Functional analysis of the microRNA genes of C. elegans

Abstract

MicroRNAs (miRNAs) were discovered in C. elegans during studies of the control of developmental timing. MicroRNAs are a large class of short non-coding RNAs found in many viruses, plants and animals that regulate gene expression through sequence-specific base-pairing with target mRNAs. Initial studies since the identification of many miRNAs only six years ago, have revealed their very diverse roles in biology. Yet, few miRNAs have been studied using loss-of-function mutations. We have generated deletion mutations in 87 miRNA genes in C. elegans, and performed an initial characterization of the 95 miRNA mutants available (86% of known C. elegans miRNAs). We found that the majority of miRNAs are not essential for the viability or development of C. elegans, and mutations in most miRNA genes do not result in grossly abnormal phenotypes. Within species, many miRNAs can be grouped into families according to their sequence similarities. We generated a collection of 12 multiply mutant C. elegans strains that each lacks an entire miRNA family. We found that at least four families display synthetic abnormalities, indicating that miRNAs within a family can have redundant functions. While single mutants are superficially wild-type, mutants deleted for all members of the mir-35 or the mir-51 families show embryonic or early larval lethality, mutants deleted for all members of the mir-58 family show an egglaying defect, and mutants deleted for some members of the let-7 family show defects in developmental timing. We developed a microarray technology suitable for detecting microRNAs and used this microarray to determine the profile of microRNAs expressed in the developing mouse brain. We observed a temporal wave of expression of microRNAs, suggesting that microRNAs play important roles in the development of the mammalian brain.

(cont.) We also performed a systematic expression analysis of 334 samples covering diverse human cancers, using a bead-based flow cytometric miRNA expression profiling method we developed. The miRNA profiles reflect the developmental lineage and differentiation state of the tumors, and reveal a general down-regulation of miRNAs in tumors compared to normal tissues.