Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 216

Abstract
Originally discovered in C. elegans, microRNAs (miRNAs) are small RNAs that
regulate fundamental cellular processes in diverse organisms. MiRNAs are encoded
within the genome and are initially transcribed as primary transcripts that can be
several kilobases in length. Primary transcripts are successively cleaved by two
RNase III enzymes, Drosha in the nucleus and Dicer in the cytoplasm, to produce
70 nucleotide (nt) long precursor miRNAs and 22 nt long mature miRNAs,
respectively. Mature miRNAs regulate gene expression post-transcriptionally by
imperfectly binding target mRNAs in association with the multiprotein RNA
induced silencing complex (RISC). The conserved sequence, expression pattern,
and function of some miRNAs across distinct species as well as the importance of
specific miRNAs in many biological pathways have led to an explosion in the study
of miRNA biogenesis, miRNA target identification, and miRNA target regulation.
Many advances in our understanding of miRNA biology have come from studies in
the powerful model organism C. elegans. This chapter reviews the current methods
used in C. elegans to study miRNA biogenesis, small RNA populations, miRNA-
protein complexes, and miRNA target regulation.
I. Introduction
microRNAs (miRNAs) play a major role in regulating many important processes
including cellular differentiation and proliferation, developmental timing, hemato-
poiesis, immune responses, apoptosis, and nervous system patterning ( Fineberg
et al., 2009; Gangaraju and Lin, 2009; Latronico and Condorelli, 2009; Negrini
et al., 2009; O ' Connell et al., 2010; Subramanian and Steer, 2010 ). Consequently,
alterations in miRNA levels, timing of expression, or target recognition can have
devastating consequences including cancer ( Kai and Pasquinelli, 2010; Medina and
Slack, 2008 ).
Most miRNAs are transcribed by RNA polymerase II as independent transcripts or
as RNAs embedded within introns of protein-coding messenger RNAs ( Davis and
Hata, 2009 ). These miRNA transcripts, called primary miRNAs, are capped, poly-
adenylated, and can be several thousand nucleotides (nt) long ( Davis and Hata,
2009 ). Primary miRNAs are successively cleaved into 70 nt hairpin precursor
miRNAs and then to 22 nt mature miRNAs by two RNase III enzymes respectively
called Drosha and Dicer (DRSH-1 and DCR-1 in C. elegans)( Davis and Hata, 2009 ).
Mature miRNAs guide the argonaute protein ALG-1/2 to a specific target mRNA
( Grishok et al., 2001; Okamura et al., 2004 ). In animals, the mature miRNA is
imperfectly complementary to the target site and though most miRNA-ALG-1/2
complexes bind to the 3 0 UTR, target binding can also occur in the 5 0 UTR, exons, and
introns ( Davis and Hata, 2009; Zisoulis et al., 2010 ). ALG-1/2 associates with the
RISC to regulate gene expression by inhibiting translation or triggering degradation
of the target mRNA ( Chekulaeva and Filipowicz, 2009 ).