Regulation of Pre-mRNA Processing Through Nascent RNA Folding
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| Publicado en: | ProQuest Dissertations and Theses (2025) |
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| Acceso en línea: | Citation/Abstract Full Text - PDF |
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| Resumen: | RNA sequence determines the formation of intramolecular base pairs. Together with tertiary contacts, base pairs define every RNA's structure, or fold. The catalytic, regulatory, or coding potential of an RNA strongly depends on its folding pattern. Because base pairing occurs much faster than RNA synthesis (transcription) in cells, early structural states can govern RNA processing events and dictate the formation of functional conformations. These co-transcriptional structural states remained largely unknown. To address this gap, I developed CoSTseq, a chemical probing and enrichment method to measure nascent RNA base pairing upon exit from RNA polymerases (Pols) transcriptome-wide in living Saccharomyces cerevisiae (yeast) cells. By monitoring each nucleotide's base pairing activity during transcription, CoSTseq reveals predominantly rapid pairing - within 25 bp of transcription after addition to the nascent chain. My data show that previously uncharacterized pre-rRNA base pairing patterns emerge during transcription, representing radically different local intermediates to mature rRNA structure and function. I find that helicases can act on rRNA directly after synthesis across the entire locus, facilitating extensive remodeling of transient structures. In contrast, nascent pre-mRNAs fold into local structures that are indistinguishable from mature mRNAs, suggesting that co-transcriptional base pairing resembles mRNA re-folding during translation.Pre-mRNA splicing relies on the recognition of sequence elements (5' splice site, branch site, 3' splice site) by protein and RNA components of the spliceosome, and occurs co-transcriptionally in yeast. Since I showed using CoSTseq that RNA base pairing also occurs rapidly, I hypothesized that structures involving the splice sites are potent modulators of splicing. I developed a massively parallel reporter assay that allows accurate quantification of splicing efficiencies modulated by RNA structure across tens of thousands of intron variants in yeast. I find that sequestering the 5'SS or branch site in base pairing interactions modulates splicing within an unexpectedly large dynamic range, which can be nearly fully explained by structure stability. In addition to known intronic recognition sites, I find new structural elements that are required for efficient splicing. Simple machine learning models are able to learn the relationship between structure and splicing, and using a genetic algorithm, can be employed to create designer introns that are spliced with any desired efficiency.In summary, this work establishes general principles of RNA folding upon synthesis through direct experimental detection of co-transcriptional base pairing inside cells. I show through systematic multiplexed experiments that such base pairing can be a potent regulator of pre-mRNA splicing, modulating protein expression levels across orders of magnitude just through varied structure stability. Given the scale of regulation, I hypothesize that intron sequence forming structures with and near splice sites is generally under evolutionary selection and expect such structures as regulatory principles to be widespread in natural genomes. |
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| ISBN: | 9798286442355 |
| Fuente: | ProQuest Dissertations & Theses Global |