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Identifying mRNA Sequence Elements for Target Recognition by Human Argonaute Proteins

ABSTRACT: It is commonly known that mammalian microRNAs guide the RNA-induced silencing complex (RISC) to target mRNAs through the seed-pairing rule. However, recent experiments by co-immunoprecipitating the argonaute proteins (AGOs), the central catalytic component of RISC, have consistently revealed extensive AGO-associated mRNAs lacking seed complementarity with microRNAs. We herein test the hypothesis that AGO has its own binding preference within target mRNAs, independent of guide microRNAs. By systematically analyzing the data from in vivo cross-linking experiments with human AGOs, we have identified a structurally accessible and evolutionarily conserved region (~10 nucleotides) that alone can accurately predict AGO-mRNA associations, independent of the presence of microRNA binding sites. Within this region, we further identified an enriched motif that is also consistently present in several independent AGO-immunoprecipitation datasets. We used RNAcompete to enumerate the RNA-binding preference of human AGO2 to all possible 7-mer RNA sequences, and validated our motif in vitro. These findings reveal a novel function of AGOs as sequence-specific RNA-binding proteins, which may aid microRNAs in recognizing their targets with high specificity.

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE55122 | GEO | 2014/02/19

SECONDARY ACCESSION(S): PRJNA238583

REPOSITORIES: GEO

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Identifying mRNA Sequence Elements for Target Recognition by Human Argonaute Proteins

Project description:It is commonly known that mammalian microRNAs guide the RNA-induced silencing complex (RISC) to target mRNAs through the seed-pairing rule. However, recent experiments by co-immunoprecipitating the argonaute proteins (AGOs), the central catalytic component of RISC, have consistently revealed extensive AGO-associated mRNAs lacking seed complementarity with microRNAs. We herein test the hypothesis that AGO has its own binding preference within target mRNAs, independent of guide microRNAs. By systematically analyzing the data from in vivo cross-linking experiments with human AGOs, we have identified a structurally accessible and evolutionarily conserved region (~10 nucleotides) that alone can accurately predict AGO-mRNA associations, independent of the presence of microRNA binding sites. Within this region, we further identified an enriched motif that is also consistently present in several independent AGO-immunoprecipitation datasets. We used RNAcompete to enumerate the RNA-binding preference of human AGO2 to all possible 7-mer RNA sequences, and validated our motif in vitro. These findings reveal a novel function of AGOs as sequence-specific RNA-binding proteins, which may aid microRNAs in recognizing their targets with high specificity. Here, we analyze the RNA-binding preference of human Argonaute 2 protein using RNAcompete assay

2014-02-19 | E-GEOD-55122 | biostudies-arrayexpress

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. Various Argonautes associated small RNA profiles were generated by deep sequencing the Agos-IP samples in HEK293 Cells transfected with corresponding Argonaute.

2012-07-12 | E-GEOD-39146 | biostudies-arrayexpress

Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

2012-07-12 | GSE39146 | GEO

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential

Project description:It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function. This series includes microRNA sequencing data for human cell lines 293, C8166, A549, SH-SY5Y, and an EBV-transformed LCL line. For each cell line, total small RNA isolated by TRIzol was sequenced for comparison to RISC-associated microRNAs as determined by sequencing small RNAs from an Argonaute immunoprecipitation.

2014-05-30 | E-GEOD-58127 | biostudies-arrayexpress

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential

2014-05-30 | GSE58127 | GEO

Identification of microRNA-668 and microRNA-489 targets by RISC immunoprecipitation

Project description:MicroRNAs inhibits their target mRNA translation by forming RISC complex. Immunoprecipitation of RISC will pull down both microRNA and their target mRNAs. To systemetically examine the potential targets of microRNA-668 and microRNA-489, HEK293 cells were overexpressed with negative control, microRNA-668, or microRNA-489 mimics. Both total RNA samples and RNA samples from RISC complex pulled down by Ago-2 immunoprecipitation were examined by RNA-seq.

2019-07-27 | GSE117805 | GEO

The Vasa homolog RDE-12 engages target mRNA and a secondary-Argonaute WAGO-1 to promote RNAi in C. elegans

Project description:Argonaute proteins (AGOs) are key nuclease effectors of RNA interference (RNAi) [1]. Although purified AGOs can mediate a single round of target-RNA cleavage in vitro, accessory factors are required for siRNA loading and to achieve multiple-target turnover [2, 3]. To identify AGO co-factors we immunoprecipitated the C. elegans AGO WAGO-1, which engages amplified small RNAs during RNAi [4]. These studies identified a robust association between WAGO-1 and a conserved Vasa ATPase-related protein RDE-12. rde-12 mutants are deficient in RNAi including viral suppression, and fail to produce amplified secondary siRNAs and certain endogenous siRNAs (endo-siRNAs). RDE-12 co-localizes with WAGO-1 in germline P-granules and to peri-nuclear cytoplasmic foci in somatic cells. These findings and our genetic studies suggest that (i) RDE-12 is first recruited to target mRNAs by upstream AGOs (RDE-1 and ERGO-1) where it promotes small-RNA amplification and/or WAGO-1 loading, and that (ii) downstream of these events, RDE-12 forms an RNase-resistant (target mRNA-independent) complex with WAGO-1 that may scan for additional target mRNAs. Examine small RNA population changes in rde-12 mutants

2014-06-24 | E-GEOD-54396 | biostudies-arrayexpress

Deciphering the cell-specific role of Arabidopsis Argonaute 3 [smallRNA-Seq]

Project description:ARGONAUTE (AGO) proteins are the major effectors of RNA silencing. In Arabidopsis, loading of 21-22-nt long small RNAs into AGOs results in post-transcriptional gene silencing (PTGS) by mRNA cleavage and/or translational repression. On the other hand, loading of 24-nt sRNAs results in transcriptional gene silencing (TGS) by RNA directed DNA methylation (RdDM). The Arabidopsis genome encodes 10 AGOs, which are known, for most, to belong to either pathway. Here we characterized the cell specific role of Arabidopsis AGO3. It is specifically expressed in tissue close to vascular termination and strongly accumulates in chalazal seed integuments. AGO3 encodes a functional AGO able to bind sRNAs with a preference for 24nt in length with a 5’ nucleotide bias for Adenosine. Down-regulation of AGO3 affects gene expression in siliques and its expression is strongly induced in the plant vasculature upon proteasome inhibition. Our results suggest that AGO3 might act to regulate gene expression by a novel RNA silencing pathway involving 24nt sRNA-directed PTGS.

2021-08-30 | GSE74722 | GEO

Deciphering the cell-specific role of Arabidopsis Argonaute 3 [RNA-seq]

2021-08-30 | GSE74721 | GEO

Analysis of RNA associated with the RISC complex (Ago2) in four day old mice carrying a mutation in miR-96

Project description:The diminuendo mouse carries a mutation in the seed region of miR-96. Homozygotes are deaf and exhibit vestibular dysfunction, heterozygotes display rapidly progressive hearing loss. Some targets of miR-96 are known, but it is likely that many still remain to be discovered. MicroRNAs function by recruiting the RISC complex to their mRNA targets, so one way to examine the mRNAs being controlled by microRNAs in a cell is to immunoprecipitate Ago2, one of the RISC proteins, and extract the RNA bound to it. This should detect all the mRNAs being targetted by microRNAs in the tissue. We extracted mRNA bound to Ago2 from the organs of Corti and olfactory bulbs of diminuendo homozygote and wildtype littermates at four days old, and carried out microarray analysis.

2016-02-26 | E-MTAB-3737 | biostudies-arrayexpress

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