Biochemical analyzes of endogenous argonaute complexes immunopurified with anti-Argonaute monoclonal antibodies.

Argonaute proteins are key factors in RNA silencing. After association with small RNAs of 20-30 -nucleotides, Argonaute proteins are targeted to homologous RNA molecules that are to be silenced. To understand the functional contributions of Argonaute proteins to RNA silencing at a biochemical level, immunoisolation of Argonaute proteins from living cells of various organisms has been performed.

Functional involvement of Tudor and dPRMT5 in the piRNA processing pathway in Drosophila germlines.

In Drosophila, the PIWI proteins, Aubergine (Aub), AGO3, and Piwi are expressed in germlines and function in silencing transposons by associating with PIWI-interacting RNAs (piRNAs). Recent studies show that PIWI proteins contain symmetric dimethyl-arginines (sDMAs) and that dPRMT5/Capsuleen/DART5 is the modifying enzyme. Here, we show that Tudor (Tud), one of Tud domain-containing proteins, associates with Aub and AGO3, specifically through their sDMA modifications and that these three proteins form heteromeric complexes.

Characterization of the miRNA-RISC loading complex and miRNA-RISC formed in the Drosophila miRNA pathway.

In Drosophila, miRNA is processed by Dicer-1 (DCR-1) from its precursor and loaded onto Argonaute1 (AGO1). AGO1 recognizes target mRNAs based on the miRNA sequence and suppresses the expression at post-transcriptional levels. GW182, a P-body component, localizes the AGO1 complex to processing bodies (P-bodies) where mRNA targets are decayed or stored.

Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells.

RNA silencing is a conserved mechanism in which small RNAs trigger various forms of sequence-specific gene silencing by guiding Argonaute complexes to target RNAs by means of base pairing. RNA silencing is thought to have evolved as a form of nucleic-acid-based immunity to inactivate viruses and transposable elements. Although the activity of transposable elements in animals has been thought largely to be restricted to the germ line, recent studies have shown that they may also actively transpose in somatic cells, creating somatic mosaicism in animals.