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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

ABSTRACT: 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

PROVIDER: PRJEB15579 | ENA |

REPOSITORIES: ENA

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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

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

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Exonuclease Requirements for Mammalian Ribosomal RNA Biogenesis and Surveillance

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Nascent strand with BrdU or lambda exonuclease

Project description:Nascent strand were purified with either the BrdU or the lambda exonuclease methods from culture human basophilic erythroblasts and sequenced/ Two methods were compared to isolated the Nascent strand, lambda exonuclease digestion or immunoprecipitation after pulse labelling with BrdU The following files were generated by combining round 1 and 2 sample files; FNY01_2_2_Ery_NS_BrdU_macs_summits.bed FNY01_2_2_Ery_NS_BrdU_macs_peaks.bed FNY01_2_2_Ery_NS_lexo_macs_peaks.bed FNY01_2_2_Ery_NS_lexo_macs_summits.bed

2014-05-01 | E-GEOD-50976 | biostudies-arrayexpress

Architectural complexity of the polyadenylated intestinal transcriptome revealed by hybrid RNA sequencing

Project description:We report the complexity of the polyadenylated transcriptome of the mouse intestinal tract assessed by a hybrid RNA-sequencing. We show that 90.72% of the polyA-derived unique reads map to known genes, but 9.28% of reads matched to nonannotated genomic regions.

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Next generation sequencing identifies polyadenylated species of hTR

Project description:The paired-end next-generation sequencing of all hTR versions of less than 200 nucleotides in length was used to analyze the 3’ end distribution of hTR associated to Flag-tagged versions of PABPN1, hTERT and Dyskerin. In total, we obtained 2,716,342, 3,152,013, and 3,077,395 hTR-specific reads for the PABPN1, hTERT, and Dyskerin purifications, respectively. We found that the majority of polyadenylated telomerase RNA recovered in the PABPN1, hTERT, and Dyskerin purifications had poly(A) tails starting immediately after the annotated hTR 3’ end. However, a greater proportion of poly(A) tails were found on genome-encoded 3’-extentions in the PABPN1-bound fraction compared to the population of hTR that was copurified with hTERT and DKC1: 15.5% for PABPN1, 6.9% for hTERT, and 6.5% for Dyskerin. Notably, the population of polyadenylated telomerase RNA recovered with PABPN1 was significantly different in terms of poly(A) tail length compared to polyadenylated hTR retrieved in the hTERT- and Dyskerin-bound fractions (P-value=5.551e-16, Kolmogorov-Smirnov test), showing a tendency toward longer poly(A) tails in the PABPN1-bound fraction. The enrichment of telomerase RNA with long poly(A) tails in the PABPN1-bound fraction is consistent with a role of PABPN1 in a maturation pathway that depends on hTR 3’ end polyadenylation. Moreover, our results indicate that a fraction of hTERT and Dyskerin are associated with polyadenylated versions of hTR.

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Effects of transcription elongation rate and Xrn2 exonuclease activity on RNA polymerase II termination suggest widespread kinetic competition

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An extended PAXT connection is required for turnover of nuclear polyadenylated RNA in human cells

Project description:Recruitment of the human ribonucleolytic RNA exosome to nuclear polyadenylated (pA+) RNA is facilitated by the Poly(A) Tail eXosome Targeting (PAXT) connection. Besides its core dimer, formed by the exosome co-factor MTR4 and the ZFC3H1 protein, the PAXT connection remains poorly defined. By characterizing nuclear pA+-RNA bound proteomes as well as MTR4-ZFC3H1 containing complexes in conditions favoring PAXT assembly, we here uncover three additional proteins required for PAXT function: ZC3H3, RBM26/RBM27 and the known PAXT-associated protein, PABPN1. The zinc-finger protein ZC3H3 interacts directly with MTR4-ZFC3H1 and loss of either identified PAXT component results in the accumulation of PAXT substrates. Collectively, our results establish new factors involved in the turnover of nuclear pA+ RNA and suggest that these are limiting for PAXT activity.

2019-10-31 | PXD016070 | JPOST Repository

Integrator restraints paraspeckles assembly by promoting the 3’-end processing of the polyadenylated lncRNA NEAT1_1

Project description:Alternative 3’-end RNA processing provides an important source of transcriptome diversification, which impacts various biological processes and the etiology of diseases, including cancer. Here, we focused on a transcript isoform switch that leads to the readthrough expression of the long noncoding RNA NEAT1_2, at the expense of the shorter polyadenylated transcript NEAT1_1. Expression of NEAT1_2 is required for the formation of paraspeckles (PS), nuclear bodies that protect cancer cells from oncogene-induced replication stress and chemotherapy. Searching for proteins that interact with endogenous NEAT1 by RNA Affinity Purification coupled to mass spectrometry (RAP-MS), we identified factors involved in the 3’-end processing of polyadenylated (pA+) RNA as well as several components of the Integrator complex, known to process the 3’-ends of RNAs lacking polyadenylation sites. Unexpectedly, genetic perturbation experiments established that Integrator restraints NEAT1_2 expression, and thereby PS assembly, by promoting the 3’-end processing of pA+ NEAT1_1. Consistently, low expression levels of several Integrator subunits correlated with poorer prognosis of cancer patients exposed to chemotherapeutics. Our study identifies Integrator as a key regulator of PS biogenesis and highlights a previously unrecognized ability of the complex to process pA+ RNA. The data also establish a link between Integrator, cancer biology and chemosensitivity, which may be exploited therapeutically.

2020-10-14 | PXD015158 | Pride

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