Hsp90 Regulates the Function of Argonaute 2 and Its Recruitment to Stress Granules and P-Bodies

Abstract

Argonaute proteins are effectors of RNA interference that function in the context of cytoplasmic ribonucleoprotein complexes to regulate gene expression. Processing bodies (PBs) and stress granules (SGs) are the two main types of ribonucleoprotein complexes with which Argonautes are associated. Targeting of Argonautes to these structures seems to be regulated by different factors. In the present study, we show that heat-shock protein (Hsp) 90 activity is required for efficient targeting of hAgo2 to PBs and SGs. Furthermore, pharmacological inhibition of Hsp90 was associated with reduced microRNA-and short interfering RNA-dependent gene silencing. Neither Dicer nor its cofactor TAR RNA binding protein (TRBP) associates with PBs or SGs, but interestingly, protein activator of the double-stranded RNAactivated protein kinase (PACT), another Dicer cofactor, is recruited to SGs. Formation of PBs and recruitment of hAgo2 to SGs were not dependent upon PACT (or TRBP) expression. Together, our data suggest that Hsp90 is a critical modulator of Argonaute function. Moreover, we propose that Ago2 and PACT form a complex that functions at the level of SGs. INTRODUCTIONThe Argonaute superfamily comprises a group of RNAbinding proteins that form the cores of ribonucleoprotein complexes (RNPs) that mediate RNA interference (RNAi) and related gene-silencing pathways (reviewed in Hannon, 2002;Peters and Meister, 2007;Hutvagner and Simard, 2008). All metazoans encode multiple Argonaute genes, the products of which have unique as well as overlapping functions. The basic functions of Argonautes in the canonical RNAi pathway are reasonably well understood. Small guide RNAs direct Argonaute-containing RNPs to homologous mRNAs, thereby providing specificity in this pathway (reviewed in Hannon, 2002;Jaronczyk et al., 2005). The Argonaute superfamily has been divided into two subgroups, Argonaute and Piwi (Bohmert et al., 1998;Cox et al., 1998;Carmell et al., 2002). Expression of the Piwi subfamily members is restricted to germline tissues and undifferentiated cells, whereas the Argonaute group members are ubiquitous. The two main types of small RNAs that associate with Argonaute subfamily members differ in origin and function. Short interfering RNAs (siRNAs) and microRNAs (miRNAs) are derived from long double-stranded (ds)RNA and hairpin RNA precursors, respectively. Successive processing of miRNA precursors in the nucleus and cytoplasm, respectively, by the RNases Drosha (Lee et al., 2003) and Dicer (Bernstein et al., 2001) produces mature miRNAs that are then transferred to an Argonaute-containing RNP complex termed RNA induced silencing complex (RISC) (Hammond et al., 2000). The biogenesis of siRNAs only requires Dicer activity. A third species of small noncoding RNAs (Piwiinteracting RNAs) that associate with Piwi subfamily members was recently discovered (Aravin et al., 2006;Girard et al., 2006). However, their expression is limited to germline tissues in which they are thought to function in gametogenesis and retrotransposon silen...

The Hsp90 Inhibitor Geldanamycin Abrogates Colocalization of eIF4E and eIF4E-Transporter into Stress Granules and Association of eIF4E with eIF4G

“…Furthermore, it should be emphasized that the Hsp90 inhibitor GA serves as a useful tool to analyze the differences between the mechanisms of assembly of stress granules and P-bodies. 7 During the preparation of this paper, Pare et al (51) reported that GA treatment of HeLa cells resulted in impairment of the association of Ago2 with stress granules (but did not affect their formation) and the biogenesis and/or stability of P-bodies.…”

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

“…Loading of the sncRNA duplex onto the AGO proteins is well described for AGO-2 and involves the heat-shock protein 90 (HSP90) (Figure 1). HSP90 aids in the recruitment [78] and stabilization [79] of unloaded AGO within processing bodies (P-bodies).…”

Multiple sensors ensure guide strand selection in human RNAi pathways

“…Loading of these RNAs is enhanced by the Hsc70/Hsp90 chaperone machinery, and in Drosophila requires Dicer/dsRBP heterodimers (Liu et al 2003;Pare et al 2009;Iki et al 2010;Iwasaki et al 2010;Johnston et al 2010;Miyoshi et al 2010). Upon loading, Ago2 cleaves and/or removes one strand of the duplex (the passenger strand), driving unwinding with its N-terminal domain and the aid of an endoribonuclease protein complex called C3PO (Matranga et al 2005;Rand et al 2005;Leuschner et al 2006;Kim et al 2007;Liu et al 5 2009; Tian et al 2011;Ye et al 2011;Kwak and Tomari 2012).…”