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Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE39146 | GEO | 2012/07/12

SECONDARY ACCESSION(S): PRJNA170090

REPOSITORIES: GEO

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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

Distinct Functions of Argonaute Slicer in siRNA Maturation and Heterochromatin Formation

Project description:Small-RNA (sRNA)-guided transcriptional gene silencing by Argonaute (Ago)-containing complexes is fundamental to genome integrity and epigenetic inheritance. The RNA cleavage (“Slicer”) activity of Argonaute has been implicated in both sRNA maturation and target RNA cleavage. Typically, Argonaute slices and releases the passenger strand of duplex sRNA to generate active silencing complexes, but it remains unclear whether slicing of target nascent RNAs, or other RNAi components, also contributes to downstream transcriptional silencing. Here, we develop a strategy for loading the fission yeast Ago1 with a single-stranded sRNA guide, which bypasses the requirement for slicer activity in generation of active silencing complexes. We show that slicer-defective Ago1 can mediate secondary sRNA generation, H3K9 methylation, and silencing similar to or better than wild-type and associates with chromatin more efficiently. The results define an ancient and minimal sRNA-mediated chromatin silencing mechanism, which resembles the germline-specific sRNA-dependent transcriptional silencing pathways in Drosophila and mammals. This SuperSeries is composed of the SubSeries listed below.

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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

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Homo sapiens

Project description:Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

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