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SKI2 mediates degradation of RISC 5’-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

ABSTRACT: SKI2 mediates degradation of RISC 5’-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

PROVIDER: PRJEB10023 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR973023.fastq.gz	Fastqsanger.gz
	ERR973024.fastq.gz	Fastqsanger.gz
	ERR973025.fastq.gz	Fastqsanger.gz
	ERR973026.fastq.gz	Fastqsanger.gz
	ERR973027.fastq.gz	Fastqsanger.gz

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SKI2 mediates degradation of RISC 5â-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

Project description:A loss-of-function mutant in the SKI2 helicase in Arabidopsis was shown to accumulate 5'-cleavage fragments of several miRNA targets. Small RNAs in ski2 single and ski2/rdr6 double mutants (two biological replicates of each genotype) were next analysed by NGS. The data show that several miRNA targets produce RDR6-dependent siRNAs upon inactivation of SKI2. Files containing these NGS data are deposited here. Seeds were surface sterilized and germinated on MS medium for 2 weeks. The seedlings were grown on soil until flowering stage in a 16h light period. The inflorescences were harvested and used to prepare RNA. Small RNA libraries were prepared using the NEBnext Multiplex small RNA library prep kit for Illumina sequencing.

2015-10-20 | E-MTAB-3781 | biostudies-arrayexpress

SKI2 mediates degradation of RISC 5'-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis.

Project description:Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20-24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5', but not 3'-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5' to the cleavage site, but several examples of 3'-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5'-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5'-cleavage fragments.

| S-EPMC4678812 | biostudies-literature

Mechanisms of RNAi: mRNA cleavage fragments may indicate stalled RISC.

Project description:The molecular mechanism of RNA interference (RNAi) is under intense investigation. We previously demonstrated the existence of inactive siRNAs and also of mRNA cleavage in vivo in human cells. Here it is shown that some siRNAs with low activity leave mRNA cleavage fragments while an siRNA with higher activity does not. The pattern is consistent with both short-term (4-24 hours) and long-term (1-4 days) time-series. Analysis of the putative 3' mRNA cleavage product showed high GC content immediately after the cleavage point. The cleavage fragments might indicate a stalled or slowed RNAi cleavage complex - possibly in the RISC enzyme restoration phase - and thus constitute a novel explanation for the existence of inactive siRNAs.

| S-EPMC2737194 | biostudies-literature

LC-MS/MS analyses of MMP cleavage fragments of tyrosyl-tRNA synthetase

Project description:Tyrosyl-tRNA synthetase was digested with MMPs after which cleavage fragments were resolved using SDS-PAGE. Major bands were digested with trypsin and analyzed by LC-MS/MS.

2019-05-30 | PXD013197 | Pride

RISC-interacting clearing 3'- 5' exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana.

Project description:RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments. The 5' cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5' products of AGO cleavage to maintain functional RISC. Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC.

| S-EPMC5451212 | biostudies-literature

Post-transcriptional 3’UTR cleavage of mRNA transcripts generates thousands of stable uncapped autonomous RNA fragments

Project description:Here, using genome wide analysis, we demonstrate that canonical mRNA is processed post-transcriptionally through an alternative cleavage and polyadenylation mechanism. As a result of this process, the downstream cleavage fragment of the 3′UTR remains uncapped and stable This finding indicates that different parts of gene mRNA are separate and independent, by re-annotating the human transcriptome using this model, we provide a new overview of the function and impact of microRNA (miRNA) Our results shed new light on the mammalian transcriptome and show that what were considered as 3′UTRs are in fact autonomous RNA fragments.

2017-11-20 | GSE84068 | GEO

Cleavage of the siRNA passenger strand during RISC assembly in human cells.

Project description:A crucial step in the RNA interference (RNAi) pathway involves the assembly of RISC, the RNA-induced silencing complex. RISC initially recognizes a double-stranded short interfering RNA (siRNA), but only one strand is finally retained in the functional ribonucleoprotein complex. The non-incorporated strand, or 'passenger' strand, is removed during the assembly process and most probably degraded thereafter. In this report, we show that the passenger strand is cleaved during the course of RISC assembly following the same rules established for the siRNA-guided cleavage of a target RNA. Chemical modifications impairing the cleavage of the passenger strand also impair the cleavage of a target RNA in vitro as well as the silencing of a reporter gene in vivo, suggesting that passenger strand removal is facilitated by its cleavage during RISC assembly. Interestingly, target RNA cleavage can be rescued if an otherwise non-cleavable passenger strand shows a nick at the scissile phosphodiester bond, which further indicates that the cleavage event per se is not essential.

| S-EPMC1456892 | biostudies-other

Human ANGIOGENIN is sufficient but not required for tRNA cleavage to generate tRNA fragments

Project description:Human ANGIOGENIN is sufficient but not required for tRNA cleavage to generate tRNA fragments

| PRJNA541296 | ENA

Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

Project description:Small RNAs regulate the genetic networks through a ribonucleoprotein complex called the RNA induced silencing complexes (RISC), which in mammals contains at its center one of four Argonaute proteins (Ago1-4). A key regulatory event in the RNAi and miRNA pathways is Ago loading, where double stranded small RNA duplexes are incorporated into RISC (pre-RISC) and then become single stranded (mature-RISC), a process that is not well understood. The Agos contain an evolutionary conserved PAZ (Piwi/Argonaute/Zwille) domain whose primary function is to bind the 3’-end of small RNAs. We created multiple Paz domain disrupted Ago mutant proteins and studied their biochemical properties and biological functionality in cells. We found that the Paz domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer substrate duplex RNAs, Paz-disrupted Agos bound duplex siRNAs but were unable to unwind/eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved Paz domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA and RNAi-based therapeutics.

2012-07-12 | GSE39146 | GEO

The Integrator complex regulates microRNA abundance through RISC loading

Project description:MicroRNA (miRNA) homeostasis is crucial for the post-transcriptional regulation of their target genes and miRNA dysregulation has been linked to multiple diseases, including cancer. The mechanistic steps of miRNA biogenesis are well understood at molecular level1,2. Subsequent miRNA duplex loading into members of the Argonaute (Ago) protein family, representing the core of the RNA-induced silencing complex (RISC), is pivotal to miRNA-mediated gene silencing3-5. The Integrator complex has been previously identified as important regulator of RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination, processes dependent on Integrator’s catalytical activity6-9. Here we report that absence of the Integrator complex leads to a global loss of mature miRNAs. By incorporating 4-Thiouridine (s4U) in nascent transcripts, we traced miRNA fate from biogenesis to stabilization and identified Integrator to be essential for proper miRNA assembly into RISC. Indeed, Integrator enhances miRNA-Ago2 interaction in vitro, which functionally translates to amplified miRNA target cleavage. These findings demonstrate for the first time cytoplasmic Integrator complex functions and stress an indirect role of Integrator in transcription regulation through modulation of miRNA abundance and stability.

2023-02-14 | GSE178127 | GEO

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