Building the Human Argonaute 2 Interaction Network

Abstract

RNA interference (RNAi) is a system that has been largely studied and defined by its ability to affect gene expression and translation in the cytoplasm. However, Argonaute (AGO) proteins, which are the major catalytic component of the RNA-induced silencing complex (RISC), have been found to be involved in nuclear roles outside of the canonical RNAi pathway. Within non-mammalian systems such as yeast and plants, AGOs have been shown to be involved in functions such as DNA methylation and heterochromatin formation. My laboratory has utilized systems involving small RNAs to target nuclear events such as transcription and splicing in human cells. In the case of transcription, we have shown that small RNAs are capable of targeting long noncoding RNAs (lncRNAs) along both the promoter and past the 3’ end of genes in order to control gene expression. We have also demonstrated that targeting of small RNAs to introns and exons of pre-mRNA can robustly alter the splicing pattern. Within these systems, we have found that AGO proteins are recruited by the small RNAs to the nuclear target. However, the protein-protein interactions and mechanisms involved remain unclear. Identification and understanding of the interactions of AGO proteins in the nucleus is essential for comprehension of the mechanisms by which these proteins act. I have studied AGO2 interactions through immunoprecipitation and a novel semi-quantitative mass spectrometry technique known as the Normalized Spectral Index Method (SINQ). Stringent screening of mass spectrometry results identified interactions with the TRNC6 proteins, and AGO3. Most cytoplasmic interacting partners were also partners for AGO2 in the nucleus, however interactions with the loading complex was impaired although it is present in nuclei. These data demonstrate that the core RNAi machinery is largely conserved between cytoplasm and nucleus. This work opens new avenues for the utilization of small RNAs in gene regulation both in the laboratory and in the clinic.