A MC motif in silkworm Argonaute 1 is indispensible for translation repression

Abstract

Small RNA-mediated gene silencing is a fundamental gene regulatory mechanism, which is conserved in many organisms. Argonaute (Ago) family proteins in the RNA-induced silencing complex (RISC) play crucial roles in RNA interference (RNAi) pathways. In the silkworm Bombyx mori, four Ago proteins have been identified, named as Ago1, Ago2, Ago3 and Siwi. Ago2 participates in double-stranded RNA (dsRNA)-induced RNAi, whereas Ago3 and Siwi are involved in the Piwi-interacting RNA (piRNA) pathway. However, there is no experimental evidence concerning silkworm Ago1 (BmAgo1) in the RNAi mechanism. In the present study, we analysed the function of BmAgo1 in the microRNA (miRNA)-mediated RNAi pathway using tethering and miRNA sensor reporter assays. These results clearly demonstrate that BmAgo1 plays an indispensable role in translation repression in silkworm. Moreover, coimmunoprecipitation data indicated that BmAgo1 interacts with BmDcp2, an orthologue of mRNA-decapping enzyme 2 (Dcp2) protein in the Drosophila processing-bodies (P-bodies). Substitutions of two conserved phenylalanines (F522 and F557) by valines in the MC motif strongly impaired the function of BmAgo1 in translation repression and its localization in P-bodies, suggesting that these two amino acid residues in the MC motif of BmAgo1 are prerequisites for mRNA translation repression in B. mori.

Genome wide screening of RNAi factors of Sf21 cells reveal several novel pathway associated proteins

“…Taking in view the finding of a recent study, we performed amino acid alignment of Sf-Ago-1 that identifies MC motif in the MID domain with two conserved phenylalanine residues at F522 and F557 like Bm-Ago-1. Such correlation establishes the fact that besides translation repression, Sf-Ago-1 being homologous to Bm-Ago-1 might influence P-body localization [ 114 ].…”

Loqs depends on R2D2 to localize in D2 body-like granules and functions in RNAi pathways in silkworm cells

“…We We found silkworm Loqs represses translation in the tethering assay (Fig. 1E) 569 and interacts with Ago1, the key protein in miRNA pathway (Zhu et al, 2013b), in 570 BiFC assay (Fig. 2F) and I2H assay (Fig.…”

“…3C). Consistently, Drosophila Loqs is Loqs is defective in translation repression in the Ago1-depleted cells, due to the 576 indispensability of Ago1 in miRNA pathway (Zhu et al, 2013b). Silkworm R2D2 577 protein shows important roles in determining Loqs in miRNA pathway, although 578 tethering assay indicate that R2D2 does not participate in silkworm miRNA pathway 579 (Fig.…”

“…123 The pENTR plasmids (pENTR-R2D2, pENTR-Dicer2, pENTR-Loqs, and mutated Table. S1. The dsRNAs for the RNAi components 154 were synthesized as described before (Zhu et subjected to RNA extraction for reverse transcription polymerase chain reaction (RT-165 PCR) as described before (Zhu et al, 2012) Osaka, Japan). The details were described in our previous report (Zhu et al, 2013b).…”

Enhancement of Larval RNAi Efficiency by Over-expressing Argonaute2 in Bombyx mori

“…Four Argonaute proteins, BmAgo1, BmAgo2, BmAgo3 and BmPIWI, have been identified in B. mori 36 . BmAgo1 and BmAgo2 are included in the Ago subfamily, which are responsible for siRNA- and miRNA-mediated post-transcriptional silencing, respectively 37 . However, BmAgo3 and BmPIWI are described as the PIWI subfamily members and express in the silkworm gonads significantly 38 .…”