MicroRNAs are small non-coding RNAs that regulate translation of target mRNA. Argonaute (AGO) proteins are the core of the microRNA Induced Silencing Complex (miRISC) that mediates microRNA function on mRNA targets.To characterize protein cofactors associated with miRISC complexes in C. elegans, we isolated Argonaute ALG-1 proteins by immunoaffinity using anti-ALG-1 antisera and performed MUDPIT analysis. A total of 110 proteins were identified as bona-fide interactors of ALG-1, including the known C. elegans ALG-1 interactors, DCR-1 and AIN-1 as well as homologs of other proteins previously shown to interact with Argonaute in other organisms.We reported previously novel antimorphic alleles (anti) of the C. elegans Argonaute gene
alg-1 that broadly impair the functions of many microRNAs, and exhibit an increased association with miRISC loading complex (miRLC) component DCR-1, and a decreased association with the miRISC effector AIN-1 [1]. To determine if interaction of ALG-1 with other proteins were affected by the
alg-1(anti) mutations, and to better understand protein dynamics associated with miRLC-to-miRISC maturation step, we also characterized the ALG-1(anti) associated proteins by MUDPIT analysis. In addition, in order to characterize complexes that may be formed around specific miRNAs, we performed oligonucleotide-mediated pull-down of individual microRNAs from WT and
alg-1(anti) worm extracts followed by MUDPIT analysis.Our results support the idea that
alg-1(anti) mutants are defective in transitioning from biogenesis (miRLC) to effector (miRISC) status. This is as evidenced by increased association, compared to wild type, with DCR-1, certain
hsp-60 and
hsp-70 family proteins and other putative chaperones, and a reduced yield of several known miRISC effector components including AIN-1, and the poly-A binding proteins
pab-1 and
pab-2, which have been shown to play critical roles in target repression. In general, our results revealed a dramatic shift in the overall composition of the ALG-1(anti) associated complexes towards a stalled miRISC assembly configuration.[1] Zinovyeva et al (2014) PLoS Genet 10(4):
e1004286.