Strand-asymmetric endogenous Tetrahymena small RNA production requires a previously uncharacterized uridylyltransferase protein partner.
Many eukaryotes initiate pathways of Argonaute-bound small RNA (sRNA) production with a step that specifically targets sets of aberrant and/or otherwise deleterious transcripts for recognition by an RNA-dependent RNA polymerase complex (RDRC). The biogenesis of 23- to 24-nt sRNAs in growing Tetrahymena occurs by physical and functional coupling of the growth-expressed Dicer, Dcr2, with one of three RDRCs each containing the single genome-encoded RNA-dependent RNA polymerase, Rdr1. Tetrahymena RDRCs contain an active uridylyltransferase, either Rdn1 or Rdn2, and Rdn1 RDRCs also contain the Rdf1 and Rdf2 proteins. Although Rdn2 is nonessential and RDRC-specific, Rdn1 is genetically essential and interacts with a non-RDRC protein of 124 kDa. Here we characterize this 124-kDa protein, designated RNA silencing protein 1 (Rsp1), using endogenous locus tagging, affinity purification, and functional assays, as well as gene-knockout studies. We find that Rsp1 associates with Rdn1-Rdf1 or Rdn1-Rdf2 subcomplexes as an alternative to Rdr1, creating Rsp1 complexes (RSPCs) that are physically separate from RDRCs. The uridylyltransferase activity of Rdn1 is greatly reduced in RSPCs compared with RDRCs, suggesting enzyme regulation by the alternative partners. Surprisingly, despite the loss of all known RDRC-generated classes of endogenous sRNAs, RSP1 gene knockout was tolerated in growing cells. A minority class of Dcr2-dependent sRNAs persists in cells lacking Rsp1 with increased size heterogeneity. These findings bring new insights about the essential and nonessential functions of RNA silencing in Tetrahymena, about mechanisms of endogenous small interfering RNA production, and about the roles of cellular uridylyltransferases.
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200, USA. kcollins@berkeley.edu
Kristin Benjamin
KB
Talsky
Kathleen
K
Collins
D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016808:Tetrahymena thermophila / Q000235:genetics / Q000378:metabolism*
D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; D034741:RNA, Small Interfering; D012316:RNA Nucleotidyltransferases; C031687:UTP-RNA uridylyltransferase; D012324:RNA-Dependent RNA Polymerase