Mutations in conserved residues of the C. elegans microRNA Argonaute ALG-1 identify separable functions in ALG-1 miRISC loading and target repression.

Anna Y Zinovyeva,Victor Ambros,Samir Bouasker,Martin J Simard,Christopher M Hammell,Xuemei Chen

doi:10.1371/journal.pgen.1004286

Abstract

microRNAs function in diverse developmental and physiological processes by regulating target gene expression at the post-transcriptional level. ALG-1 is one of two Caenorhabditis elegans Argonautes (ALG-1 and ALG-2) that together are essential for microRNA biogenesis and function. Here, we report the identification of novel antimorphic (anti) alleles of ALG-1 as suppressors of lin-28(lf) precocious developmental phenotypes. The alg-1(anti) mutations broadly impair the function of many microRNAs and cause dosage-dependent phenotypes that are more severe than the complete loss of ALG-1. ALG-1(anti) mutant proteins are competent for promoting Dicer cleavage of microRNA precursors and for associating with and stabilizing microRNAs. However, our results suggest that ALG-1(anti) proteins may sequester microRNAs in immature and functionally deficient microRNA Induced Silencing Complexes (miRISCs), and hence compete with ALG-2 for access to functional microRNAs. Immunoprecipitation experiments show that ALG-1(anti) proteins display an increased association with Dicer and a decreased association with AIN-1/GW182. These findings suggest that alg-1(anti) mutations impair the ability of ALG-1 miRISC to execute a transition from Dicer-associated microRNA processing to AIN-1/GW182 associated effector function, and indicate an active role for ALG/Argonaute in mediating this transition.

Highlights

Development of complex organisms requires execution of molecular and cellular events with precise temporal control
We propose that these mutant ALG-1 proteins assemble nonfunctional complexes that effectively compete with the paralogous ALG-2 for critical microRNA Induced Silencing Complexes (miRISCs) components, including mature microRNAs
To identify factors that function in conjunction with let-7-Family microRNAs, we performed a forward genetic screen for suppressors of lin-28(lf) phenotypes. lin-28(lf) animals are 100% egg laying defective, owing to an abnormal vulval morphology that results from their precocious cell lineage defects [18,34]

Summary

Introduction

Development of complex organisms requires execution of molecular and cellular events with precise temporal control. In C. elegans, the heterochronic gene network controls the stage-specific execution of larval developmental events. C. elegans heterochronic mutants display either precocious or retarded development, due to skipping or reiteration of certain stage-specific cell fate programs. The let7-Family microRNAs, let-7, mir-241, mir-48, and mir-84, are key players in the heterochronic genetic network that controls progression through the C. elegans larval development [1,2,3]. Deletion of the let-7 microRNA or its sisters miR-48, miR-241 and miR-84 results in reiteration of larval stage-specific cell fates, delaying the adoption of adult cell fates [1,2,3]

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Conclusion