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MiRNAs associated with the different human Argonaute proteins

ABSTRACT: microRNAs (miRNAs) are small non-coding RNAs that function in literally all cellular processes. miRNAs interact with Argonaute (Ago) proteins and guide them to specific target sites located in the 3’ untranslated region (UTR) of target mRNAs leading to translational repression and deadenylation-induced mRNA degradation. Most miRNAs are processed from hairpin-structured precursors by the consecutive action of the RNase III enzymes Drosha and Dicer. However, processing of miR-451 is Dicer-independent and cleavage is mediated by the endonuclease Ago2. Here we have characterized miR-451 sequence and structure requirements for processing as well as sorting of miRNAs into different Ago proteins. Pre-miR-451 appears to be optimized for Ago2 cleavage and changes result in reduced processing. In addition, we show that the mature miR-451 only associates with Ago2 suggesting that mature miRNAs are not exchanged between different members of the Ago protein family. Based on cloning and deep sequencing of endogenous miRNAs associated with Ago1-3, we do not find evidence for miRNA sorting in human cells. However, Ago identity appears to influence the length of some miRNAs, while others remain unaffected. Examination of miRNAs associated with endogenous human Ago1-4 in HeLa cells

ORGANISM(S): Homo sapiens

SUBMITTER: Anne Dueck

PROVIDER: E-GEOD-45506 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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miRNAs associated with the different human Argonaute proteins

Project description:microRNAs (miRNAs) are small non-coding RNAs that function in literally all cellular processes. miRNAs interact with Argonaute (Ago) proteins and guide them to specific target sites located in the 3’ untranslated region (UTR) of target mRNAs leading to translational repression and deadenylation-induced mRNA degradation. Most miRNAs are processed from hairpin-structured precursors by the consecutive action of the RNase III enzymes Drosha and Dicer. However, processing of miR-451 is Dicer-independent and cleavage is mediated by the endonuclease Ago2. Here we have characterized miR-451 sequence and structure requirements for processing as well as sorting of miRNAs into different Ago proteins. Pre-miR-451 appears to be optimized for Ago2 cleavage and changes result in reduced processing. In addition, we show that the mature miR-451 only associates with Ago2 suggesting that mature miRNAs are not exchanged between different members of the Ago protein family. Based on cloning and deep sequencing of endogenous miRNAs associated with Ago1-3, we do not find evidence for miRNA sorting in human cells. However, Ago identity appears to influence the length of some miRNAs, while others remain unaffected.

2013-03-29 | GSE45506 | GEO

PARN mediates 3'-end trimming of Argonaute-cleaved precursor microRNAs

Project description:microRNAs (miRNAs) are typically generated as ~22-nucleotide double-stranded RNAs via processing of precursor hairpins by the RNase III enzyme Dicer, after which they are loaded into Argonaute (Ago) proteins to form RNA-induced silencing complex (RISC). However, the biogenesis of miR-451, an erythropoietic miRNA conserved in vertebrates, does not require Dicer processing. Instead, the short pre-miR-451 precursor hairpin is directly loaded into Ago, followed by cleavage of the 3' arm and trimming of the 3' end to the mature length by PARN. Here we show the in vivo activity of miR-430 Ago2-hairpin, a canonical microRNA engineered to fit the structure of miR-451 and hence become Ago2-dependent. Moreover, we test a modified miR-430 Ago2-haipin with 3x phoshorothioate bonds that impairs trimmng. Surprisingly, our data show that trimming of Ago-cleaved pre-miRNAs is not essential for target silencing, indicating that RISC is functional with miRNAs longer than 22-nucleotides. Rescue of MZdicer zebrafish mutant with the injection of trimmable and nontrimmable miR-430 Ago2 hairpins: Transcriptome of wild type, MZdicer mutant, and MZdicer mutant micoinjected with miR-430 duplex, miR-430 (Ago2-haripin), miR-430 (Ago2-haripin 3xPhosphorothioate)

2013-11-13 | E-GEOD-51036 | biostudies-arrayexpress

PARN mediates 3'-end trimming of Argonaute-cleaved precursor microRNAs

2013-11-13 | GSE51036 | GEO

A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity

Project description:Here we identify a Dicer-independent miRNA biogenesis pathway that employs the slicer catalytic activity of Argonaute2 (Ago2). To uncover Dicer-independent miRNAs, we sequenced small RNAs in wild type, maternal-zygotic dicer (MZdicer) and MZago2 mutants, using zebrafish as a model system. We find that, in contrast to other miRNAs, miR-451 levels were increased in MZdicer but drastically reduced in the MZago2 mutants. We show that pre-miR-451 processing requires Ago2 catalytic activity in vivo. MZago2 mutant embryos display delayed erythrocyte maturation that can be rescued by wild type Ago2 or miR-451 duplex but not catalytically dead Ago2. We propose that Ago2-mediated cleavage of a subset of pre-miRNAs, followed by uridylation and trimming, generates functional miRNAs in a Dicer-independent manner.

2010-07-01 | GSE21503 | GEO

A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity

2010-07-01 | E-GEOD-21503 | biostudies-arrayexpress

MCMV infection of cultured mouse cells induces expression of miR-7a.

Project description:One goal of viral infection is to reprogram the host cell to optimize viral replication. As part of this process, viral miRNAs may compete for components of the miRNA/siRNA pathway as well as regulate cellular targets. Mouse Cytomegalovirus has been described to generate large numbers of viral miRNAs during lytic infection and was therefore used to analyze the impact of viral miRNAs on the host cell small RNA system as well as to check for sorting of viral small RNAs into specific Ago-proteins. Deep sequencing analysis of MCMV infected cells revealed that viral miRNAs represent only app. 13% of all detected miRNAs. All previously described MCMV miRNAs with the exception of miR-m88-1* were confirmed and for the MCMV miR-m01-1 hairpin an additional miRNA, designated miR-m01-1-3p, was found. Its presence was confirmed by qPCR and Northern Blot. Deep sequencing after RISC IP with antibodies specific for either Ago1 or Ago2 showed that all MCMV miRNAs are loaded into both RISC complexes. The ratio of MCMV to mouse miRNAs was not increased after immunoprecipitation of Ago-proteins. Viral miRNAs therefore do not overwhelm the host miRNA processing system nor are they preferentially incorporated into RISC. We found that 3 mouse miRNAs showed an altered expression due to MCMV infection. Down-regulation of miR-27a, as previously described, could be confirmed. In addition, miR-26a was down-regulated and an up-regulation of miR-7a dependent on viral protein expression could be observed. Examination of small RNA expression in uninfected vs. infected cells, immunoprecipitation + sequencing of Ago1 and Ago2 loaded small RNAs in infected cells

2012-06-30 | E-GEOD-36919 | biostudies-arrayexpress

Argonaute proteins couple chromatin silencing to alternative splicing (exon array)

Project description:While Argonaute (AGO) proteins play a major role in transcriptional gene silencing (TGS) in many organisms, their role in the nucleus of somatic mammalian cells remains elusive. Here, we have purified AGO1 and AGO2 chromatin-embedded complexes, and found these proteins associated with previously described partners, but also with chromatin modifiers and, rather unexpectedly, with different splicing factors. Using the CD44 gene as a model for alternative splicing, we show that both AGO1 and AGO2 are required for Protein Kinase C (PKC)-dependent variant exon inclusion. AGO proteins facilitate the spliceosome recruitment and modulate the elongation rate of RNA polymerase II (RNAPII). The recruitment of AGO proteins to CD44 transcribed region is dependent on both the endonuclease Dicer and the chromodomain-containing protein HP1g, and results in locally increased levels of histone H3 lysine 9 (H3K9) methylation on variant exons. Genome wide analysis of splicing in either AGO2 or Dicer null cells showed that the two proteins have similar effects on many splicing events. Finally, sRNAs associated with nuclear AGO2 are mostly in sense orientation relative to protein-coding genes, supporting a role for intragenic antisense non-coding RNAs in the recruitment AGO and splicing factors. Together, our data demonstrate for the first time that the endogenous RNAi pathway is involved in alternative splicing decisions, unravelling a new model in which AGO proteins couple RNAPII elongation and chromatin modification. Study of AGO2 or Dicer knock-out on gene expression and splicing regulation in MEF cells Transcriptome analysis of AGO2 and Dicer null MEF cells on GeneChipM-BM-. Mouse Exon 1.0 ST Arrays (Affymetrix). Dicer null MEF cells and wild-type MEF cells were from M. Otsuka. AGO2 null MEF cells were from A. Tarakhovsky. Experiment has been done in experimental triplicates. 9 Total samples were analyzed.

2012-09-10 | E-GEOD-39746 | biostudies-arrayexpress

ChIP-Seq of human endogenous small RNAs associated to Argonaute 1 and 2

Project description:Small non-coding RNAs function in concert with Argonaute (Ago) proteins to regulate gene expression at the level of transcription, mRNA stability or translation. Ago proteins bind small RNAs and form the core of silencing complexes. Here we report the analysis of small RNAs associated with human Ago1 and Ago2 revealed by immunoprecipitation and deep sequencing. Among the reads we find small RNAs originating from the small nucleolar RNA (snoRNA) ACA45. Moreover, processing of ACA45 requires Dicer activity but is independent of Drosha/DGCR8. Using bio-informatic prediction algorithms and luciferase reporter assays, we uncover the mediator subunit CDC2L6 as one potential mRNA target of ACA45 small RNAs suggesting a role for ACA45 processing products in post-transcriptional gene silencing. We further identify a number of human snoRNAs with microRNA (miRNA)- like processing signatures. We have therefore identified a novel class of small RNAs in human cells that originate from snoRNAs and can function like miRNAs. Two samples examined. Small RNAs associated to human Argonaute 1 and human Argonaute 2

2008-11-20 | E-GEOD-13370 | biostudies-arrayexpress

Sites for Argonaute Binding within Human Nuclear RNA and their Impact on Alternative Splicing

Project description:Mammalian RNA interference (RNAi) is often linked to regulation in the cytoplasm. While synthetic RNAs regulate transcription and splicing, endogenous functions for nuclear RNAi have been obscure. Using enhanced crosslinking immunoprecipitation (eCLIP), we mapped AGO2 binding sites within nuclear RNA. The strongest AGO binding sites mapped to miRNAs. The prevalence of individual miRNAs was similar in the cytoplasm and the nucleus. Most statistically-significant AGO2 binding was within intron boundaries. RNAseq comparing AGO1, AGO2, AGO1/2, and AGO1/2/3 knockout to wild-type cells revealed that binding was associated with both up- and down-regulation of gene expression. We observed changes of splicing at genes with significant AGO binding at exon/intron junctions or within introns. Splicing changes were confirmed by RT-PCR and could be recapitulated by synthetic duplex RNAs that target the sites of AGO binding. Inhibition of miRNA action by addition of an anti-miR reversed miRNA-mediated control of splicing. We conclude: 1) That AGO binding sites occur throughout nuclear RNA but are often within introns; 2) The distribution of miRNAs is similar in the nucleus and cytoplasm; and 3) miRNAs can control gene splicing. Nuclear RNAi has the potential to be a natural regulatory mechanism but careful study will be necessary to identify critical RNA drivers of normal physiology and disease.

2021-02-10 | GSE161559 | GEO

Argonaute proteins couple chromatin silencing to alternative splicing (RNA IP-Seq)

Project description:While Argonaute (AGO) proteins play a major role in transcriptional gene silencing (TGS) in many organisms, their role in the nucleus of somatic mammalian cells remains elusive. Here, we have purified AGO1 and AGO2 chromatin-embedded complexes, and found these proteins associated with previously described partners, but also with chromatin modifiers and, rather unexpectedly, with different splicing factors. Using the CD44 gene as a model for alternative splicing, we show that both AGO1 and AGO2 are required for Protein Kinase C (PKC)-dependent variant exon inclusion. AGO proteins facilitate the spliceosome recruitment and modulate the elongation rate of RNA polymerase II (RNAPII). The recruitment of AGO proteins to CD44 transcribed region is dependent on both the endonuclease Dicer and the chromodomain-containing protein HP1g, and results in locally increased levels of histone H3 lysine 9 (H3K9) methylation on variant exons. Genome wide analysis of splicing in either AGO2 or Dicer null cells showed that the two proteins have similar effects on many splicing events. Finally, sRNAs associated with nuclear AGO2 are mostly in sense orientation relative to protein-coding genes, supporting a role for intragenic antisense non-coding RNAs in the recruitment AGO and splicing factors. Together, our data demonstrate for the first time that the endogenous RNAi pathway is involved in alternative splicing decisions, unravelling a new model in which AGO proteins couple RNAPII elongation and chromatin modification. Deep sequencing of small RNAs (approx. 15-80 nucleotides) bound to either cytoplasmic or chromatin-associated AGO2 complex.

2012-09-10 | E-GEOD-39748 | biostudies-arrayexpress

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