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 approximately 500 kDa, also assemble on all other miRNAs used in our experiments. Two smaller complexes, M1 ( approximately 160 kDa) and M2 ( approximately 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 5'' 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.