Generation of small RNA complexity requires specialization of RNA-dependent RNA polymerase 1 and RNA silencing protein 1 by shared protein partners

RNA-dependent RNA polymerases (RdRPs) induce sequence-specific gene silencing through the production of double-stranded RNA (dsRNA) from a single-stranded RNA template. In Tetrahymena thermophila, dsRNA products are synthesized by the only genome-encoded RdRP (Rdr1) and then cleaved by an associated Dicer endonuclease (Dcr2) into 23-24 nt small RNAs (sRNAs). These sRNAs accumulate constitutively, dependent on Rdr1 activity. They are derived from endogenous transcripts and map in clusters to specific genomic loci. Rdr1 activity is robust on all templates in vitro, yet the silencing process remains selective in the sequences it targets in the cell. RNA targeting specificity is likely an absolute requirement for cell viability, and, until now, the factors controlling it have not been clearly defined. This dissertation details (1) the roles that Rdr1- interacting proteins play in the biogenesis of multiple classes of 23-24 nt sRNA, (2) the in vitro template selectivity and initiation mechanisms by distinct Rdr1 complexes (RDRCs) and (3) the identification of a new RNA silencing protein (Rsp1) that, along with Rdr1, is required for bulk sRNA accumulation. Rsp1 shares several protein partners with Rdr1, including the uridyltransferase Rdn1 and two factors (Rdf1 and Rdf2) that control RNA target specificity in vivo through an unknown mechanism. Characterization of Rdn1, Rdf1 and Rdf2 functions in vitro revealed that Rdf1 and Rdf2 are adaptors that activate the polyuridylation of RNA by Rsp1-bound Rdn1. The data suggest that Rsp1 functions upstream of Rdr1 in sRNA generation, as discussed in greater detail in Chapter 3. These findings demonstrate that sRNA generation requires the coordination of a complex array of protein machines. Future studies on how these proteins mediate RNA targeting in vivo will offer exciting insights into the molecular mechanisms that are central to controlling the specificity of RNA fates in the cell