Rate of transcription elongation and sequence-specific pausing by RNA polymerase I directly influence ribosomal RNA processing
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ABSTRACT:
PROVIDER: PRJNA655915 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR12412945.fastq.gz | Fastqsanger.gz | |||
| SRR12412946.fastq.gz | Fastqsanger.gz | |||
| SRR12412947.fastq.gz | Fastqsanger.gz | |||
| SRR12412948.fastq.gz | Fastqsanger.gz | |||
| SRR12412949.fastq.gz | Fastqsanger.gz |
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Project description:One of the first steps in ribosome biogenesis is transcription of the ribosomal DNA (rDNA) by RNA polymerase I (Pol I). Processing of the resultant ribosomal RNA (rRNA) begins co-transcriptionally and perturbation of Pol I transcription elongation results in defective rRNA processing. Mechanistic insight regarding the link between transcription elongation and ribosome assembly is lacking due to limited in vivo methods to assay Pol I transcription. Here, we use native elongating transcript sequencing (NET-seq) with a strain of Saccharomyces cerevisiae containing a point mutation in Pol I, rpa190-F1205H, which results in impaired rRNA processing and ribosome assembly. We previously demonstrated that this mutation caused a mild reduction in the transcription elongation rate of Pol I in vitro, however, transcription elongation by the mutant has not been characterized in vivo. Here, our findings demonstrate that the mutant Pol I has an increased pause propensity during processive transcription elongation both in vitro and in vivo. NET-seq reveals that rpa190-F1205H Pol I displays alternative pause site preferences in vivo. Specifically, the mutant is sensitized to A/G residues in the RNA:DNA hybrid and at the last incorporated nucleotide position. Furthermore, both NET-seq and EM analysis of Miller chromatin spreads reveal pileups of rpa190-F1205H Pol I throughout the rDNA, particularly at the 5' end of the 35S gene. This combination of in vitro and in vivo analyses of the Pol I mutant provides novel insights into Pol I elongation properties and indicates how these properties are crucial for efficient co-transcriptional rRNA processing and ribosome assembly.
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Project description:We report altered pause profiles and sensitivity to nucleotide composition in the rpa190-f1205h RNA polymerase I mutant in vivo and in vitro
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Project description:The objective of this study was to investigate the relationship between Pol I transcription elongation rate and ribosomal RNA processing in vivo by using a yeast strain containing a mutation in Pol I. This mutation, rpa190-F1205H, has been previously characterized to have a reduced elongation rate in vitro. Here, we used native elongating transcript sequencing (NET-seq) to determine the effect of this mutation on Pol I occupancy in vivo. Our findings demonstrate that this mutation induces increased Pol I pausing, especially in the first half of the 35S gene, and causes sequence-specific changes in Pol I occupancy.
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Project description:Yeast mRNAs are polyadenylated at multiple sites in their 3' untranslated regions (3' UTRs), and poly(A) site usage is regulated by the rate of transcriptional elongation by RNA polymerase II (Pol II). Slow Pol II derivatives favor upstream poly(A) sites, and fast Pol II derivatives favor downstream poly(A) sites. Transcriptional elongation and polyadenylation are linked at the nucleotide level, presumably reflecting Pol II dwell time at each residue that influences the level of polyadenylation. Here, we investigate the effect of Pol II elongation rate on pausing patterns and the relationship between Pol II pause sites and poly(A) sites within 3' UTRs. Mutations that affect Pol II elongation rate alter sequence preferences at pause sites within 3' UTRs, and pausing preferences differ between 3' UTRs and coding regions. In addition, sequences immediately flanking the pause sites show preferences that are largely independent of Pol II speed. In wild-type cells, poly(A) sites are preferentially located < 50 nucleotides upstream from Pol II pause sites, but this spatial relationship is diminished in cells harboring Pol II speed mutants. Based on a random forest classifier, Pol II pause sites are modestly predicted by the distance to poly(A) sites but are better predicted by the chromatin landscape in Pol II speed derivatives. Transcriptional regulatory proteins can influence the relationship between Pol II pausing and polyadenylation but in a manner distinct from Pol II elongation rate derivatives. These results indicate a complex relationship between Pol II pausing and polyadenylation.
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Project description:Transcription by RNA polymerase I (RNAPI) represents the majority of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal rRNAs (rRNAs). As several rRNA maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. We show here that the conserved fission yeast RNA-binding protein Seb1 associates with the RNAPI transcription machinery and promotes RNAPI pausing states along the rDNA. The overall faster progression of RNAPI at the rDNA in Seb1-deficient cells impaired cotranscriptional rRNA processing and the production of mature rRNAs. Given that Seb1 also influences pre-mRNA processing by modulating RNAPII progression, our findings unveil Seb1 as a pausing-promoting factor for RNA polymerases I and II to control cotranscriptional RNA processing.
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