The nuclear cap-binding complex regulates subcellular RNA processing and surveillance of coding and non-coding RNAs
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ABSTRACT:
PROVIDER: PRJNA1236297 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR32699200_1.fastq.gz | Fastqsanger.gz | |||
| SRR32699200_2.fastq.gz | Fastqsanger.gz | |||
| SRR32699201_1.fastq.gz | Fastqsanger.gz | |||
| SRR32699201_2.fastq.gz | Fastqsanger.gz | |||
| SRR32699202_1.fastq.gz | Fastqsanger.gz |
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Project description:RNA cleavage is essential for processing and regulating all classes of RNA. Current methods to profile genome-wide RNA cleavage are biased towards cytoplasmic events and ignore compartmentalized differences. Here, we couple subcellular RNA fractionation with degradome profiling to detect genome-wide nucleoplasmic and cytoplasmic RNA cleavage in Arabidopsis thaliana. While mRNA cleavage dominated cytoplasmic fractions, we captured a diverse array of nucleoplasm-enriched RNA cleavage events. This included pre-mRNA cleavage and non-coding RNA processing, including microRNAs, ribosomal RNAs, small nucleolar RNAs, enhancer-associated RNAs, and retrotransposon-derived RNAs. Furthermore, we revealed that CAP-BINDING PROTEIN80/ABA HYPERSENSITIVE1 regulates RNA surveillance at the nuclear periphery during the pioneer round of translation. Our data also emphasized its role in stabilizing nucleoplasmic RNAs (e.g. mRNA-associated anti-sense RNAs) and affecting cytoplasmic mRNA cleavage. Overall, our results highlight the diversity of compartmentalized RNA cleavage and reveal that the nuclear cap-binding complex has numerous functions in subcellular RNA processing and surveillance.
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Project description:The nuclear cap-binding complex (CBC) stimulates multiple steps in several RNA maturation pathways, but how it functions in humans is incompletely understood. For small capped RNAs like pre-snRNAs, the CBC recruits PHAX. Here, we identify the CBCAP complex, composed of CBC, Ars2 and PHAX, and show that both CBCAP and CBC-Ars2 complexes can be reconstituted from recombinant proteins. Ars2 stimulates PHAX binding to the CBC as well as snRNAs 3'-end processing, hereby coupling maturation with export. In vivo, CBC and Ars2 bind similar capped non-coding and coding RNAs, and stimulate their 3M-bM-^@M-^Y-end processing. Strongest effects are displayed for cap-proximal polyadenylation sites, thereby favoring premature transcription termination. Ars2 functions in part through the mRNA 3M-bM-^@M-^Y-end cleavage factor CLP1, which binds RNA Polymerase II through PCF11. Ars2 is thus a major CBC effector that stimulates functional and cryptic 3'-end processing sites. In total 12 samples; 4 control IP, 2 Flag- Ars2 IP and 2 Flag-CBP20 IP; 2 control FFL siRNA and 2 siRNA Ars2
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Project description:Recent advances in next generation sequencing have improved human genome annotations and revealed thousands of previosly unknown long non-coding RNA loci. Here, we characterized immune-responsive long non-coding RNAs (lncRNAs) and determined their subcellular localization and co-sedimentation with protein complexes in primary human macrophages. To this end, we profiled LPS-responsive lncRNAs, isolated cytoplasmic and nuclear RNA fractions from mock- and LPS-treated cells and seperated cell lysates on 10-60 % glycerol gradients, followed by gradient fractionation. All samples were subjected to RNA-Seq analysis. LPS-responsive lncRNAs were found to be mostly cytoplasmic. Glycerol gradient datasets revealed that a substantial fraction of LPS-responsive lncRNAs, similar to mRNAs, co-sediments with ribosomal RNAs and also ribosomal proteins, as confirmed by mass-spectrometry analysis. LncRNAs not co-sedimenting with ribosomes displayed a highly heterogenous gradient distriubtion. Among these truly non-coding RNAs we identified lncRNA MaIL1 as a novel element of the macrophage TLR-TRIF signaling pathway contributing to antibacterial defense.
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Project description:The nuclear cap-binding complex (CBC) stimulates multiple steps in several RNA maturation pathways, but how it functions in humans is incompletely understood. For small capped RNAs like pre-snRNAs, the CBC recruits PHAX. Here, we identify the CBCAP complex, composed of CBC, Ars2 and PHAX, and show that both CBCAP and CBC-Ars2 complexes can be reconstituted from recombinant proteins. Ars2 stimulates PHAX binding to the CBC as well as snRNAs 3'-end processing, hereby coupling maturation with export. In vivo, CBC and Ars2 bind similar capped non-coding and coding RNAs, and stimulate their 3’-end processing. Strongest effects are displayed for cap-proximal polyadenylation sites, thereby favoring premature transcription termination. Ars2 functions in part through the mRNA 3’-end cleavage factor CLP1, which binds RNA Polymerase II through PCF11. Ars2 is thus a major CBC effector that stimulates functional and cryptic 3'-end processing sites.
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Project description:Competing exonucleases that promote 3’ end maturation or degradation direct quality control of small non-coding RNAs, but how these enzymes distinguish normal from aberrant RNAs is poorly understood. The Pontocerebellar Hypoplasia 7 (PCH7)-associated 3’ exonuclease TOE1 promotes maturation of canonical small nuclear RNAs (snRNAs). Here, we demonstrate that TOE1 achieves specificity towards canonical snRNAs by recognizing Sm complex assembly and cap trimethylation, two features that distinguish snRNAs undergoing correct biogenesis from other small non-coding RNAs. Indeed, disruption of Sm complex assembly via snRNA mutations or protein depletions obstructs snRNA processing by TOE1, and in vitro snRNA processing by TOE1 is stimulated by a trimethylated cap. An unstable snRNA variant that normally fails to undergo maturation becomes fully processed by TOE1 when its degenerate Sm binding motif is converted into a canonical one. Our findings uncover the molecular basis for how TOE1 distinguishes snRNAs from other small non-coding RNAs and explain how TOE1 promotes maturation specifically of canonical snRNAs undergoing proper processing.
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