Guide RNAs:

Hammond et al. determined that the endogenous genes of Drosophila S2 cells could be targeted in a sequence- specific manner by transfection with dsRNA, and loss-of-function phenotypes were created in cultured Drosophila cells. The inability of cellular extracts treated with a Ca2+-dependent nuclease (micrococcal nuclease, which can degrade both DNA and RNA) to degrade the cognate mRNAs and the absence of this effect with DNase I treatment showed that RNA was an essential component of the nuclease activity. The sequence-specific nuclease activity observed in the cellular extracts responsible for ablating target mRNAs was termed the RNA-induced silencing complex (RISC).

After partial purification of crude extracts through differential centrifugation and anion exchange chromatography, the nucleaseco fractionated with a discrete =25- nucleotide RNA species. These results suggested that small RNAs were associated with sequence-specific nuclease and served as guides to target specific messages based upon sequence recognition.

In another report, the multicomponent RNAi nuclease was purified to homogeneity as a ribonucleoprotein complex of = 500 kDa. One of the protein components of this complex was identified as a member of the Argonaute family of proteins and was termed Argonaute2 (AG02). AG02 is homologous to RDE1, a protein required for dsRNA-mediated gene silencing in C. elegans. AG02 is a =130-kDa protein containing polyglutamine residues, PAZ, and PIWI domains characteristic of members of the Argonaute gene family.

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The Argonaute family members have been linked both to the gene-silencing phenomenon and to the control of development in diverse species. The first link betweenArgonaute protein and RNAi was shown by isolation of rde 1 mutants of C. elegans in a screen for RNAi-deficient mutants. Argonautefamily members have been shown to be involved in RNAi in Neurospora crassa (QDE3) as well as in A. thaliana (AGOl).