scholarly journals Spt4 Promotes Pol I Processivity and Transcription Elongation

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 413
Author(s):  
Abigail K. Huffines ◽  
Yvonne J. K. Edwards ◽  
David A. Schneider
Keyword(s):  
Mammalian Cells ◽  
Transcription Elongation ◽  
Eukaryotic Cell ◽  
Yeast Cells ◽  
Single Nucleotide ◽  
Pol I ◽  
In Vivo Analysis ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

RNA polymerases (Pols) I, II, and III collectively synthesize most of the RNA in a eukaryotic cell. Transcription by Pols I, II, and III is regulated by hundreds of trans-acting factors. One such protein, Spt4, has been previously identified as a transcription factor that influences both Pols I and II. Spt4 forms a complex with Spt5, described as the Spt4/5 complex (or DSIF in mammalian cells). This complex has been shown previously to directly interact with Pol I and potentially affect transcription elongation. The previous literature identified defects in transcription by Pol I when SPT4 was deleted, but the necessary tools to characterize the mechanism of this effect were not available at the time. Here, we use a technique called Native Elongating Transcript Sequencing (NET-seq) to probe for the global occupancy of Pol I in wild-type (WT) and spt4△ Saccharomyces cerevisiae (yeast) cells at single nucleotide resolution in vivo. Analysis of NET-seq data reveals that Spt4 promotes Pol I processivity and enhances transcription elongation through regions of the ribosomal DNA that are particularly G-rich. These data suggest that Spt4/5 may directly affect transcription elongation by Pol I in vivo.

scholarly journals No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5’-OH ends phosphorylated by Trl1

2018 ◽  
Author(s):  
Albertas Navickas ◽  
Sébastien Chamois ◽  
Rénette Saint-Fort ◽  
Julien Henri ◽  
Claire Torchet ◽  
...  
Keyword(s):  
Single Nucleotide ◽  
Endonucleolytic Cleavage ◽  
Naturally Occurring ◽  
Mrna Surveillance ◽  
Rna Fragments ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

AbstractThe No-Go Decay (NGD) mRNA surveillance pathway degrades mRNAs containing stacks of stalled ribosomes. Although an endoribonuclease has been proposed to initiate cleavages upstream of the stall sequence, the production of two RNA fragments resulting from a unique cleavage has never been demonstrated. We have used mRNAs expressing a 3’-ribozyme to produce truncated transcripts in vivo to mimic naturally occurring truncated mRNAs known to trigger NGD. This technique allows us to analyse endonucleolytic cleavage events at single-nucleotide resolution starting at the third collided ribosome, which we show to be Hel2-dependent. These cleavages map precisely in the mRNA exit tunnel of the ribosome, 8 nucleotides upstream of the first P-site residue and release 5’-hydroxylated RNA fragments requiring 5’-phosphorylation prior to digestion by the exoribonuclease Xrn1, or alternatively by Dxo1. Finally, we identify the RNA kinase Trl1, alias Rlg1, as an essential player in the degradation of NGD RNAs.

Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq

2015 ◽  
Vol 10 (7) ◽  
pp. 1050-1066 ◽  
Author(s):  
Yiliang Ding ◽  
Chun Kit Kwok ◽  
Yin Tang ◽  
Philip C Bevilacqua ◽  
Sarah M Assmann
Keyword(s):  
Rna Structure ◽  
Single Nucleotide ◽  
Genome Wide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

scholarly journals FICC-Seq: a method for enzyme-specified profiling of methyl-5-uridine in cellular RNA

2019 ◽  
Vol 47 (19) ◽  
pp. e113-e113 ◽  
Author(s):  
Jean-Michel Carter ◽  
Warren Emmett ◽  
Igor Rdl Mozos ◽  
Annika Kotter ◽  
Mark Helm ◽  
...  
Keyword(s):  
Mapping Method ◽  
Single Nucleotide ◽  
Genome Wide ◽  
A Genome ◽  
Target Sites ◽  
Reliable Detection ◽  
Resolution Mapping ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

Abstract Methyl-5-uridine (m5U) is one the most abundant non-canonical bases present in cellular RNA, and in yeast is found at position U54 of tRNAs where modification is catalysed by the methyltransferase Trm2. Although the mammalian enzymes that catalyse m5U formation are yet to be identified via experimental evidence, based on sequence homology to Trm2, two candidates currently exist, TRMT2A and TRMT2B. Here we developed a genome-wide single-nucleotide resolution mapping method, Fluorouracil-Induced-Catalytic-Crosslinking-Sequencing (FICC-Seq), in order to identify the relevant enzymatic targets. We demonstrate that TRMT2A is responsible for the majority of m5U present in human RNA, and that it commonly targets U54 of cytosolic tRNAs. By comparison to current methods, we show that FICC-Seq is a particularly robust method for accurate and reliable detection of relevant enzymatic target sites. Our associated finding of extensive irreversible TRMT2A-tRNA crosslinking in vivo following 5-Fluorouracil exposure is also intriguing, as it suggests a tangible mechanism for a previously suspected RNA-dependent route of Fluorouracil-mediated cytotoxicity.

scholarly journals No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5′-OH ends phosphorylated by Trl1

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Albertas Navickas ◽  
Sébastien Chamois ◽  
Rénette Saint-Fort ◽  
Julien Henri ◽  
Claire Torchet ◽  
...  
Keyword(s):  
Single Nucleotide ◽  
Endonucleolytic Cleavage ◽  
Naturally Occurring ◽  
Mrna Surveillance ◽  
Rna Fragments ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

AbstractThe No-Go Decay (NGD) mRNA surveillance pathway degrades mRNAs containing stacks of stalled ribosomes. Although an endoribonuclease has been proposed to initiate cleavages upstream of the stall sequence, the production of two RNA fragments resulting from a unique cleavage has never been demonstrated. Here we use mRNAs expressing a 3′-ribozyme to produce truncated transcripts in vivo to mimic naturally occurring truncated mRNAs known to trigger NGD. This technique allows us to analyse endonucleolytic cleavage events at single-nucleotide resolution starting at the third collided ribosome, which we show to be Hel2-dependent. These cleavages map precisely in the mRNA exit tunnel of the ribosome, 8 nucleotides upstream of the first P-site residue and release 5′-hydroxylated RNA fragments requiring 5′-phosphorylation prior to digestion by the exoribonuclease Xrn1, or alternatively by Dxo1. Finally, we identify the RNA kinase Trl1, alias Rlg1, as an essential player in the degradation of NGD RNAs.

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