Yeast PAF1 complex counters the pol III accumulation and replication stress on the tRNA genes

Bhalla, Pratibha; Shukla, Avanish; Vernekar, Dipti Vinayak; Arimbasseri, Aneeshkumar G.; Sandhu, Kuljeet Singh; Bhargava, Poonam

doi:10.1038/s41598-019-49316-5

Cited by 12 publications

(12 citation statements)

References 75 publications

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“…Consistent with this, Spt16 occupancy on the tRNA genes goes up during replication stress ( Fig. 7 E), while Pol III levels are reported to go down significantly under replication stress ( Bhalla et al 2019b ). The above results indicate Spt16 involvement in cellular stress-sensing/response mechanisms.…”

Section: Resultssupporting

confidence: 77%

“…Though Pol II is found near many tRNA genes (Supplemental Fig. S2C; Bhalla et al 2019b ); the average Spt16 profile was not influenced by the presence of Pol II-transcribed genes within 150 or 300 bp of both the gene ends (Supplemental Fig. S2D).…”

Section: Resultsmentioning

confidence: 99%

“…We had earlier reported that FACT does not affect the transcription of a chromatinized tRNA gene in vitro ( Mahapatra et al 2011 ). However, Pol III-transcribed genes generally elicit gene-specific behavior under different conditions ( Foretek et al 2016 ; Turowski et al 2016 ; Shukla and Bhargava 2018 ; Bhalla et al 2019b ). A gene-specific effect of Spt16 depletion, showing an increased or decreased transcription of some of the tested Pol II-transcribed genes was reported earlier ( Jimeno-González et al 2006 ).…”

Section: Resultsmentioning

confidence: 99%

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Transcription-dependent enrichment of the yeast FACT complex influences nucleosome dynamics on the RNA polymerase III-transcribed genes

Shukla

Bhalla

Potdar

et al. 2020

RNA

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The FACT (FAcilitates Chromatin Transactions) complex influences transcription initiation and enables passage of RNA polymerase (Pol) II through gene body nucleosomes during elongation. In the budding yeast, ∼280 noncoding RNA genes highly transcribed in vivo by Pol III are found in the nucleosome-free regions bordered by positioned nucleosomes. The downstream nucleosome dynamics was found to regulate transcription via controlling the gene terminator accessibility and hence, terminator-dependent Pol III recycling. As opposed to the enrichment at the 5′-ends of Pol II-transcribed genes, our genome-wide mapping found transcription-dependent enrichment of the FACT subunit Spt16 near the 3′-end of all Pol III-transcribed genes. Spt16 physically associates with the Pol III transcription complex and shows gene-specific occupancy levels on the individual genes. On the non-tRNA Pol III-transcribed genes, Spt16 facilitates transcription by reducing the nucleosome occupany on the gene body. On the tRNA genes, it maintains the position of the nucleosome at the 3′ gene end and affects transcription in a gene-specific manner. Under nutritional stress, Spt16 enrichment is abolished in the gene downstream region of all Pol III-transcribed genes and reciprocally changed on the induced or repressed Pol II-transcribed ESR genes. Under the heat and replicative stress, its occupancy on the Pol III-transcribed genes increases significantly. Our results show that Spt16 elicits a differential, gene-specific and stress-responsive dynamics, which provides a novel stress-sensor mechanism of regulating transcription against external stress. By primarily influencing the nucleosomal organization, FACT links the downstream nucleosome dynamics to transcription and environmental stress on the Pol III-transcribed genes.

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Section: Resultssupporting

confidence: 77%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Transcription-dependent enrichment of the yeast FACT complex influences nucleosome dynamics on the RNA polymerase III-transcribed genes

Shukla

Bhalla

Potdar

et al. 2020

RNA

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“…On the other hand, the loss of PAF1 complex function through the deletion of rtf1 attenuated the suppression by RBP3‐TAP , suggesting that the PAF1 complex functions in the same pathway (Fig EV3E). This is not surprising given that Paf1 itself interacts with RNAPII (Mueller & Jaehning, 2002) and possibly with RNAPIII (Bhalla et al , 2019). Interestingly, during prolonged growth on high levels of HU (0.2 M), we also see mec1‐S1991A sensitivity to HU on rich media (YPAD) and its suppression by RPB3‐TAP (Fig EV3F), ruling out media conditions as a factor in this suppression pathway.…”

Section: Resultsmentioning

confidence: 95%

A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress

et al. 2021

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Upon replication stress, budding yeast checkpoint kinase Mec1 ATR triggers the downregulation of transcription, thereby reducing the level of RNA polymerase (RNAP) on chromatin to facilitate replication fork progression. Here, we identify a hydroxyurea-induced phosphorylation site on Mec1, Mec1-S1991, that contributes to the eviction of RNAPII and RNAPIII during replication stress. The expression of the non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII through a TAP fusion to its Rpb3 subunit, suggesting that lethality in mec1-S1991A mutants arises from replication-transcription conflicts. Coincident with a failure to repress gene expression on hydroxyurea in mec1-S1991A cells, highly transcribed genes such as GAL1 remain bound at nuclear pores. Consistently, we find that nuclear pore proteins and factors controlling RNAPII and RNAPIII are phosphorylated in a Mec1-dependent manner on hydroxyurea. Moreover, we show that Mec1 kinase also contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase by promoting degradation of the Rpb1 subunit.

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“…We also identified four components of the PAF1 complex, which binds to Pol II to regulate transcriptional elongation and termination, as well as mRNA processing and export [ 63 ]. Interestingly, recent findings indicate that PAF1 also participates in Pol I and Pol III transcription [ 64 ]. Among the PAF1 subunits, a putative ortholog of RTF1 (HHpred E-value of 0.0011) was identified.…”

Section: Discussionmentioning

confidence: 99%

Participation of TFIIIB Subunit Brf1 in Transcription Regulation in the Human Pathogen Leishmania major

Florencio-Martínez

Cano-Santiago

Mondragón-Rosas

et al. 2021

Genes

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In yeast and higher eukaryotes, transcription factor TFIIIB is required for accurate initiation of transcription by RNA Polymerase III (Pol III), which synthesizes transfer RNAs (tRNAs), 5S ribosomal RNA (rRNA), and other essential RNA molecules. TFIIIB is composed of three subunits: B double prime 1 (Bdp1), TATA-binding protein (TBP), and TFIIB-related factor 1 (Brf1). Here, we report the molecular characterization of Brf1 in Leishmania major (LmBrf1), a parasitic protozoan that shows distinctive transcription characteristics, including the apparent absence of Pol III general transcription factors TFIIIA and TFIIIC. Although single-knockout parasites of LmBrf1 were obtained, attempts to generate LmBrf1-null mutants were unsuccessful, which suggests that LmBrf1 is essential in promastigotes of L. major. Notably, Northern blot analyses showed that the half-lives of the messenger RNAs (mRNAs) from LmBrf1 and other components of the Pol III transcription machinery (Bdp1 and Pol III subunit RPC1) are very similar (~40 min). Stabilization of these transcripts was observed in stationary-phase parasites. Chromatin immunoprecipitation (ChIP) experiments showed that LmBrf1 binds to tRNA, small nuclear RNA (snRNA), and 5S rRNA genes. Unexpectedly, the results also indicated that LmBrf1 associates to the promoter region of the 18S rRNA genes and to three Pol II-dependent regions here analyzed. Tandem affinity purification and mass spectrometry analyses allowed the identification of a putative TFIIIC subunit. Moreover, several proteins involved in transcription by all three RNA polymerases co-purified with the tagged version of LmBrf1.

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Yeast PAF1 complex counters the pol III accumulation and replication stress on the tRNA genes

Cited by 12 publications

References 75 publications

Transcription-dependent enrichment of the yeast FACT complex influences nucleosome dynamics on the RNA polymerase III-transcribed genes

Transcription-dependent enrichment of the yeast FACT complex influences nucleosome dynamics on the RNA polymerase III-transcribed genes

A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress

Participation of TFIIIB Subunit Brf1 in Transcription Regulation in the Human Pathogen Leishmania major

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