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. This has enabled the analysis of Argonaute-associated proteins and RNAs. Identifying the small RNAs that associate with individual Argonaute proteins, for instance, could help to elucidate the silencing pathways in which particular Argonaute proteins are involved. However, it is also necessary to note that the results obtained through such biochemical analyzes are greatly affected by the quality and properties of the antibodies used, as well as by the immunoprecipitation conditions employed, including buffer contents and/or salt concentration. In this chapter, we describe fundamental methods for immunoprecipitating Argonaute proteins using monoclonal antibodies as well as for detecting associated proteins and small RNAs. Furthermore, we will also explain how various parameters, such as antibody properties and buffer conditions, can alter the production and interpretation of experimental data.

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. piRNA precursor-like molecules are detected in these complexes. The expression levels of Aub and AGO3, along with their degree of sDMA modification, were not changed by tud mutations. However, the population of transposon-derived piRNAs associated with Aub and AGO3 was altered by tud mutations, whereas the total amounts of small RNAs on Aub and AGO3 was increased. Loss of dprmt5 did not change the stability of Aub, but impaired its association with Tud and lowered piRNA association with Aub. Thus, in germline cells, piRNAs are quality-controlled by dPRMT5 that modifies PIWI proteins, in tight association with Tud.

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. However, the details of the pathway remain elusive. In this study, two distinct types of AGO1-containing complexes from Drosophila Schneider2 (S2) cells were isolated and compared at the molecular level. The AGO1 complex with DCR-1 contained neither mature miRNA nor GW182 but exhibited pre-miRNA processing activity. The resultant mature RNA was loaded onto AGO1 within the complex. The AGO1 complex with GW182 excluded DCR-1, but possessed mature miRNA and showed no pre-miRNA processing activity. Thus, the AGO1-DCR-1 and AGO1-GW182 complexes correspond to miRLC (miRISC loading complex) and miRISC, respectively. The requirement for various domains of AGO1 in miRNA-loading and DCR-1/GW182 interaction was also examined. The Mid domain mutant (F2V2) interacted with DCR-1 but not with mature miRNA and GW182. The AGO1-PAZ mutant lacks the mature miRNA-binding ability but associates with either DCR-1 or GW182. The AGO1-PIWI mutant showed no Slicer activity but associates with mature miRNA. These results indicate that these domains are required differently for miRLC and miRISC formation in the miRNA pathway.

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. In the Drosophila germ line, Piwi-interacting RNAs arise from repetitive intergenic elements including retrotransposons by a Dicer-independent pathway and function through the Piwi subfamily of Argonautes to ensure silencing of retrotransposons. Here we show that, in cultured Drosophila S2 cells, Argonaute 2 (AGO2), an AGO subfamily member of Argonautes, associates with endogenous small RNAs of 20-22 nucleotides in length, which we have collectively named endogenous short interfering RNAs (esiRNAs). esiRNAs can be divided into two groups: one that mainly corresponds to a subset of retrotransposons, and the other that arises from stem-loop structures. esiRNAs are produced in a Dicer-2-dependent manner from distinctive genomic loci, are modified at their 3' ends and can direct AGO2 to cleave target RNAs. Mutations in Dicer-2 caused an increase in retrotransposon transcripts. Together, our findings indicate that different types of small RNAs and Argonautes are used to repress retrotransposons in germline and somatic cells in Drosophila.