Identification of Novel Argonaute-Associated Proteins

Abstract

RNA silencing processes are guided by small RNAs known as siRNAs and microRNAs (miRNAs) . They reside in ribonucleoprotein complexes, which guide the cleavage of complementary mRNAs or affect stability and translation of partial complementary mRNAs . Argonaute (Ago) proteins are at the heart of silencing effector complexes and bind the single-stranded siRNA and miRNA . Our biochemical analysis revealed that Ago2 is present in a pre-miRNA processing complex that is able to transfer the miRNA into a target-mRNA cleaving complex. To gain insight into the function and composition of RNA silencing complexes, we purified Ago1- and Ago2-containing complexes from human cells. Several known Ago1- and/or Ago2-associated proteins including Dicer were identified, but also two novel factors, the putative RNA helicase MOV10, and the RNA recognition motif (RRM)-containing protein TNRC6B/KIAA1093. The new proteins localize, similar to Ago proteins, to mRNA-degrading cytoplasmic P bodies, and they are functionally required to mediate miRNA-guided mRNA cleavage.

NMDAR ‐dependent Argonaute 2 phosphorylation regulates mi RNA activity and dendritic spine plasticity

“…Co‐immunoprecipitation of endogenous GW182 with endogenous Ago2 from cultured cortical neurons was significantly increased 10 min after bath application of NMDA (Fig 1A). We also analysed the association of Ago2 with the RNA helicase DDX6, which associates with Ago2 via GW182, and MOV10, which is recruited to RISC by an unknown mechanism (Meister et al , 2005; Chen et al , 2014). While Ago2‐DDX6 interactions were significantly increased by NMDAR stimulation, binding to MOV10 was unaffected (Fig 1A).…”

Principles and effects of microRNA-mediated post-transcriptional gene regulation

“…The functions of these co-purified factors are largely unknown yet. In human cells Gemin 3 and 4 have been identified in complex together with hAgo2 (Mourelatos et al, 2002;Meister et al, 2005). Both hAgo1 and hAgo2 co-purify with the helicase Mov10, the fly orthologue of which is required for RISC assembly, Tncr6 Bisoform1 and Prmt5 proteins with unknown function (Tomari et al, 2004a;Meister et al, 2005).…”

“…There is an increasing number of reports that Argonautes associates with complexes that contain Dicer, the key enzyme in siRNA and miRNA maturation (Tabara et al, 2002;Sasaki et al, 2003;Okamura et al, 2004;Gregory et al, 2005;Maniataki and Mourelatos, 2005;Meister et al, 2005;Duchaine et al, 2006). The co-association of RISC and the miRNA processing is not surprising.…”

“…The miRNA specific fly Dicer-1 requires Loquacious, for proper miRNA processing, Dicer in worms coimmunoprecipitates with RDE-4, a dsRNA-binding protein essential for RNAi initiated with long dsRNA, and human Dicer requires two R2D2 orthologues, TRBP and PACT, for miRNA-mediated gene silencing (Tabara et al, 2002;Forstemann et al, 2005;Haase et al, 2005;Jiang et al, 2005;Saito et al, 2005;Lee et al, 2006). Furthermore, Argonautes seem to interact physically with the miRNA maturation and loading machinery as immunoprecipitated hAgo2 complexes contain functional Dicer activity that can asymmetrically process exogenous pre-miRNAs, generating mature RISC that can cleave a perfect complementary target RNA Maniataki and Mourelatos, 2005;Meister et al, 2005). Together, the data suggest that RISC formation is a highly dynamic process that requires the coordinated interaction of complexes involved in distinct steps of siRNA and miRNA maturation (Figure 1).…”

“…Human Argonaute proteins and C. elegans ALG-1, a worm Argonaute protein required for let-7-directed gene regulation, colocalize, co-purify and interact with proteins also located in PBs (Ding et al, 2005;Jakymiw et al, 2005;Sen and Blau, 2005;Liu et al, 2005a, b;Meister et al, 2005). The localization of Argonaute proteins to PBs requires that the protein bind small RNAs; the target mRNA is transported to the PB only if it contains miRNA-binding sites, suggesting that miRNAs bound to Argonaute proteins direct the mRNA to the PBs (Agbottah et al, 2005;Pillai et al, 2005;Liu et al, 2005a, b).…”