The biogenesis and function of small RNAs in C. elegans

RNAi is the process by which double-stranded RNA (dsRNA) induces sequence-specific mRNA degradation. DsRNA is diced into small interfering RNAs (siRNAs) of ~21-23 nt by a complex containing the RNaseIII enzyme DICER. The mature siRNAs are subsequently bound by Argonaute proteins and incorporated into an RNA-induced silencing complex (RISC), which cleaves homologous mRNAs. This process is involved in viral resistance and transposon silencing. Here we describe the identification of two proteins involved in the siRNA pathway; RRF-2 and RDE-2. The first is an RNA-directed RNA polymerase, the latter is a Caenorhabditae-specific protein, binding to MUT-7. Both RRF-2 and RDE-2 are required for an efficient RNAi response in C. elegans. A process closely related to the RNAi pathway is the microRNA (miRNA) pathway. MiRNAs are transcribed from the genome as long transcripts that can snap-back on themselves (primary miRNA or pri-miRNA) and are processed into ~70 nt stem-loop structures (precursor miRNA or pre-miRNA) by Drosha in the nucleus (Lee et al. 2003). The pre-miRNA serves as a template for Dicer which processes the pre-miRNA into the double-stranded miRNA of ~21 nt. These miRNAs are, like siRNAs, bound by Argonaute proteins and can bind to mRNAs (usually to the 3' UTR), but unlike siRNA that degrade mRNAs, miRNAs primarily inhibit translation. MiRNAs play a role in developmental timing, stress responses and fine-tuning gene-regulation. Here we describe the identification of Pasha (partner of Drosha) in Drosophila and C. elegans. Pasha is an essential protein, that associates with Drosha in the nucleus and is required for processing of pri-miRNAs into pre-miRNAs. We also discuss the role of the two Argonaute proteins, ALG-1 and ALG-2, that function in the miRNA pathway in C. elegans and present data supporting specialized roles for these Argonautes in this pathway. We show that a cluster of miRNAs, mir-35-41, regulates sex-determination in C. elegans. These miRNAs re-enforce decisions at several levels in the sex-determination pathway, that result in hermaphrodite-specific development. One of the targets of mir-35-41 is the fem-2 gene. Finally we discuss the pros and cons of the methods currently used to predict/verify miRNA targets, illustrated by a C. elegans example.