Transcription elongation rate affects nascent histone pre-mRNA folding and 3’ end processing
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ABSTRACT:
PROVIDER: PRJNA435673 | ENA |
REPOSITORIES: ENA
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Project description:We report that slow transcription by mutant RNA pol II caused accumulation of polyadenylated histone mRNAs that extend past the stem loop processing site. UV irradiation, which decelerates pol II elongation, also induced long poly(A)+ histone transcripts. Inhibition of 3’ processing by slow pol II correlates with failure to recruit SLBP to histone genes. Chemical probing of nascent RNA structure showed that the stem loop fails to fold in transcripts made by slow pol II, thereby explaining the absence of SLBP and failure to process 3’ ends. These results show that regulation of transcription speed can modulate pre-mRNA processing by changing nascent RNA structure, and suggest a mechanism by which alternative processing could be controlled
2018-02-23 | GSE109652 | GEO
Project description: Not available
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Project description:Transcription elongation rates influence RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S. cerevisiae. Mapping RNAPI by Miller chromatin spreads or UV crosslinking revealed 5′ enrichment and strikingly uneven local polymerase occupancy along the rDNA, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution: folding energy and GC content in the transcription bubble. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high GC in the transcription bubble slows elongation. A mathematical model for RNAPI elongation confirmed the importance of nascent RNA folding in transcription. RNAPI from S. pombe was similarly sensitive to transcript folding, as were S. cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site use, indicating regulatory significance for transcript folding.
2020-06-24 | GSE136056 | GEO
Project description:Most RNA processing occurs co-transcriptionally. We interrogated nascent pol II transcripts by chemical and enzymatic probing, and determined how the “nascent RNA structureome” relates to splicing, A-I editing and transcription speed. RNA folding within introns and steep structural transitions at splice sites are associated with efficient co-transcriptional splicing. A slow pol II mutant elicits extensive remodeling into more folded conformations with increased A-I editing. Introns that become more structured at their 3’ splice sites get co-transcriptionally excised more efficiently. Slow pol II altered folding of intronic Alu elements where cryptic splicing and intron retention are stimulated, an outcome mimicked by UV which decelerates transcription. Slow transcription also remodeled RNA folding around alternative exons in distinct ways that predict whether skipping or inclusion is favored, even though it occurs post-transcriptionally. Hence co-transcriptional RNA folding modulates post-transcriptional alternative splicing. In summary the plasticity of nascent transcripts has widespread effects on RNA processing.
2021-02-09 | GSE149018 | GEO
Project description:Folding newly synthesized proteins relies on the ribosome intricately coordinating mRNA translation with a network of ribosome-associated machinery. The principles that drive the coordination of this diverse machinery remain poorly understood. Here, we use selective ribosome profiling to determine how the essential chaperonin TRiC/CCT and the Hsp70 Ssb are recruited to ribosome-nascent chain complexes to mediate cotranslational protein folding. Whereas substrate localization and nascent chain sequence are the major determinants of cotranslational recruitment of Ssb, we found that temporal and structural elements drive TRiC engagement. For both chaperones, however, local slowdowns in translation enhance chaperone enrichment. This work helps define the principles that dictate the coordinated activity of ribosome-associated factors to perform their critical role in maintaining a properly folded nascent proteome.
2019-08-22 | GSE114882 | GEO
Project description:The folding of most proteins occurs during the course of their translation while their tRNA-bound C-termini are embedded in the ribosome. How the close proximity of nascent proteins to the ribosome influences their folding thermodynamics remains poorly understood. Here, we have developed a mass spectrometry-based approach for determining the stabilities of nascent polypeptide chains using methionine oxidation as a folding probe. This approach enables quantitative measurements sub-global folding stabilities of ribosome nascent chains (RNCs) within complex protein mixtures and extracts. To validate the methodology, we analyzed the folding thermodynamics of three model proteins (DHFR, CheY and DinB) in soluble and ribosome-bound states. The data indicated that the ribosome can significantly alter the stability of nascent polypeptides. Ribosome-induced stability modulations were highly variable among different folding domains and were dependent on localized charge distributions within nascent polypeptides. The results implicated electrostatic interactions between the ribosome surface and nascent polypeptides as the cause of ribosome-induced stability modulations. The study establishes a robust proteomic methodology for analyzing localized stabilities within ribosome-bound nascent polypeptides and sheds light on how the ribosome influences the thermodynamics of protein folding.
2023-08-09 | PXD037995 | Pride
Project description:Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and has been proposed to regulate translation efficiency, accuracy and protein folding based on the assumption that codon usage affects translation dynamics. The role of codon usage in regulating translation, however, is not clear and has been challenged by recent ribosome profiling studies. Here we used a Neurospora cell-free translation system to directly monitor the velocity of mRNA translation. We demonstrated that the use of preferred codons enhances the rate of translation elongation, whereas non-optimal codons slow translation. In addition, codon usage regulates ribosome traffic on the mRNA. These conclusions were supported by ribosome profiling results in vitro and in vivo with substrate mRNAs manipulated to increase signal over background noise. We further show that codon usage plays an important role in regulating protein function by affecting co-translational protein folding. Together, these results resolve a long-standing fundamental question and demonstrate the importance of codon usage on protein folding.
2015-07-18 | GSE71032 | GEO
Project description:The yeast Hsp70 chaperone Ssb interacts with ribosomes and nascent chains to co-translationally assist protein folding. Here, we present a proteome-wide analysis of Hsp70 function during translation, based on in vivo selective ribosome profiling, that reveals mechanistic principles coordinating translation with chaperone-assisted protein folding. Ssb binds most cytosolic, nuclear, and mitochondrial proteins and a subset of ER proteins, supporting its general chaperone function. Position-resolved analysis of Ssb engagement reveals compartment- and protein-specific nascent chain binding profiles that are coordinated by emergence of positively charged peptide stretches enriched in aromatic amino acids. Ssbs’ function is temporally coordinated by RAC but independent from NAC. Analysis of ribosome footprint densities along orfs reveals that ribosomes translate faster at times of Ssb binding. This is coordinated by biases in mRNA secondary structure, and codon usage as well as the action of Ssb, suggesting chaperones may allow higher protein synthesis rates by actively coordinating protein synthesis with co-translational folding.
2017-07-17 | GSE93830 | GEO
Project description:Nascent Transcript Folding Plays a Major Role in Determining RNA Polymerase Elongation Rates
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Project description:Nascent Polypeptide Domain Topology and Elongation Rate Direct the Cotranslational Hierarchy of Hsp70 and TRiC/CCT
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