Abstract

The C. elegans heterochronic gene pathway orchestrated by regulation of several miRNAs and their targets regulates larval stage-specific development. The let-7 miRNA controls cell cycle exit and terminal differentiation at the larva-to-adult transition. Low expression of let-7 results in abnormal vulva development and attenuated terminal differentiation of hypodermal seam cells in C. elegans, causing vulva bursting and reiterated seam cell division in adult worms, and also has been associated with a variety of cancers in humans. Previously, the scaffolding receptor of activated C kinase (RACK-1) has been proposed to facilitate the recruitment of miRNA-induced silencing complex (miRISC) to the polysome and the let-7 function in C. elegans and humans. However, here we found that RACK-1 knockdown by RNAi suppressed the abnormal vulva and seam cell phenotypes caused by a hypomorphic let-7(n2853) allele or lack of the let-7 paralogs miR-48 and miR-241, suggesting that, opposing to the previous studies, depletion of RACK-1 may enhance let-7 function. Moreover, the incomplete precocious phenotypes caused by hbl-1(mg285), the null allele of one of several important let-7 targets, was enhanced by RACK-1 knockdown. This might be explained by a synthetic effect of the hbl-1(mg285) mutation and enhanced let-7 down-regulation on other targets. In addition, we found that the suppression on let-7 mutants by RACK-1 knockdown is dependent on the Argonaute protein ALG-1, the core of miRISC, suggesting that RACK-1 functions via the miRNA regulation machinery. We further demonstrated that depletion of RACK-1 caused increased levels of mature let-7 that may explain the mutant suppression effect. In RACK-1/Dicer double RNAi experiments, we observed that depletion of RACK-1 reinforced the accumulation of pre-let-7 caused by Dicer knockdown, suggesting that RACK-1 affects a let-7 biogenesis step upstream of the Dicer processing. We examined the role of ADAR, Tudor-SN and KSRP proteins that have been implicated with post-transcriptional modification of pre-miRNAs in the putative RACK-1 function regarding miRNA biogenesis and found that depletion of RACK-1 in an ADR-2 null mutant caused no increase in the pre-let-7 levels. This might lead to the involvement of ADR-2 with RACK-1 function in miRNA biogenesis. The detailed mechanism awaits future elucidation.

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