Functions and mechanisms of new components in RNA silencing

The long-term goal of the PI Zhang’s laboratory is to decipher intertwined regulatory layers and mechanisms that control RNA silencing and processing, and to eventually manipulate the regulatory components and pathways in biotechnological applications. Here this proposal is to describe how the PI Zhang will investigate unknown mechanisms and regulations of several newly identified components within two master regulatory hubs centered on Serrate (SE) and Suppressor of Gene Silencing 3 (SGS3) for RNA silencing in Arabidopsis. RNA silencing clears endogenous and invasive transcripts, and this process is directed by microRNAs (miRNAs) and small interfering RNAs (siRNAs) in eukaryotes. MicroRNAs are produced from hairpin-structured primary miRNA substrates (pri-miRNAs) by microprocessor that comprises of Dicer-like protein 1 (DCL1), dsRNA binding protein 1 (DRB1), and SE. By contrast, siRNA production entails the initial conversion of single-stranded (ss) RNA to double-stranded (ds) RNA via a complex composed of RNA-dependent RNA polymerase 6 (RDR6) and SGS3 before its entry into DCL4/DRB4 complex. Since levels of miRNAs and siRNAs are critical for their proper functionality in biology, the processing and accumulation of small RNAs need to be tightly controlled. In research area 1, the PI Zhang will study how the components in the SE-centered hub regulate miRNA production. Briefly, the PI Zhang laboratory has recently discovered that RNA secondary structure (RSS) of pri-miRNAs can be remodeled to regulate DCL1/HYL1 activity. Importantly, several RNA helicases (RHs) have been newly recovered from the hub, but how they sensor and re-wire RSS of pri-miRNAs to control miRNA production is unknown. Furthermore, extensive preliminary data also identify key connections between reactive oxygen species (ROS) and microprocessor components that modulate miRNA production. Thus, the PI Zhang will systematically investigate the novel functions and mechanisms of the RHs and catalases from SE hub in miRNA biogenesis in Area 1. In research Area 2, the PI Zhang focuses on the major gap in the siRNA production that is how ssRNA substrates are specified and loaded onto SGS3/RDR6 for dsRNA synthesis. Through a newly developed genetic screening, numerous novel components exemplified by U1-70K and BICE1 among others have been recovered to be engaged in siRNA- mediated RNA silencing. Remarkably, the components interact with RNA polymerases in nucleus and ribosomes in cytoplasm as well as the shared SGS3 protein. In this setting, the PI Zhang would comprehensively study how these factors help SGS3 to fetch the nascent transcripts from chromatin, and to set up the platform for SGS3/RDR6 nearby ribosomes to synthesize dsRNAs. The proposed study will not only reveal new regulatory layers and mechanisms of RNA silencing, but also enable us to explore the highly conserved components to control small RNA production and activities in biological processes, and to eventually to cure physiological disorders that arise from dysfunctions of RNA processing in biotechnology.