Mutations in conserved residues of the C. elegans microRNA Argonaute ALG-1 identify separable functions in ALG-1 miRISC loading and target repression.
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.
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
Anna Y
AY
Zinovyeva
St-Patrick Research Group in Basic Oncology, Hôtel-Dieu de Québec (Centre Hospitalier Universitaire de Québec), Laval University Cancer Research Centre, Quebec City, Québec, Canada.
Samir
S
Bouasker
St-Patrick Research Group in Basic Oncology, Hôtel-Dieu de Québec (Centre Hospitalier Universitaire de Québec), Laval University Cancer Research Centre, Quebec City, Québec, Canada.
Martin J
MJ
Simard
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America.
Christopher M
CM
Hammell
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
Victor
V
Ambros
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics*; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics*; D017124:Conserved Sequence; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics; D016415:Sequence Alignment
C504319:AIN-1 protein, C elegans; D060565:Argonaute Proteins; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; C452195:LIN-28 protein, C elegans; D035683:MicroRNAs; D012097:Repressor Proteins