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Argonaute proteins interact with small RNAs to mediate gene silencing. C.
elegans contains 27 Argonaute homologs, raising the question of what roles these genes play in RNAi and related gene-silencing pathways. Through our collaborator, Dr. Shohei Mitani, we have obtained a set of 30 deletion alleles representing all of the previously uncharacterized Argonaute genes.
Analysis of single- and multiple-Argonaute mutant strains reveals essential functions in several pathways including: (i) chromosome segregation, (ii) fertility, and (iii) at least two separate steps in the RNAi pathway. We show that RDE-1 interacts with trigger-derived sense and antisense RNAs to initiate RNAi, while several other Argonaute proteins interact with amplified antisense siRNAs to mediate downstream silencing. Overexpression of downstream Argonautes enhances silencing, suggesting that these proteins are limiting for RNAi. These downstream Argonautes also function in
endogenous RNAi (endo-RNAi) pathways of unknown function. A distinct
Argonaute, ERGO-1, appears to function in a manner analogous to RDE-1 at an upstream step in the endo-RNAi pathway. The ERGO-1 and RDE-1 mediated pathways appear to compete for the downstream secondary Argonautes which lack key residues required for mRNA cleavage. Thus our findings support a two-step model for RNAi, in which Argonaute proteins function sequentially, and downstream silencing is mediated by a set of Argonautes unlikely to harbor catalytic-slicer activity.
Nobel Prize recipient Craig C. Mello, PhD, is the Blais University Chair in Molecular Medicine at the University of Massachusetts Medical School (UMMS). He was also designated an Investigator of the Howard Hughes Medical Institute in 2000, the third HHMI researcher selected at UMMS. HHMI is a $13 billion medical research organization that employs more than 350 eminent researchers at 72 medical schools, universities and research institutes worldwide. Dr. Mello holds his BS in biochemistry from Brown University and his PhD in Cellular and Developmental Biology from Harvard University. He was a postdoctoral fellow at the Fred Hutchinson Cancer Research Center before coming to UMMS in 1995. He is also a 1995 Pew Scholar in the Biomedical Sciences. Mello and his colleague Andrew Fire, PhD, formerly of the Carnegie Institution of Washington, received the 2006 Nobel Prize in Medicine for their discovery of RNA interference (RNAi). They demonstrated that a certain form of RNA had the unanticipated property of silencingor interfering withthe expression of a gene whose coding sequence of DNA was similar to that of the RNA they tested. The RNAi mechanisma natural response of an organism to double-stranded RNA, of which many viruses are compriseddestroys the gene products that a virus needs to replicate itself, essentially halting the progression of the invading viral infection.
The discovery, which offers astounding potential for understanding and manipulating the cellular basis of human disease, has had two extraordinary impacts on biological science. One is as a research tool: RNAi is now the state-of-the-art method by which scientists can knock down the expression of specific genes in cells, to thus define the biological functions of those genes. But just as important has been the finding that RNA interference is a normal process of genetic regulation that takes place during development.
Thus, RNAi has provided not only a powerful research tool for experimentally knocking out the expression of specific genes, but has opened a completely new and totally unanticipated window on developmental gene regulation.