Characterization of components of cytoplasmic Ago2-complexes

RNA interference is an only recently discovered pathway, which is mediating negative gene regulation by small RNA molecules. These can derive either from endogenous microRNA precursors, long double stranded RNAs, or from exogenous sources like viruses or synthetic small interfering RNAs. After processing a single stranded RNA, the so called guide strand, is incorporated into ribo-nucleoprotein complexes also known as RISC (RNA induced silencing complexes), which are able to recognize complementary mRNA sequences. These targets are then either degraded or their translation is inhibited. Essential components of RISC are the Argonaute proteins. There are eight members of this family known in humans, only one of which, Argonaute 2 (Ago2 or eIF2C2), is able to mediate the cleavage of target RNAs, rendering it the active "slicer". Previous studies have shown, that Ago2 is interacting with Dicer, the upstream RNase III type enzyme, which processes precursor microRNAs or long double stranded RNA to smaller pieces of approximately 21 nucleotides. Furthermore, also the HIV-1 transactivating region RNA-binding protein (TRBP), which is misused by HIV for viral multiplication, is part of the small minimal RISC. But the larger native "holo" RISC also contains various other proteins, which are not well characterized until now. In the proposed project we intend to purify Ago2-containing complexes from cytoplasmic human cell extracts and to identify the single components by fractionation and mass spectrometry. Cloning of the corresponding cDNA sequences into expression vectors will allow us to produce recombinant proteins, and subsequently to raise specific antibodies. These will be used to cross-check the interactions with Ago2 by co-immunoprecipitation, immuno- colocalization and Western blot analysis of size fractions. Verified members of Ago2-complexes will be further analyzed for their importance in RNAi by investigation of the efficacy of miRNA and siRNA-mediated silencing during concomitant knockdown of the corresponding candidate protein. Additionally, also the miRNAs contained in Ago2-complexes will be analyzed by cloning and sequencing. By thoroughly analysing the Ago2-complex-components and their interactions, a new improved model for RISC- function should be developed and exploited for enhancement of RNAi-efficiency.