Program of the 2004 East Coast Worm Meeting - Caenorhabditis ...

15. Multiple, dynamic microRNA ribonucleoprotein complexes with selective microRNA
cargos in C. elegans
Gopalakrishna Ramaswamy, Eun-Young Choi, Frank J. Slack
Department of Molecular, Cellular and Developmental Biology, Yale University, P.O. Box 208103,
New Haven, CT 06520
Hundreds of small, non-translated regulatory RNAs, known as microRNAs (miRNAs) are
expressed from the genomes of animals and plants. miRNAs as well as small interfering RNAs
(siRNAs) are associated with protein components of the RNA induced silencing complex (RISC),
including Argonaute and VIG proteins, but little is known about miRNA mechanism of action or
miRNA ribonucleoprotein (miRNP) assembly. Here we demonstrate the presence of multiple,
distinct miRNPs in C. elegans extracts. These complexes form in a temporal sequence, which
suggests that a series of smaller intermediates form into a larger, functional miRNP. All of these
complexes require Argonaute-like-2 (ALG-2) to form, and are thus, likely to be related to RISC.
We detected two large complexes (~ 500 kDa and > 669 kDa), which are able to bind both let-7
and lin-4 miRNAs, while two smaller complexes of ~160 kDa and ~250 kDa, show selectivity and
bind let-7, but not lin-4. We identified two proteins, p41 and p14 that bind directly to the let-7
miRNA in all complexes, suggesting that these might be core miRNP factors. p41 is similar in size
to C. elegans VIG-1 and we demonstrate that VIG-1 can bind directly to let-7 miRNA. We
conclude that multiple miRNP complexes exist in C. elegans, and that these may have different
miRNA cargos and roles.