scholarly journals Genome-wide R-loop analysis defines unique roles for DDX5, XRN2, and PRMT5 in DNA/RNA hybrid resolution

2020 ◽  
Vol 3 (10) ◽  
pp. e202000762
Author(s):  
Oscar D Villarreal ◽  
Sofiane Y Mersaoui ◽  
Zhenbao Yu ◽  
Jean-Yves Masson ◽  
Stéphane Richard
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Transcription Termination ◽  
Start Site ◽  
Loop Gain ◽  
Transcription Start ◽  
Genome Wide ◽  
Rna Polymerase Ii Transcription

DDX5, XRN2, and PRMT5 have been shown to resolve DNA/RNA hybrids (R-loops) at RNA polymerase II transcription termination sites at few genomic loci. Herein, we perform genome-wide R-loop mapping using classical DNA/RNA immunoprecipitation and high-throughput sequencing (DRIP-seq) of loci regulated by DDX5, XRN2, and PRMT5. We observed hundreds to thousands of R-loop gains and losses at transcribed loci in DDX5-, XRN2-, and PRMT5-deficient U2OS cells. R-loop gains were characteristic of highly transcribed genes located at gene-rich regions, whereas R-loop losses were observed in low-density gene areas. DDX5, XRN2, and PRMT5 shared many R-loop gain loci at transcription termination sites, consistent with their coordinated role in RNA polymerase II transcription termination. DDX5-depleted cells had unique R-loop gain peaks near the transcription start site that did not overlap with those of siXRN2 and siPRMT5 cells, suggesting a role for DDX5 in transcription initiation independent of XRN2 and PRMT5. Moreover, we observed that the accumulated R-loops at certain loci in siDDX5, siXRN2, and siPRMT5 cells near the transcription start site of genes led to antisense intergenic transcription. Our findings define unique and shared roles of DDX5, XRN2, and PRMT5 in DNA/RNA hybrid regulation.

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

scholarly journals Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

2020 ◽  
Author(s):  
Zhe Sun ◽  
Alexander Yakhnin ◽  
Peter C. FitzGerald ◽  
Carl E. Mclntosh ◽  
Mikhail Kashlev
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Environmental Cues ◽  
Start Site ◽  
Transcription Start ◽  
E Coli ◽  
Eukaryotic Genes ◽  
Genome Wide ◽  
Transcript Cleavage

ABSTRACTPromoter-proximal pausing regulates expression of many eukaryotic genes and serves as checkpoints for assembly of elongation/splicing machinery. Little is known how broadly the pausing is employed in transcriptional regulation in bacteria. We applied NET-seq combined with RNase I footprinting for genome-wide analysis of σ70-dependent transcription pauses in Escherichia coli. Many E. coli genes appear to contain clusters of strong backtracked pauses at 10-20-bp distance from the transcription start site caused by retention of σ70 subunit in RNA polymerase. The pauses in 10-15-bp register of the promoter are dictated by binding of σ70 to canonical −10 element, 6-7 nt spacer and “YR+1Y” motif centered at transcription start site all characteristic for strong E. coli promoters. The promoters for the pauses in 16-20-bp register contain an additional −10-like sequence positioned on the same face of the DNA duplex as the original −10 element suggesting that σ70 hopping was responsible for these pauses. Our in vitro analysis reveals that RNA polymerase backtracking and DNA scrunching are involved in these pauses that are relieved by Gre transcript cleavage factors. The genes coding for transcription factors are enriched in these pauses suggesting that σ70 and Gre proteins regulate transcription in response to changing environmental cues.

scholarly journals RNA polymerase II-independent recruitment of SPT6L at transcription start sites in Arabidopsis

2019 ◽  
Vol 47 (13) ◽  
pp. 6714-6725 ◽  
Author(s):  
Chen Chen ◽  
Jie Shu ◽  
Chenlong Li ◽  
Raj K Thapa ◽  
Vi Nguyen ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Elongation Factor ◽  
Proper Function ◽  
Gene Body ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Genome Wide ◽  
Yeast Mutants

Abstract SPT6 is a conserved elongation factor that is associated with phosphorylated RNA polymerase II (RNAPII) during transcription. Recent transcriptome analysis in yeast mutants revealed its potential role in the control of transcription initiation at genic promoters. However, the mechanism by which this is achieved and how this is linked to elongation remains to be elucidated. Here, we present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNAPII occupancy across transcribed genes. We also further demonstrate that SPT6L enrichment is unexpectedly shifted, from gene body to transcription start site (TSS), when its association with RNAPII is disrupted. Protein domains, required for proper function and enrichment of SPT6L on chromatin, are subsequently identified. Finally, our results suggest that recruitment of SPT6L at TSS is indispensable for its spreading along the gene body during transcription. These findings provide new insights into the mechanisms underlying SPT6L recruitment in transcription and shed light on the coordination between transcription initiation and elongation.

scholarly journals The sua8 suppressors of Saccharomyces cerevisiae encode replacements of conserved residues within the largest subunit of RNA polymerase II and affect transcription start site selection similarly to sua7 (TFIIB) mutations.

1994 ◽  
Vol 14 (1) ◽  
pp. 226-237 ◽  
Author(s):  
R W Berroteran ◽  
D E Ware ◽  
M Hampsey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Start Site ◽  
Site Selection ◽  
Synthetic Lethality ◽  
Single Amino Acid ◽  
Start Site ◽  
Transcription Start ◽  
Conserved Residues

Mutations in the Saccharomyces cerevisiae sua8 gene were found to be suppressors of an aberrant ATG translation initiation codon in the leader region of the cyc1 gene. Analysis of cyc1 transcripts from sua8 mutants revealed that suppression is a consequence of diminished transcription initiation at the normal start sites in favor of initiation at downstream sites, including a site between the aberrant and normal ATG start codons. This effect is not cyc1 gene specific since initiation at other genes, including ADH1, CYC7, and HIS4, was similarly affected, although initiation at HIS3 and SPT15 was unaffected. The SUA8 gene was cloned and partially sequenced, revealing identity to RPB1, which encodes the largest subunit of RNA polymerase II. The sua8 suppressors are the result of single amino acid replacements of highly conserved residues. Three replacements were found either within or immediately preceding homology block D, and a fourth was found adjacent to homology block H, indicating that these regions play a role in defining start sites in vivo. Nearly identical effects on start site selection were observed for sua7 suppressors, which encode altered forms of TFIIB. Synthetic lethality was associated with double sua7 sua8 suppressor mutations, and recessive sua7 mutants failed to fully complement recessive sua8 mutants in heterozygous diploids (nonallelic noncomplementation). These data indicate that the largest subunit of RNA polymerase II and TFIIB are important determinants of transcription start site selection in S. cerevisiae and suggest that this function might be conferred by interaction between these two proteins.

scholarly journals Histone modifications at the transcription start site of non-coding RNA revealed regulatory patterns of activation in Caenorhabditis elegans embryo

2020 ◽  
Author(s):  
Megan A. Bandeira ◽  
Max E. Boeck
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Start Site ◽  
Histone Modifications ◽  
Principal Component ◽  
Integrated Analysis ◽  
Regulation Of Transcription ◽  
Start Site ◽  
Transcription Start ◽  
Non Coding Rna

AbstractHistone modifications play an essential role in regulating recruitment of RNA polymerase II and through this regulation of transcription itself. Which modifications are essential for regulating the transcription of non-coding RNA (ncRNA) species and how these patterns differ between the different types of ncRNA remains less studied compared to mRNA. We performed a principal component analysis (PCA) of histone modifications patterns surrounding the transcription start site (TSS) of ncRNA in an attempt to understand how histone modifications predict polymerase recruitment and transcription of ncRNA in early C. elegans development We found that our first PCA axis was a better predictor of polymerase recruitment and expression than any single histone modification for ncRNA and miRNA. This indicates an integrated analysis of many histone modifications is essential for predicting expression based on histone modifications and that each ncRNA species have unique regulation of RNA polymerase recruitment through histone modifications.

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