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Role of XRN2 ribonucleolytic activity in RNA metabolism

ABSTRACT: We analysed the effect of depriving the human cell of the catalytic activity of the nuclear 5’ to 3’ exoribonuclease XRN2. Catalytic amino acids in this protein had been defined previously, so it was possible to design a mutated catalytically inactive form of the protein (XRN2D233A-D235A) (PMID: 19194460). We created 293 Flp-In T-REx stable cell lines that induciby silence endogenous XRN2, and concomitantly express wild-type or inactive XRN2 in fusion with EGFP at the C-terminus. Thus, complementation of silencing of endogenous XRN2 with the expression of mutant version of the protein allows to directly link potential phenotypes with the lack of XRN2 enzymatic activity. To this end we isolated total RNA from tetracycline-treated cells, depleted it from rRNA and conducted strand-specific deep sequencing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE99421 | GEO | 2018/04/17

REPOSITORIES: GEO

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XRN2 Autoregulation and Control of Polycistronic Gene Expression in Caenorhabditis elegans

Project description:XRN2 is a conserved 5â??-->3â?? exoribonuclease that complexes with XTB-domain containing proteins. Thus, in Caenorhabditis elegans (C. elegans), the XTBD-protein PAXT-1 stabilizes XRN2 to retain its activity. XRN2 activity is also promoted by 3'(2'),5'-bisphosphate nucleotidase 1 (BPNT1) through its hydrolysis of 3â??-phosphoadenosine-5'-bisphosphate (PAP), an endogenous XRN inhibitor. Here, we find through unbiased screening that loss of bpnt-1 function suppresses lethality caused by paxt-1 deletion. This unexpected finding is explained by XRN2 autoregulation, which occurs through repression of a cryptic promoter activity and destabilization of the xrn-2 transcript. Autoregulation appears to be triggered at different thresholds of XRN2 inactivation, such that more robust XRN2 perturbation, by elimination of both PAXT-1 and BPNT1, is less detrimental to worm viability than absence of PAXT-1 alone. Like more than 15% of C. elegans genes, xrn-2 occurs in an operon, and we identify additional operons under its control, consistent with a broader function of XRN2 in polycistronic gene regulation. Regulation occurs through intercistronic regions that link genes in an operon, but similar mechanisms may allow XRN2 to operate on monocistronic genes in organisms lacking operons. Wild-type C. elegans worms were subjected to mock or xrn-2 RNAi from L1 to L4 stage at 20Â°C. Total RNA was extracted from the worms, and polyadenylated RNA was analyzed.

2016-08-25 | E-GEOD-79994 | biostudies-arrayexpress

XRN2 Autoregulation and Control of Polycistronic Gene Expression in Caenorhabditis elegans

Project description:XRN2 is a conserved 5’-->3’ exoribonuclease that complexes with XTB-domain containing proteins. Thus, in Caenorhabditis elegans (C. elegans), the XTBD-protein PAXT-1 stabilizes XRN2 to retain its activity. XRN2 activity is also promoted by 3'(2'),5'-bisphosphate nucleotidase 1 (BPNT1) through its hydrolysis of 3’-phosphoadenosine-5'-bisphosphate (PAP), an endogenous XRN inhibitor. Here, we find through unbiased screening that loss of bpnt-1 function suppresses lethality caused by paxt-1 deletion. This unexpected finding is explained by XRN2 autoregulation, which occurs through repression of a cryptic promoter activity and destabilization of the xrn-2 transcript. Autoregulation appears to be triggered at different thresholds of XRN2 inactivation, such that more robust XRN2 perturbation, by elimination of both PAXT-1 and BPNT1, is less detrimental to worm viability than absence of PAXT-1 alone. Like more than 15% of C. elegans genes, xrn-2 occurs in an operon, and we identify additional operons under its control, consistent with a broader function of XRN2 in polycistronic gene regulation. Regulation occurs through intercistronic regions that link genes in an operon, but similar mechanisms may allow XRN2 to operate on monocistronic genes in organisms lacking operons.

2016-08-25 | GSE79994 | GEO

XRN2

Project description:XRN2

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A NOVEL EPIGENETIC SILENCING PATHWAY INVOLVING the HIGHLY CONSERVED 5'-3' EXORIBONUCLEASE Dhp1/Rat1/Xrn2 in SCHIZOSACCHAROMYCES POMBE [CRAC-seq]

Project description:Epigenetic gene silencing plays a critical role in regulating gene expression and contributes to organismal development and cell fate acquisition in eukaryotes. In fission yeast, Schizosaccharomyces pombe, heterochromatin-associated gene silencing is known to be mediated by RNA processing pathways including RNA interference (RNAi) and a 3’-5’ exoribonuclease complex exosome. Here, we report a new RNA-processing pathway that contributes to epigenetic gene silencing and assembly of heterochromatin mediated by 5’-3’ exoribonuclease Dhp1/Xrn2. Dhp1 mutation causes defective gene silencing both at peri-centromeric regions and at the silent mating type locus. Intriguingly, mutation of either one of two well-characterized Dhp1-interacting proteins, the Din1 pyrophosphohydrolase or the Rhn1 transcription termination factor, does not show silencing defects at the main heterochromatic regions. Dhp1 is essential in the sequential steps of establishing silencing in a manner independent of both RNAi and the exosome. Genomic and genetic analysis suggest that Dhp1 is involved in post-transcriptional silencing of repetitive regions through its catalytic activity. Our study is the first investigation into an unexpected role of Dhp1/Rat1/Xrn2 in chromatin-based silencing. These results elucidate how various RNA-processing pathways, acting together or independently, contribute to epigenetic regulation of the eukaryotic genome.

2016-01-28 | GSE77290 | GEO

A NOVEL EPIGENETIC SILENCING PATHWAY INVOLVING the HIGHLY CONSERVED 5'-3' EXORIBONUCLEASE Dhp1/Rat1/Xrn2 in SCHIZOSACCHAROMYCES POMBE [gene expression]

2016-01-28 | GSE77289 | GEO

Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity

Project description:Termination is a ubiquitous phase in every transcription cycle but is incompletely understood and a subject of debate. We have used gene editing as a new approach to address its mechanism through engineered conditional depletion of the 5’-3’ exonuclease, Xrn2, or the polyadenylation signal (PAS) endonuclease, CPSF73. The ability to rapidly control Xrn2 reveals a clear and general role for it in co-transcriptional degradation of 3’ flanking region RNA and transcriptional termination. This defect is characterised genome-wide, at high resolution, using native elongating transcript sequencing (mNET-seq). An Xrn2 effect on termination requires prior RNA cleavage and we provide evidence for this by showing that catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73. Notably, Xrn2 plays no significant role in either Histone or snRNA gene termination even though both RNA classes undergo 3’ end cleavage. In sum, efficient termination on most protein-coding genes involves CPSF73 mediated RNA cleavage and co-transcriptional degradation of polymerase-associated RNA by Xrn2. However, as CPSF73 loss caused more extensive read-through transcription than Xrn2 elimination, it likely plays a more underpinning role in termination.

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XRN2

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XRN2 Suppresses Aberrant Entry of tRNA Trailers into Argonaute in Humans and Arabidopsis

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2023-04-24 | GSE184124 | GEO

PP1-NIPP1 wild type and mutant overexpression in Flip-In™ T-Rex Hela cells

Project description:RNAi-seq of stable Flp-In™ T-Rex Hela cells that inducibly expressed PP1-NIPP1 and mutants.

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2018-04-06 | PXD008731 | Pride

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