Identification of specific let-7 microRNA binding complexes in Caenorhabditis elegans

Abstract

Little is known about the protein complexes required for microRNA formation and function. Here we used native gel electrophoresis to identify miRNA ribonucleoprotein complexes (miRNPs) in Caenorhabditis elegans. Our data reveal multiple distinct miRNPs that assemble on the let-7 miRNA in vitro. The formation of these complexes is affected but not abolished by alg-1 or alg-2 null mutations. The largest complex (M*) with an estimated molecular mass of >669 kDa cofractionates with the known RISC factors ALG-1, VIG-1, and TSN-1. The M* complex and two complexes, M3 and M4, with similar molecular weights of ;500 kDa, also assemble on all other miRNAs used in our experiments. Two smaller complexes, M1 (;160 kDa) and M2 (;250 kDa), assemble on the members of the let-7 miRNAs family but not lin-4 or mir-234, and their formation is highly dependent on specific sequences in the 59 seed region of let-7. Moreover, an unidentified protein, p40, which only appears in the M1 and M2 complexes, was detected by UV triggered cross-linking to let-7 but not to lin-4. The cross-linking of p40 to let-7 is also dependent on the let-7 sequence. Another unidentified protein, p13, is detected in all let-7 binding complexes and lin-4 cross-linked products. Our data suggest that besides being present in certain large miRNPs with sizes similar to reported RISC, the let-7 miRNA also assembles with specific binding proteins and forms distinct small complexes.

Functional relevance of “seed” and “non-seed” sequences in microRNA-mediated promotion of C. elegans developmental progression

“…The variety of observations specific to C. elegans let-7 in this work and those of Vella et al (2004a,b) indicate that this may be a particularly complex microRNA in the diversity of its interactions and functions. Consistent with this possibility are studies in which specialized RNA (Cevec et al 2010) and RNP complexes (Chan et al 2008) have been observed in association with this microRNA. let-7 is a highly conserved microRNA, with substantial (and somewhat consistent) diversity in both seed and non-seed regions.…”

Analysis of microRNA Expression and Function

“…Gel filtration has been used to fractionate cell extracts from C. elegans , Drosophila, and human cells to determine the sizes of complexes corresponding to RNAi activity, siRNAs, miRNAs, or miRNP/RISC (Caudy et al , 2002, 2003; Chan et al , 2008; Gregory et al , 2005; Pham et al , 2004). Gel filtration can also be used to detect cofractionation of proteins of interest with miRNAs or to purify fractions that possess miRNA-related activities, that is miRNA binding or pre-miRNA processing.…”

Cell shape and Wnt signaling redundantly control the division axis ofC. elegansepithelial stem cells

“…Although ALG-1 probably acts with miRNAs to control the division axis, it remains unclear which miRNAs are involved. Recent studies identified large numbers of high-confidence miRNA-mRNA interactions, which are associated with various biological processes (Chan et al, 2008;Zhang et al, 2007;Zisoulis et al, 2010). Genetic confirmation of such interactions is hampered by redundancies between miRNAs and, in general, by the modest inhibition of target genes by individual miRNAs (Baek et al, 2008;Inui et al, 2010).…”