Structural biology on RNA silencing suppressors and their potential targets

Abstract

RNA silencing, which is triggered by small RNAs, is a powerful gene expression regulation mechanism and results in sequence specific inhibition of gene expression by translational repression and/or mRNA degradation. Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are processed by RNase III enzymes and subsequently loaded into Argonaute (AGO) proteins, a key component of the RNA induced silencing complex (RISC). RISC is a multi-protein complex that incorporates Argonautes, the bound small RNA, and other AGOs interacting proteins. Among these RISC components, kiaa1093 is a poorly understood protein. In this thesis (chapter 3), we solve the crystal structure of kiaa1093 C-terminal RNA recognition motif (RRM) and establish the physical association between TRBP and kiaa1093 via its C-terminal RRM domain in vitro. Compared with canonical RRMs, kiaa1093 RRM is composed of an additional C-terminal a helix, which is important to bridge TRBP and kiaa1093¿s interaction. Remarkably, kiaa1093 RRM enhance TRBP¿s RNA affinity. Therefore we hypothesize that kiaa1093 may function as a scaffold protein to strengthen TRBP/siRNA interaction and help TRBP to recruits Dicer complex to Argonaute 2 for gene silencing events. Argonaute proteins in RISC are also potential targets for viral suppressors to suppress the host RNA silencing. For example, CMV 2b, encoded by cucumovirus, is targeting AGO1 in Arabidopsis. However, its homolog Tomato aspermy virus (TAV2b), which is also encoded by cucumovirus, may suppress host RNA silencing through binding small RNAs on the basis of our work. In chapter 4, we report the crystal complex structure of TAV2b bound to a 19 bp siRNA duplex. We observe that TAV2b adopts an all a-helix structure and forms a homodimer to measure siRNA duplex major groove in a length-preference mode, which is different from the binding modes adopted by either Tomato bushy stunt virus (TBSV)/Carnation Italian ringspot virus (CIRV) p19 or flockhouse.