Impairment of RNA polymerase I elongation rate induces defects in ribosomal RNA processing and ribosome biogenesis
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA803468 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17885933.fastq.gz | Fastqsanger.gz | |||
| SRR17885934.fastq.gz | Fastqsanger.gz | |||
| SRR17885935.fastq.gz | Fastqsanger.gz | |||
| SRR17885936.fastq.gz | Fastqsanger.gz | |||
| SRR17885937.fastq.gz | Fastqsanger.gz |
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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: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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