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Determining the effect of RPA49 on RNA polymerase I occupancy in vivo

ABSTRACT: The central goal of this project was to determine the role of the RNA polymerase I (Pol I) subunits RPA34 and RPA49 in rRNA synthesis. Previous studies by various groups have demonstrated that these two subunits may play a role in transcription initiation and elongation by on Pol I occupancy in vivo, but this is still not clearly defined. Here, we deployed a high-resolution technique, native elongating transcript sequencing (NET-seq), to test the effects of RPA34 and RPA49 on Pol I occupancy in vivo. We found that when RPA34 was deleted, there was a significant reduction in Pol I occupancy across the rDNA template. Additionally, when RPA49 was deleted, Pol I occupancy was even further reduced across the template. Collectively, our findings suggest that perhaps RPA34 acts to stabilize RPA49 and these subunits may act primarily as transcription initiation factors for Pol I.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE232731 | GEO | 2023/05/22

REPOSITORIES: GEO

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Investigating the role of Hmo1 in transcription by Pol I in yeast.

Project description:The purpose of this study was to investigate the role of the yeast protein, high mobility group protein (Hmo1), in rRNA synthesis. For over twenty years, Hmo1 has been proposed to be an activator of transcription by Pol I, but its regulatory mechanism remains largely undefined. Therefore, we used native elongating transcript sequencing (NET-seq) to explore the role of this factor in transcription by Pol I. Our NET-seq results demonstrated that in cells lacking Hmo1 (hmo1D), Pol I occupancy was significantly increased across the rDNA template, indicating an increased pause propensity in these cells. These data suggest that Hmo1 is an important transcription elongation factor for Pol I.

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Defining the influence of the A12 subunit on transcription elongation and termination by rna polymerase I

Project description:we performed Native Elongating Transcript Sequencing (NET-seq) on an rpa12Δ strain of S. cerevisiae and evaluated the resultant change in Pol I occupancy throughout the 35S gene and the IGS. Compared to WT, we observed template sequence-specific changes in Pol I occupancy throughout the 35S gene. We also observed rpa12Δ Pol I occupancy downstream of both termination sites and throughout most of the IGS, including the 5S gene. Occupancy of rpa12Δ Pol I increased just upstream of the promoter proximal Reb1 binding site and dropped significantly after, implicating this site as a third terminator for Pol I transcription.

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Testing the usage of divalent metal cations and nucleases during NET-seq for Pol I

Project description:The goal of this study was to determine the effects of commonly used nuclease treatments and their divalent metal cation cofactors on Pol I occupancy during NET-seq. Other groups have previously used micrococcal nuclease (MNase) + CaCl2 and deoxyribonuclease I (DNase I) + MnCl2 in NET-seq protocols probing for Pol II occupancy. However, the effect of these treatments on polymerase occupancy has not been fully explored. Therefore, we tested the impact of these treatments (CaCl2, CaCl2 + MNase, MnCl2, and MnCl2 + DNase I) on Pol I occupancy in vivo. We found that the exposure of Pol I to either CaCl2 or MnCl2 caused a significant reduction in occupancy on the rDNA template. Nuclease treatment (with either MNase or DNase I) did not cause an additional significant effect on Pol I occupancy in vivo beyond that of either CaCl2 or MnCl2. Finally, we found that MnCl2 treatment caused a more significant reduction in Pol I occupancy as compared to CaCl2, suggesting that perhaps MnCl2 stimulates the intrinsic cleavage activity of Pol I more robustly vs. CaCl2 in vivo. Altogether, these findings indicate that the inclusion of these treatments (especially that of divalent metal cations) in NET-seq protocols for Pol I cause a significant change in the polymerase occupancy.

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2021-02-18 | GSE166983 | GEO

A remodeled Pol II complex on RNA template

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The Rio1 kinase downregulates RNA polymerase I to promote rDNA stability and segregation

Project description:To sustain growth, budding yeast actively transcribes its ribosomal gene array (rDNA) in the nucoleolus to produce ribosomes and proteins. However, intense transcription during rDNA replication may provoke collisions between RNA polymerase I (Pol I) and the replisome, may cause replication fork instability, double-strand breaks, local recombinations and rDNA instability. The latter is manifested by rDNA array expansion or reduction and the formation of extrachromosomal rDNA circles, anomalies that accelerate aging in yeast. Transcription also interferes with the resolution, condensation and segregation of the sister chromatid rDNA arrays. As a consequence, rDNA segregation lags behind the rest of the yeast genome and occurs in late anaphase when rDNA transcription is temporarily shut off. How yeast promotes the stability and transmission of its rDNA array while satisfying a constant need for ribosomes remains unclear. Here we show that the downregulation of Pol I by the conserved cell cycle kinase Rio1 spatiotemporally coordinates rDNA transcription, replication and segregation. More specifically, Rio1 activity promotes copy-number stability of the replicating rDNA array by curtailing Pol I activity and by localising the histone deacetylase Sir2, which establishes a heterochromatic state that silences rDNA transcription. At anaphase entry, Rio1 and the Cdc14 phosphatase target Pol I subunit Rpa43 to dissociate Pol I from the 35S rDNA promoter. The rDNA locus then condensates and segregates, thereby concluding the genome transmission process. Rio1 is involved in ribosome maturation in the cytoplasm of budding yeast and human cells. Additional engagements in the cytoplasm or roles in the nucleus are unknown. Our study describes its first nuclear engagement as a Pol I silencing kinase. This activity may prove highly relevant as dysregulated RNA polymerase I activity has been associated with cancer initiation and proliferation.

2017-02-10 | GSE54930 | GEO

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2022-01-15 | GSE193569 | GEO

Sir2 suppresses transcription-mediated displacement of Mcm2-7 replicative helicases at the ribosomal DNA repeats

Project description:Repetitive DNA sequences within eukaryotic heterochromatin are poorly transcribed and replicate late in S-phase. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA arrays (rDNA). Despite the widespread association between transcription and replication, it remains unclear how transcription might impinge on replication, or vice versa. Here we show that, when silencing of an RNA polymerase II (RNA Pol II)-transcribed non-coding RNA at the rDNA is disrupted by SIR2 deletion, RNA polymerase pushes and thereby relocalizes replicative Mcm2-7 helicases away from their loading sites to an adjacent region with low nucleosome occupancy, and this relocalization is associated with increased rDNA origin efficiency. Our results suggest a model in which two of the major defining features of heterochromatin, transcriptional silencing and late replication, are mechanistically linked through suppression of polymerase-mediated displacement of replication initiation complexes.

2019-05-08 | GSE130273 | GEO

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