Target specificity of human RNA-induced silencing complex

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Summary/Abstract of Parent Award Project In humans, microRNAs (miRNAs) are loaded into four Argonaute (AGO) proteins to form RNA-induced silencing complexes (RISC), which target mRNAs for gene silencing. The finding that the four AGOs share ~80% amino acid identity and even 75% of their suite of bound miRNAs inspired the notion that the four AGOs target the same set of RNAs redundantly. However, in addition to RNA interference, each AGO contributes to specialized roles in neural development, antiviral response, and cancer. A clear knowledge gap in the field is the molecular bases for the distinctive functions of each AGO. This void is due to an incomplete understanding of the target-recognition determinants of each AGO. To date, all prediction algorithms of guide-binding sites only consider the guide-target complementarity but not the type of AGO in which the guide is loaded. We will test the hypothesis that each AGO's nuanced structural features and interacting partners shape the inventory of targets for each of the four AGO-RISC complexes. Our long-term goal is to establish an algorithm capable of predicting guide-binding sites by taking account of the unique properties of human AGOs. Toward this overarching objective, we will pursue the following three specific Aims. In Aim 1, we will determine the cryo-electron microscopy and crystal structures of all four human AGOs bound to the same guide and target RNAs. We have already purified homogeneous AGO1-, AGO2-, and AGO3-RISCs (i.e., miR-20a-loaded AGO) and obtained initial structures of AGO1-RISC and AGO2-RISC bound to a 25-nt target RNA. We have also obtained preliminary crystals for these complexes. We will use a filter-binding assay to measure the fraction of target RNAs bound by different homogeneous AGO-RISC complexes. The outcome from Aim 1 will provide the structural basis for the differential target recognition by the four human AGOs. In Aim 2, we will use Selective 2’ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) to elucidate how each of the four human AGOs recognizes the guide-binding site and the flanking regions on target RNAs. To understand how RISCs recognize the guide-binding site buried in a highly structured target RNA, we will perform SHAPE studies with a 352- nt human immunodeficiency virus 5’ untranslated region RNA and a 176-nt fragment of ribosomal internal entry site RNA. The outcome from Aim 2 will quantify the dynamic target recognition of the four AGOs. In Aim 3, we will create AGO1-AGO2 chimeras and test the mutants for interaction with USP34 and SART3 to identify their binding site located only in AGO1. Then, we will co-express FLAG-AGO1 with Myc-USP40 or -SART3 and perform a tandem immunoprecipitation using anti-FLAG and anti-Myc beads, followed by RNA sequencing to identify bound RNAs. This strategy will be applied to the other AGOs to determine their respective inventory of specific partners and the bound RNAs. The outcome from Aim 3 will reveal how protein partners of AGOs contribute to their target specificity. Altogether, the proposed projects will provide a solid foundation for fields and aid new therapeutic strategies to design a new class of guide RNA drugs customized for each AGO.