehmann G, Kullmann L, Eichner N, Strauss D, Meister G, Schneuwly S, Krahn MP, and Medenbach J
- Subjects
- Animals, Cells, Cultured, Drosophila Proteins biosynthesis, Exons genetics, Female, Gene Expression Profiling, Male, RNA, Messenger genetics, RNA, Messenger metabolism, RNA-Binding Proteins biosynthesis, Regulatory Sequences, Ribonucleic Acid genetics, Alternative Splicing genetics, Drosophila Proteins genetics, Drosophila Proteins metabolism, Drosophila melanogaster genetics, Drosophila melanogaster metabolism, Gene Expression Regulation, Developmental, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Sex Characteristics
- Abstract
In Drosophila, female development is governed by a single RNA-binding protein, Sex-lethal (Sxl), that controls the expression of key factors involved in dosage compensation, germline homeostasis and the establishment of female morphology and behaviour. Sxl expression in female flies is maintained by an auto-regulatory, positive feedback loop with Sxl controlling splicing of its own mRNA. Until now, it remained unclear how males prevent accidental triggering of the Sxl expression cascade and protect themselves against runaway protein production. Here, we identify the protein Sister-of-Sex-lethal (Ssx) as an inhibitor of Sxl auto-regulatory splicing. Sxl and Ssx have a comparable RNA-binding specificity and compete for binding to RNA regulatory elements present in the Sxl transcript. In cultured Drosophila cells, Sxl-induced changes to alternative splicing can be reverted by the expression of Ssx. Moreover, in adult male flies ablation of the ssx gene results in a low level of productive Sxl mRNA splicing and Sxl protein production in isolated, clonal cell populations. In sum, this demonstrates that Ssx safeguards male animals against Sxl protein production to reinforce a stable, male-specific gene expression pattern., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2019
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17. Purification of cross-linked RNA-protein complexes by phenol-toluol extraction.
- Author
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Urdaneta EC, Vieira-Vieira CH, Hick T, Wessels HH, Figini D, Moschall R, Medenbach J, Ohler U, Granneman S, Selbach M, and Beckmann BM
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- Animals, Base Sequence, Brain, HEK293 Cells, Humans, Mice, Mice, Inbred C57BL, Multiprotein Complexes isolation & purification, Proteome chemistry, Proteomics methods, RNA chemistry, RNA, Messenger, RNA-Binding Proteins chemistry, Ribonucleoproteins chemistry, Ribonucleoproteins isolation & purification, Salmonella typhimurium, Sensitivity and Specificity, Molecular Biology methods, Phenol chemistry, RNA-Binding Proteins isolation & purification, Toluene chemistry
- Abstract
Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium.
- Published
- 2019
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18. Insights into the evolutionary conserved regulation of Rio ATPase activity.
- Author
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Knüppel R, Christensen RH, Gray FC, Esser D, Strauß D, Medenbach J, Siebers B, MacNeill SA, LaRonde N, and Ferreira-Cerca S
- Subjects
- Adenosine Triphosphatases chemistry, Adenosine Triphosphatases metabolism, Adenosine Triphosphate metabolism, Archaeal Proteins chemistry, Archaeal Proteins metabolism, Binding Sites, Cloning, Molecular, Conserved Sequence, Escherichia coli genetics, Escherichia coli metabolism, Evolution, Molecular, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Haloferax volcanii genetics, Isoenzymes chemistry, Isoenzymes genetics, Isoenzymes metabolism, Kinetics, Models, Molecular, Nucleic Acid Conformation, Protein Binding, Protein Biosynthesis, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Protein Serine-Threonine Kinases chemistry, Protein Serine-Threonine Kinases metabolism, RNA, Ribosomal, 18S chemistry, RNA, Ribosomal, 18S metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Ribosomes chemistry, Ribosomes metabolism, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins metabolism, Adenosine Triphosphatases genetics, Adenosine Triphosphate chemistry, Archaeal Proteins genetics, Haloferax volcanii enzymology, Protein Serine-Threonine Kinases genetics, RNA, Ribosomal, 18S genetics, Saccharomyces cerevisiae Proteins genetics
- Abstract
Eukaryotic ribosome biogenesis is a complex dynamic process which requires the action of numerous ribosome assembly factors. Among them, the eukaryotic Rio protein family members (Rio1, Rio2 and Rio3) belong to an ancient conserved atypical protein kinase/ ATPase family required for the maturation of the small ribosomal subunit (SSU). Recent structure-function analyses suggested an ATPase-dependent role of the Rio proteins to regulate their dynamic association with the nascent pre-SSU. However, the evolutionary origin of this feature and the detailed molecular mechanism that allows controlled activation of the catalytic activity remained to be determined. In this work we provide functional evidence showing a conserved role of the archaeal Rio proteins for the synthesis of the SSU in archaea. Moreover, we unravel a conserved RNA-dependent regulation of the Rio ATPases, which in the case of Rio2 involves, at least, helix 30 of the SSU rRNA and the P-loop lysine within the shared RIO domain. Together, our study suggests a ribosomal RNA-mediated regulatory mechanism enabling the appropriate stimulation of Rio2 catalytic activity and subsequent release of Rio2 from the nascent pre-40S particle. Based on our findings we propose a unified release mechanism for the Rio proteins.
- Published
- 2018
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19. Drosophila Sister-of-Sex-lethal is a repressor of translation.
- Author
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Moschall R, Strauss D, García-Beyaert M, Gebauer F, and Medenbach J
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- Amino Acid Motifs, Animals, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Drosophila Proteins chemistry, Drosophila Proteins genetics, Drosophila melanogaster genetics, Nuclear Proteins genetics, Nuclear Proteins metabolism, RNA, Messenger chemistry, RNA, Messenger metabolism, RNA-Binding Proteins chemistry, RNA-Binding Proteins genetics, Transcription Factors genetics, Transcription Factors metabolism, Uracil analysis, Drosophila Proteins metabolism, Protein Biosynthesis, RNA-Binding Proteins metabolism
- Abstract
The RNA-binding protein Sex-lethal (Sxl) is an important post-transcriptional regulator of sex determination and dosage compensation in female Drosophila To prevent the assembly of the MSL dosage compensation complex in female flies, Sxl acts as a repressor of male-specific lethal-2 (msl-2) mRNA translation. It uses two distinct and mutually reinforcing blocks to translation that operate on the 5' and 3' untranslated regions (UTRs) of msl-2 mRNA, respectively. While 5' UTR-mediated translational control involves an upstream open reading frame, 3' UTR-mediated regulation strictly requires the co-repressor protein Upstream of N-ras (Unr), which is recruited to the transcript by Sxl. We have identified the protein Sister-of-Sex-lethal (Ssx) as a novel repressor of translation with Sxl-like activity. Both proteins have a comparable RNA-binding specificity and can associate with uracil-rich RNA regulatory elements present in msl-2 mRNA. Moreover, both repress translation when bound to the 5' UTR of msl-2 However, Ssx is inactive in 3' UTR-mediated regulation, as it cannot engage the co-repressor protein Unr. The difference in activity maps to the first RNA-recognition motif (RRM) of Ssx. Conversion of three amino acids within this domain into their Sxl counterpart results in a gain of function and repression via the 3' UTR, allowing detailed insights into the evolutionary origin of the two proteins and into the molecular requirements of an important translation regulatory pathway., (© 2018 Moschall et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2018
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20. Promiscuity in post-transcriptional control of gene expression: Drosophila sex-lethal and its regulatory partnerships.
- Author
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Moschall R, Gaik M, and Medenbach J
- Subjects
- Alternative Splicing genetics, Alternative Splicing physiology, Animals, Drosophila, Drosophila Proteins genetics, Drosophila Proteins metabolism, Female, Gene Expression Regulation, Developmental genetics, Male, Models, Biological, RNA, Messenger genetics, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Gene Expression Regulation, Developmental physiology
- Abstract
The Drosophila RNA-binding protein Sex-lethal (Sxl) is a potent post-transcriptional regulator of gene expression that controls female development. It regulates the expression of key factors involved in sex-specific differences in morphology, behavior, and dosage compensation. Functional Sxl protein is only expressed in female flies, where it binds to U-rich RNA motifs present in its target mRNAs to regulate their fate. Sxl is a very versatile regulator that, by shuttling between the nucleus and the cytoplasm, can regulate almost all aspects of post-transcriptional gene expression including RNA processing, nuclear export, and translation. For these functions, Sxl employs multiple interactions to either antagonize RNA-processing factors or to recruit various coregulators, thus allowing it to establish a female-specific gene expression pattern. Here, we summarize the current knowledge about Sxl function and review recent mechanistic and structural studies that further our understanding of how such a seemingly 'simple' RNA-binding protein can exert this plethora of different functions., (© 2017 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
- Published
- 2017
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21. CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs.
- Author
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Schneider T, Hung LH, Schreiner S, Starke S, Eckhof H, Rossbach O, Reich S, Medenbach J, and Bindereif A
- Subjects
- Computational Biology, HeLa Cells, Humans, Immunoprecipitation, Protein Binding, RNA genetics, RNA, Circular, RNA metabolism, RNA-Binding Proteins metabolism, Sequence Analysis, RNA methods
- Abstract
Circular RNAs (circRNAs) constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing. Here we investigated in mammalian cells the association of circRNAs with proteins. Using glycerol gradient centrifugation, we characterized in cell lysates circRNA-protein complexes (circRNPs) of distinct sizes. By polysome-gradient fractionation we found no evidence for efficient translation of a set of abundant circRNAs in HeLa cells. To identify circRNPs with a specific protein component, we focused on IMP3 (IGF2BP3, insulin-like growth factor 2 binding protein 3), a known tumor marker and RNA-binding protein. Combining RNA-seq analysis of IMP3-co-immunoprecipitated RNA and filtering for circular-junction reads identified a set of IMP3-associated circRNAs, which were validated and characterized. In sum, our data suggest that specific circRNP families exist defined by a common protein component. In addition, this provides a general approach to identify circRNPs with a given protein component.
- Published
- 2016
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22. The expanding universe of ribonucleoproteins: of novel RNA-binding proteins and unconventional interactions.
- Author
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Beckmann BM, Castello A, and Medenbach J
- Subjects
- Animals, Binding Sites, Humans, Protein Binding, RNA chemistry, RNA genetics, Ribonucleoproteins chemistry, Ribonucleoproteins genetics, RNA metabolism, Ribonucleoproteins metabolism
- Abstract
Post-transcriptional regulation of gene expression plays a critical role in almost all cellular processes. Regulation occurs mostly by RNA-binding proteins (RBPs) that recognise RNA elements and form ribonucleoproteins (RNPs) to control RNA metabolism from synthesis to decay. Recently, the repertoire of RBPs was significantly expanded owing to methodological advances such as RNA interactome capture. The newly identified RNA binders are involved in diverse biological processes and belong to a broad spectrum of protein families, many of them exhibiting enzymatic activities. This suggests the existence of an extensive crosstalk between RNA biology and other, in principle unrelated, cell functions such as intermediary metabolism. Unexpectedly, hundreds of new RBPs do not contain identifiable RNA-binding domains (RBDs), raising the question of how they interact with RNA. Despite the many functions that have been attributed to RNA, our understanding of RNPs is still mostly governed by a rather protein-centric view, leading to the idea that proteins have evolved to bind to and regulate RNA and not vice versa. However, RNPs formed by an RNA-driven interaction mechanism (RNA-determined RNPs) are abundant and offer an alternative explanation for the surprising lack of classical RBDs in many RNA-interacting proteins. Moreover, RNAs can act as scaffolds to orchestrate and organise protein networks and directly control their activity, suggesting that nucleic acids might play an important regulatory role in many cellular processes, including metabolism.
- Published
- 2016
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23. Special issue: RNA biology in physiology and disease.
- Author
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Kretz M, Medenbach J, and Meister G
- Subjects
- Animals, Cardiovascular Diseases genetics, Humans, Neoplasms genetics, Neurodegenerative Diseases genetics, RNA genetics, Cardiovascular Diseases metabolism, Neoplasms metabolism, Neurodegenerative Diseases metabolism, RNA metabolism
- Published
- 2016
- Full Text
- View/download PDF
24. Translational control via protein-regulated upstream open reading frames.
- Author
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Medenbach J, Seiler M, and Hentze MW
- Subjects
- 5' Untranslated Regions, Animals, Binding Sites, DNA-Binding Proteins genetics, Drosophila genetics, Drosophila Proteins genetics, Drosophila melanogaster genetics, Drosophila melanogaster metabolism, Female, Male, Nuclear Proteins genetics, Protein Biosynthesis, Ribosomes metabolism, Transcription Factors genetics, Drosophila metabolism, Drosophila Proteins metabolism, Gene Expression Regulation, Open Reading Frames, Peptide Chain Initiation, Translational, RNA-Binding Proteins metabolism
- Abstract
Analysis of the regulation of msl-2 mRNA by Sex lethal (SXL), which is critical for dosage compensation in Drosophila, has uncovered a mode of translational control based on common 5' untranslated region elements, upstream open reading frames (uORFs), and interaction sites for RNA-binding proteins. We show that SXL binding downstream of a short uORF imposes a strong negative effect on major reading frame translation. The underlying mechanism involves increasing initiation of scanning ribosomes at the uORF and augmenting its impediment to downstream translation. Our analyses reveal that SXL exerts its effect controlling initiation, not elongation or termination, at the uORF. Probing the generality of the underlying mechanism, we show that the regulatory module that we define experimentally functions in a heterologous context, and we identify natural Drosophila mRNAs that are regulated via this module. We propose that protein-regulated uORFs constitute a systematic principle for the regulation of protein synthesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
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25. RNA-Seq analysis in mutant zebrafish reveals role of U1C protein in alternative splicing regulation.
- Author
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Rösel TD, Hung LH, Medenbach J, Donde K, Starke S, Benes V, Rätsch G, and Bindereif A
- Subjects
- Animals, Embryo, Nonmammalian physiology, Genetic Complementation Test, HeLa Cells, Humans, Molecular Sequence Data, Mutation, RNA Precursors chemistry, RNA Precursors metabolism, Ribonucleoproteins, Small Nuclear genetics, Sequence Analysis, DNA, Zebrafish genetics, Zebrafish Proteins genetics, Alternative Splicing, Gene Expression Regulation, Ribonucleoproteins, Small Nuclear metabolism, Zebrafish physiology, Zebrafish Proteins metabolism
- Abstract
Precise 5' splice-site recognition is essential for both constitutive and regulated pre-mRNA splicing. The U1 small nuclear ribonucleoprotein particle (snRNP)-specific protein U1C is involved in this first step of spliceosome assembly and important for stabilizing early splicing complexes. We used an embryonically lethal U1C mutant zebrafish, hi1371, to investigate the potential genomewide role of U1C for splicing regulation. U1C mutant embryos contain overall stable, but U1C-deficient U1 snRNPs. Surprisingly, genomewide RNA-Seq analysis of mutant versus wild-type embryos revealed a large set of specific target genes that changed their alternative splicing patterns in the absence of U1C. Injection of ZfU1C cRNA into mutant embryos and in vivo splicing experiments in HeLa cells after siRNA-mediated U1C knockdown confirmed the U1C dependency and specificity, as well as the functional conservation of the effects observed. In addition, sequence motif analysis of the U1C-dependent 5' splice sites uncovered an association with downstream intronic U-rich elements. In sum, our findings provide evidence for a new role of a general snRNP protein, U1C, as a mediator of alternative splicing regulation.
- Published
- 2011
- Full Text
- View/download PDF
26. Human initiation factor eIF3 subunit b interacts with HCV IRES RNA through its N-terminal RNA recognition motif.
- Author
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Pérard J, Rasia R, Medenbach J, Ayala I, Boisbouvier J, Drouet E, and Baudin F
- Subjects
- Amino Acid Motifs, Eukaryotic Initiation Factor-3 chemistry, Eukaryotic Initiation Factor-3 genetics, Humans, Protein Conformation, Protein Subunits chemistry, Protein Subunits genetics, Protein Subunits metabolism, RNA, Viral chemistry, 5' Untranslated Regions, Eukaryotic Initiation Factor-3 metabolism, Hepacivirus, RNA, Viral metabolism
- Abstract
Many viral mRNAs contain a 5'-UTR RNA element called internal ribosome-entry site (IRES), which bypasses the requirement of some canonical initiation factors allowing cap-independent translation. The IRES of hepatitis-C virus drives translation by directly recruiting 40S ribosomal subunits and binds to eIF3 which plays a critical role in both cap-dependent and cap-independent translation. However, the molecular basis for eIF3 activity in either case remains enigmatic. Here we report that subunit b of the eIF3 complex directly binds to HCV IRES domain III via its N-terminal-RRM. Because eIF3b was previously shown to be involved in eIF3j binding, biological implications are discussed.
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- 2009
- Full Text
- View/download PDF
27. 3'-cyclic phosphorylation of U6 snRNA leads to recruitment of recycling factor p110 through LSm proteins.
- Author
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Licht K, Medenbach J, Lührmann R, Kambach C, and Bindereif A
- Subjects
- Antigens, Neoplasm, Autoantigens metabolism, Base Sequence, HeLa Cells, Humans, Molecular Sequence Data, RNA Splicing, RNA, Small Nuclear chemistry, Ribonucleoproteins metabolism, SS-B Antigen, Nuclear Proteins metabolism, RNA, Small Nuclear metabolism, RNA-Binding Proteins metabolism, Ribonucleoprotein, U4-U6 Small Nuclear metabolism
- Abstract
Pre-mRNA splicing proceeds through assembly of the spliceosome complex, catalysis, and recycling. During each cycle the U4/U6.U5 tri-snRNP is disrupted and U4/U6 snRNA base-pairing unwound, releasing separate post-spliceosomal U4, U5, and U6 snRNPs, which have to be recycled to the splicing-competent tri-snRNP. Previous work implicated p110--the human ortholog of the yeast Prp24 protein--and the LSm2-8 proteins of the U6 snRNP in U4/U6 recycling. Here we show in vitro that these proteins bind synergistically to U6 snRNA: Both purified and recombinant LSm2-8 proteins are able to recruit p110 protein to U6 snRNA via interaction with the highly conserved C-terminal region of p110. Furthermore, the presence of a 2',3'-cyclic phosphate enhances the affinity of U6 snRNA for the LSm2-8 proteins and inversely reduces La protein binding, suggesting a direct role of the 3'-terminal phosphorylation in RNP remodeling during U6 biogenesis.
- Published
- 2008
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28. Network of coregulated spliceosome components revealed by zebrafish mutant in recycling factor p110.
- Author
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Trede NS, Medenbach J, Damianov A, Hung LH, Weber GJ, Paw BH, Zhou Y, Hersey C, Zapata A, Keefe M, Barut BA, Stuart AB, Katz T, Amemiya CT, Zon LI, and Bindereif A
- Subjects
- Animals, Genes, Lethal, Mutagenesis, Organ Specificity genetics, Phenotype, RNA Splicing Factors, Ribonucleoprotein, U4-U6 Small Nuclear genetics, Ribonucleoprotein, U4-U6 Small Nuclear metabolism, Thymus Gland abnormalities, DEAD-box RNA Helicases genetics, DEAD-box RNA Helicases metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Spliceosomes physiology, Zebrafish genetics, Zebrafish metabolism, Zebrafish Proteins genetics, Zebrafish Proteins metabolism
- Abstract
The spliceosome cycle consists of assembly, catalysis, and recycling phases. Recycling of postspliceosomal U4 and U6 small nuclear ribonucleoproteins (snRNPs) requires p110/SART3, a general splicing factor. In this article, we report that the zebrafish earl grey (egy) mutation maps in the p110 gene and results in a phenotype characterized by thymus hypoplasia, other organ-specific defects, and death by 7 to 8 days postfertilization. U4/U6 snRNPs were disrupted in egy mutant embryos, demonstrating the importance of p110 for U4/U6 snRNP recycling in vivo. Surprisingly, expression profiling of the egy mutant revealed an extensive network of coordinately up-regulated components of the spliceosome cycle, providing a mechanism compensating for the recycling defect. Together, our data demonstrate that a mutation in a general splicing factor can lead to distinct defects in organ development and cause disease.
- Published
- 2007
- Full Text
- View/download PDF
29. Human U4/U6 snRNP recycling factor p110: mutational analysis reveals the function of the tetratricopeptide repeat domain in recycling.
- Author
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Medenbach J, Schreiner S, Liu S, Lührmann R, and Bindereif A
- Subjects
- Animals, Antigens, Neoplasm metabolism, DNA Mutational Analysis, HeLa Cells, Humans, Models, Genetic, Protein Binding, Protein Structure, Tertiary, RNA-Binding Proteins metabolism, Ribonucleoprotein, U4-U6 Small Nuclear genetics, Spliceosomes metabolism, Two-Hybrid System Techniques, Antigens, Neoplasm genetics, RNA Splicing, RNA-Binding Proteins genetics, Repetitive Sequences, Nucleic Acid, Ribonucleoprotein, U4-U6 Small Nuclear metabolism
- Abstract
After each spliceosome cycle, the U4 and U6 snRNAs are released separately and are recycled to the functional U4/U6 snRNP, requiring in the mammalian system the U6-specific RNA binding protein p110 (SART3). Its domain structure is made up of an extensive N-terminal domain with at least seven tetratricopeptide repeat (TPR) motifs, followed by two RNA recognition motifs (RRMs) and a highly conserved C-terminal sequence of 10 amino acids. Here we demonstrate under in vitro recycling conditions that U6-p110 is an essential splicing factor. Recycling activity requires both the RRMs and the TPR domain but not the highly conserved C-terminal sequence. For U6-specific RNA binding, the two RRMs with some flanking regions are sufficient. Yeast two-hybrid assays reveal that p110 interacts through its TPR domain with the U4/U6-specific 90K protein, indicating a specific role of the TPR domain in spliceosome recycling. On the 90K protein, a short internal region (amino acids 416 to 550) suffices for the interaction with p110. Together, these data suggest a model whereby p110 brings together U4 and U6 snRNAs through both RNA-protein and protein-protein interactions., (Copyright 2004 American Society for Microbiology)
- Published
- 2004
- Full Text
- View/download PDF
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