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Defective XRN3-mediated transcription termination in Arabidopsis affects the expression of protein-coding genes [array]

ABSTRACT: Arabidopsis thaliana contains two nuclear XRN2/3 5'-3' exonucleases that are homologs of yeast and human Rat1/Xrn2 proteins involved in the processing and degradation of several classes of nuclear RNAs and in transcription termination of RNA polymerase II. Using strand-specific short read sequencing we show that knockdown of XRN3 leads to an altered expression of hundreds of genes and the accumulation of uncapped and polyadenylated read-through transcripts generated by inefficiently terminated Pol II. Our data support the notion that XRN3-mediated changes in the expression of a subset of genes are caused by upstream read-through transcription and these effects are enhanced by RNA-mRNA chimeras generated in xrn3 plants. In turn, read-through transcripts that are antisense to downstream genes may trigger production of siRNA. Our results highlight the importance of XRN3 exoribonuclease in Pol II transcription termination in plants and show that disturbance in this process may significantly alter gene expression.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE95396 | GEO | 2018/01/24

REPOSITORIES: GEO

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Defective XRN3-mediated transcription termination in Arabidopsis affects the expression of protein-coding genes [seq]

Project description:Arabidopsis thaliana contains two nuclear XRN2/3 5'-3' exonucleases that are homologs of yeast and human Rat1/Xrn2 proteins involved in the processing and degradation of several classes of nuclear RNAs and in transcription termination of RNA polymerase II. Using strand-specific short read sequencing we show that knockdown of XRN3 leads to an altered expression of hundreds of genes and the accumulation of uncapped and polyadenylated read-through transcripts generated by inefficiently terminated Pol II. Our data support the notion that XRN3-mediated changes in the expression of a subset of genes are caused by upstream read-through transcription and these effects are enhanced by RNA-mRNA chimeras generated in xrn3 plants. In turn, read-through transcripts that are antisense to downstream genes may trigger production of siRNA. Our results highlight the importance of XRN3 exoribonuclease in Pol II transcription termination in plants and show that disturbance in this process may significantly alter gene expression.

2018-01-24 | GSE95473 | GEO

Transcription profiling by array of a stably RNAi-silenced nuclear 5’-3’ exonuclease XRN3 mutant line in Arabidopsis thaliana after transcriptional inhibition with cordycepin

Project description:In Arabidopsis thaliana two nuclear 5'-3' exonucleases, XRN2 & 3, are involved in the degradation and processing of several classes of nuclear RNAs and in transcription termination of RNA polymerase II. The role of XRN3 in Pol II transcription termination is investigated by combining two different types of RNA-seq data for Col-0 background WT and xrn3-8 mutant A. thaliana seedlings. The data for the experiment comprises single molecule Direct RNA Sequencing of unamplified RNA (3x WT, 4x xrn3-8 mutant) and high-depth Illumina strand-specific short read sequencing of PCR amplified ribosomal-RNA depleted RNA (Illumina TruSeq stranded ribozero Plant kit, 3x WT, 3x xrm3-8 mutant). In the xrn3 mutant, these data reveal a widespread accumulation of non-coding intergenic transcripts (xrn3-associated transcripts - XATs) generated by Pol II read-through transcription that are usually polyadenylated and lack the 5' cap structure. This data highlights the important role of exoribonucleases in the torpedo mechanism of Pol II transcription termination and show that a global disturbance in this process significantly impacts both gene expression and transcriptome integrity.

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Direct RNA Sequencing of polyadenylated RNA & high-depth Illumina strand-specific, short read RNA-seq of a stably RNAi-silenced nuclear 5'-3' exonuclease XRN3 mutant line in Arabidopsis thaliana

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Genome-wide analysis of RNA polymerase II termination at protein-coding genes

Project description:At the end of protein-coding genes, RNA polymerase (Pol) II undergoes a concerted transition that involves 3’-processing of the pre-mRNA and transcription termination. Here we conduct a genome-wide analysis of this 3’-transition in yeast. We find that the 3’-transition globally requires the Pol II elongation factor Spt5 and factors involved in the recognition of the poly-adenylation (pA) site and in endonucleolytic RNA cleavage. Pol II release from DNA occurs in a narrow termination window downstream of the pA site and generally requires the ‘torpedo’ exonuclease Rat1 (human XRN2). The Rat1-interacting factor Rai1 contributes to RNA degradation downstream of the pA site. Defects in the 3’-transition result in transcription interference at downstream genes, which is attenuated by a back-up termination mechanism.

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DNA-directed termination of RNA Polymerase II transcription II

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DNA-directed termination of RNA Polymerase II transcription I

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DSIF factor Spt5 coordinates transcription, maturation and exoribonucleolysis of RNA polymerase II transcripts

Project description:The timely termination of RNA polymerase II (Pol II) transcription is essential for recycling of this enzyme and preventing transcriptional interference. While 5'-3' exonuclease Xrn2 is known to degrade nascent RNA after poly(A) site cleavage to promote Pol II release, its molecular mechanism remains not fully understood. Spt5 is one of highly conserved transcription factors that controls and regulates multiple aspects of Pol II transcription. Here, using mass spectrometry analysis of immunoprecipitated Flag-tagged Spt5-WT and Spt5-mutant complexes from fission yeast cells, we show that Xrn2 is highly enriched in these samples. Through in vitro BS3 crosslinking experiments with purified components, we further demonstrated that Xrn2 associates with transcription machinery and we probed interfaces between different subunits of transcription machinery

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Project description:Transcription termination determines the ends of transcriptional units and thereby ensures integrity of gene regulation. Here we perform genome-wide investigation of RNA polymerase II (Pol II) transcription termination in Caenorhabditis elegans and observe two distinct modes of termination. Whereas a subset of genes requires the exoribonuclease XRN2, a known termination factor, termination of other genes appears independent of, and refractory to, XRN2. Unexpectedly, promoters instruct the choice of termination mode, but XRN2-independent termination additionally requires a compatible region downstream of the 3’ end cleavage site. Hence, different termination mechanisms may affect different configurations of Pol II complexes dictated by promoters.

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