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Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts

ABSTRACT: RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5´ peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential “housekeeping” roles. Many tRNA genes were found to generate long, 3´-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of T-residues in the coding strand, and multiple, functional terminators can be located far downstream. The steady-state levels of 3´-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery; especially the RNA-binding protein Nab2, cofactors for the nuclear exosome and the 5´-exonuclease Rat1.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE77863 | GEO | 2016/05/18

SECONDARY ACCESSION(S): PRJNA311752

REPOSITORIES: GEO

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Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts

Project description:RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5Â´ peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential âhousekeepingâ roles. Many tRNA genes were found to generate long, 3Â´-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of T-residues in the coding strand, and multiple, functional terminators can be located far downstream. The steady-state levels of 3Â´-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery; especially the RNA-binding protein Nab2, cofactors for the nuclear exosome and the 5Â´-exonuclease Rat1. CRAC protocol performed on samples containing HTP-tagged proteins: Rpc160(Rpo31), Nab2, Rrp44(Dis3), Rrp6 and Mtr4. Rpc-160-bound RNAs were analyzed in wildtype and maf1 mutant cells and after a shift to medium containing glycerol.

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Expression profile of the M-bM-^@M-^\heat-inducible N-degronM-bM-^@M-^] of Nab2 (nab2-td)

Project description:Gene expression in eukaryotes is an essential process that includes transcription, pre-RNA processing and RNA export. All these steps are coupled and normally, any failure in one step affects the other steps and could cause nuclear mRNA retention. One important player in this interface is the poly(A)-RNA binding protein Nab2, which regulates the poly(A)-tail length of mRNAs protecting their 3M-bM-^@M-^Y-ends from a second round of polyadenylation and facilitating their nucleo-cytoplasmic export. Interestingly, here we show that Nab2 has additional roles in mRNA transcription elongation, tRNA metabolism and rRNA export. We use Genome-wide analysis of expression of a conditional degron nab2 mutant that allows the depletion of the protein in 15 minutes, to demosntrate that the role of Nab2 in RNAPII transcription and RNAPIII metabolism is not the result of a secondary effect. Our results identify primary targets of nab2 revealing novel functions for Nab2 in transcription and metabolism of most types of RNAs, not only poly(A) mRNAs, indicating that Nab2 function is more ubiquitous than previously anticipated, being a central player in the general and coordinated control of gene expression from transcription elongation to translation initiation. Two and three repeats of the wild type control and the nab2-td mutant respectively at time 0, 30 and 75 minutes after the degron induction.

2011-12-01 | E-GEOD-19302 | biostudies-arrayexpress

Expression profile of nab2-1 mutant

Project description:Gene expression in eukaryotes is an essential process that includes transcription, pre-RNA processing and RNA export. All these steps are coupled and normally, any failure in one step affects the other steps and could cause nuclear mRNA retention. One important player in this interface is the poly(A)-RNA binding protein Nab2, which regulates the poly(A)-tail length of mRNAs protecting their 3’-ends from a second round of polyadenylation and facilitating their nucleo-cytoplasmic export. Interestingly, here we show that Nab2 has additional roles in mRNA transcription elongation, tRNA metabolism and rRNA export. We use Genome-wide analysis of expression of nab2-1 mutant to demosntrate that the role of Nab2 in RNAPII transcription and RNAPIII metabolism is not the result of a secondary effect. Our results identify primary targets of nab2 revealing novel functions for Nab2 in transcription and metabolism of most types of RNAs, not only poly(A) mRNAs, indicating that Nab2 function is more ubiquitous than previously anticipated, being a central player in the general and coordinated control of gene expression from transcription elongation to translation initiation.

2011-12-01 | GSE26303 | GEO

Expression profile of the “heat-inducible N-degron” of Nab2 (nab2-td)

Project description:Gene expression in eukaryotes is an essential process that includes transcription, pre-RNA processing and RNA export. All these steps are coupled and normally, any failure in one step affects the other steps and could cause nuclear mRNA retention. One important player in this interface is the poly(A)-RNA binding protein Nab2, which regulates the poly(A)-tail length of mRNAs protecting their 3’-ends from a second round of polyadenylation and facilitating their nucleo-cytoplasmic export. Interestingly, here we show that Nab2 has additional roles in mRNA transcription elongation, tRNA metabolism and rRNA export. We use Genome-wide analysis of expression of a conditional degron nab2 mutant that allows the depletion of the protein in 15 minutes, to demosntrate that the role of Nab2 in RNAPII transcription and RNAPIII metabolism is not the result of a secondary effect. Our results identify primary targets of nab2 revealing novel functions for Nab2 in transcription and metabolism of most types of RNAs, not only poly(A) mRNAs, indicating that Nab2 function is more ubiquitous than previously anticipated, being a central player in the general and coordinated control of gene expression from transcription elongation to translation initiation.

2011-12-01 | GSE19302 | GEO

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Project description:Transcription of tRNA genes by RNA Polymerase III (RNAPIII) is tuned by signaling cascades. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulators have not been described. Decoding the proteome of a native tRNA gene in yeast revealed reprogramming of the RNAPIII transcription machinery upon nutrient perturbation. Among the dynamic proteins, we identified Fpt1, a protein of unknown function that uniquely occupied RNAPIII regulated genes. Fpt1 binding at tRNA genes correlated with the efficiency of RNAPIII eviction upon nutrient perturbation and required the transcription factors TFIIIB and TFIIIC but not RNAPIII. In the absence of Fpt1, eviction of RNAPIII was reduced and the shutdown of ribosome biogenesis genes was impaired upon nutrient perturbation. Our findings provide support for a chromatin-associated mechanism required for RNAPIII eviction from tRNA genes and for tuning the physiological response to changing metabolic demands.

2023-11-28 | PXD045463 | Pride

Cdk1 Gates Cell Cycle-Dependent tRNA Synthesis by Regulating RNA Polymerase III Activity

Project description:tRNA genes are transcribed by RNA polymerase III (RNAPIII). During recent years it has become clear that RNAPIII activity is strictly regulated by the cell in response to environmental cues and the homeostatic status of the cell. However, the molecular mechanisms that control RNAPIII activity to regulate the amplitude of tDNA transcription in normally cycling cells are not well understood. Here, we show that tRNA levels fluctuate during the cell cycle and reveal the underlying molecular mechanism. The cyclin Clb5 recruits the cyclin dependent kinase Cdk1 to tRNA genes to boost tDNA transcription during S phase. At tDNA genes, Cdk1 promotes the recruitment of TFIIIC, stimulates the interaction between TFIIIB and TFIIIC, and increases the dynamics of RNA polymerase III in vivo. Furthermore, we identified Bdp1 as an important Cdk1 substrate in this process. Preventing Bdp1 phosphorylation prevented cell cycle-dependent recruitment of TFIIIC and abolished the cell cycle-induced increase in tDNA transcription. Our findings demonstrate that under optimal growth conditions Cdk1 gates tRNA synthesis in S phase by regulating the RNAPIII machinery, revealing a direct link between the cell cycle and RNAPIII activity.

2018-10-19 | PXD011084 | Pride

TORC1 shapes the SUMO proteome to control RNA Polymerase III activity

Project description:Loss of nutrient supply elicits alterations of the SUMO proteome and sumoylation is crucial to various cellular processes including transcription. However, the physiological significance of sumoylation of transcriptional regulators is unclear. To begin clarifying this, we mapped the SUMO proteome under nitrogen-limiting conditions in Saccharomyces cerevisiae. Interestingly, several RNA polymerase III (RNAPIII) components are major SUMO targets under normal growth conditions, including Rpc53, Rpc82, and Ret1, and nutrient starvation results in rapid desumoylation of these proteins. These findings are supported by ChIP-seq experiments that show that SUMO is highly enriched at tDNA genes. Furthermore, RNA-seq experiments revealed that preventing sumoylation results in significantly decreased tRNA transcription. TORC1 inhibition resulted in the same effect, and our data indicate that the SUMO and TORC1 pathways are both required for robust tDNA expression. Importantly, tRNA transcription was strongly reduced in cells expressing a non-sumoylatable Rpc82-4KR mutant, which correlated with a misassembled RNAPIII transcriptional complex. Our data suggest that in addition to TORC1 activity, sumoylation of RNAPIII is key to reaching full translational capacity under optimal growth conditions.

2017-01-21 | PXD005142 | Pride

TORC1 shapes the SUMO proteome to control RNA polymerase III activity

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Project description:Nascent RNA sequencing has recently revealed that pre-mRNA splicing can occur shortly after the intron emerges from RNA polymerase II (Pol II). Differences in co-transcriptional splicing profiles suggest regulation by cis- and/or trans-acting factors. Here we used Single Molecule Intron Tracking (SMIT) in budding yeast to identify a cohort of regulators by machine learning. One candidate, Nab2, displayed reduced co-transcriptional splicing of some pre-mRNAs when depleted. Unexpectedly, these splicing defects were attributable to readthrough transcription, which was revealed by long read sequencing of nascent RNA; individual readthrough transcripts induced by Nab2 depletion sometimes spanned multiple genes. Thus, Nab2 regulation of splicing was indirect. Moreover, unspliced transcripts displayed downstream readthrough in both control and Nab2-depleted cells, highlighting the coupling between splicing and 3′ end formation. We conclude that Nab2 is required for proper 3′ end processing, which ensures both transcription termination as well as proper splicing of downstream genes.

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