miRISC recruits decapping factors to miRNA targets to enhance their degradation

Tadashi Nishihara 0 Latifa Zekri 0 Joerg E. Braun 0 Elisa Izaurralde 0 0 Department of Biochemistry, Max Planck Institute for Developmental Biology , Spemannstrasse 35, 72076 Tu bingen, Germany MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 50-to-30 messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 50 cap structure by decapping triggers irreversible decay of the mRNA body. Here, we demonstrate that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation. - MicroRNAs (miRNAs) are a large family of endogenous non-coding RNAs that post-transcriptionally silence the expression of messenger RNA (mRNA) targets containing complementary sequences and are implicated in nearly all developmental and cellular processes that have been investigated thus far (1). To exert their regulatory functions, miRNAs associate with Argonaute (AGO) proteins in effector complexes known as miRNA-induced silencing complexes (miRISCs). These complexes induce endonucleolytic cleavage of fully complementary targets or translational repression, mRNA deadenylation and 50-to-30 exonucleolytic decay of targets with partially complementary binding sites (13). Silencing of mRNA targets containing partially complementary miRNA-binding sites requires the association of AGOs with a protein of the GW182 family, which mediates the translational repression and degradation of these targets (2,4). The mechanism of translational repression has yet to be elucidated, although increasing evidence points to an inhibition of translation initiation (3). In contrast, the mechanism of miRNA target degradation is relatively well understood. It is known that miRNAs accelerate target degradation through the 50-to-30 mRNA decay pathway (2). In this pathway, mRNAs are first deadenylated, then decapped and finally degraded by the major cytoplasmic 50-to-30 exonuclease XRN1 (5,6). mRNA deadenylation is catalyzed by the sequential action of two cytoplasmic deadenylase complexes (the PAN2-PAN3 and the CCR4-NOT complexes) (6). These complexes are recruited to miRNA targets through interactions with GW182 proteins (79). Depending on the cell type and/or specific target involved, the deadenylated mRNA target can be stored in a translationally repressed state, as observed, for example, in Caenorhabditis elegans embryos (10). However, in diverse organisms and cell types, deadenylated miRNA targets are rapidly decapped and degraded by XRN1 (1119). Decapping is catalyzed by the decapping enzyme DCP2, which requires additional co-factors for full activity/ stability (5). These include DCP1, HPat, EDC4 and the DEAD-box protein Me31B (also known as DDX6 or RCK/p54). A role for decapping activators in miRNAmediated mRNA destabilization is supported by the observation that the abundance of predicted and validated miRNA targets increases when decapping activators are depleted or when dominant-negative forms of decapping factors are overexpressed (1220). A question that remains open is whether decapping of miRNA targets occurs exclusively as a consequence of deadenylation or whether miRISCs can also recruit components of the decapping machinery independently of ongoing deadenylation. Evidence for the existence of a specific interaction between decapping factors and miRISC stems from the following observations. First, AGO proteins co-immunoprecipitate with the catalytic subunit of the decapping complex, DCP2 and other decapping factors including DCP1, RCK and EDC4 (also known as Ge-1 or Hedls) in human cells (2024). Second, GW182 co-immunoprecipitates with HPat in Drosophila melanogaster (Dm) Schneider cells (Dm S2 cells) (25). Third, EDC4 was identified as a suppressor of miRNA-mediated gene silencing in Dm cells and in Arabidopsis thaliana (15,26), and it co-localizes with miRNA targets in human cells (23). Fourth, RCK associates with HIV-1 mRNA in the presence of miR-29a (27). Finally, miRNAs and their targets localize to P-bodies wherein decapping factors, AGOs and GW182 proteins accumulate (21,22,2830). However, it is unknown whether the interactions between decapping factors and miRISC components are direct and at which step of miRNA-mediated repression decapping activators are recruited to the mRNA target. In this study, we demonstrate that miRISCs promote the association of DCP1, HPat and Me31B (the D. melanogaster RCK ortholog) with miRNA targets. This association was recapitulated on RNA polymerase III (Pol III)-transcribed targets lacking a 50 cap structure, an open reading frame (ORF) and a poly(A) tail, suggesting that decapping factors are recruited by miRISC onto the target mRNA independently of ongoing deadenylation and decapping. We further show that miRNA targets lacking a poly(A) tail are degraded through decapping. Together with previous studies (13), our results indicate that miRISCs accelerate the irreversible degradation of miRNA targets by promoting decapping independently of their effects on deadenylation. MATERIALS AND METHODS Luciferase reporters and plasmids for the expression of miRNAs and epitope-tagged proteins in D. melanogaster cells have been described elsewhere (17,3135). Plasmids encoding the Alu and hammerhead ribozyme (HhR) reporters are described in the Supplementary Figure S1. To generate plasmids expressing V5-MBP and V5-DCP1, the corresponding cDNA sequences were amplified by PCR using pAc5.1B- N-HA-MBP and pAc5.1B- N-HADCP1 as templates (34) and cloned between the KpnI and XbaI sites of vector pAc5.1B (Invitrogen). To obtain plasmids for the expression of HA-glutathione S-transferase (GST)-tagged proteins, the corresponding cDNAs were cloned into pAc5.1B- N-HA-GST, and the region encoding the N peptide was deleted (31). Antibodies and western blotting The protein co-immunoprecipitations shown in Supplementary Figure S5 were performed as described previously (19). Polyclonal anti-eIF4E and PABPC1 antibodies were generated by immunizing rabbits with purified recombinant Dm eIF4E (full-length) and poly(A)-binding protein 1 (PABP) (amino acids 501 634). For western blotting, these antibodies were used at the following dilutions: eIF4E (1:3000) and PABPC1 (1:10 000). HA-tagged proteins were imm (...truncated)