Strand-asymmetric endogenous Tetrahymena small RNA production requires a previously uncharacterized uridylyltransferase protein partner

Abstract

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.

New perspectives on the diversification of the RNA interference system: insights from comparative genomics and small RNA sequencing

“…Despite this diversification, to this point no kinetoplastid TRF members have been linked to RNAi; however, they could be involved in the distinctive RNA editing of kinetoplastids, which involves additions of polyuridine tracts. The ciliate Rdn1 and Rdn2 cluster together with other representatives from ciliates and several alveolates, stramenopiles, and Naegleria along with the GLD‐2/PAPD4‐like and ZCCHC6/ZCCHC11 repeat 2‐like clades. The ZCCHC6/ZCCHC11 repeat 2‐like clade, present across crown‐group eukaryotes, appears to retain the 3′ RNA uridylation observed in more basal versions typified by the ciliate enzymes.…”

“…MUT68/HESO1 uridylyltransferases are also implicated in 3′ modification of RISC‐cleaved fragments . In Tetrahymena , the uridylyltransferases Rdn1 and Rdn2 form distinct complexes with the RdRP to mediate processing/amplification of distinct groups of 23–25 nt RNAs . Related TRF nucleotidyltransferases also appear to be involved in highly selective modification of other subsets of RNA transcripts in animals; TUT1 is implicated in polyadenylation of specific mRNAs and polyuridylation of specific snRNA transcripts in the nucleus, while MTPAP polyadenylates mitochondrial transcripts …”

“…215,261 In Tetrahymena, the uridylyltransferases Rdn1 and Rdn2 form distinct complexes with the RdRP to mediate processing/amplification of distinct groups of 23-25 nt RNAs. 251 Related TRF nucleotidyltransferases also appear to be involved in highly selective modification of other subsets of RNA transcripts in animals; TUT1 is implicated in polyadenylation of specific mRNAs 230 and polyuridylation of specific snRNA 232 transcripts in the nucleus, while MTPAP polyadenylates mitochondrial transcripts. 233 A detailed analysis performed on the TRF polymerase family revealed that two members of the family can be confidently traced to LECA, one belonging to the classical TRF4/5 subfamily found across eukaryotes and involved in nuclear RNA surveillance that tags various ncRNA through 3 polyadenylation for degradation by the exosome 269 and another belonging to the functionally uncharacterized AT3G56320-like subfamily found in Giardia, kinetoplastids, Naegleria, chromalveolates, and plants (Table 3).…”

Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci

“…Some PGC transcripts may also be degraded by the Rdf2 RDRC (Fig. 9A, dashed arrow), based on more comprehensive PGC sRNA loss in cells with combined RDN2 and RDF2 knockout (Couvillion et al 2009;Talsky and Collins 2012). RDN2 mRNA level is maximal during sexual reproduction (Lee et al 2009).…”

“…On the other hand, Twi2p and Twi8p are loaded more canonically by the Tetrahymena Dicer Dcr2p, which generates 23-to 24-nt sRNAs distinct from the conjugation-specific 28-to 30-nt scnRNAs Collins 2006, 2007). Dcr2p substrate specificity derives largely from its physical association with three Tetrahymena RDRCs, which share the catalytic subunit Rdr1p but have unique combinations of accessory subunits (Rdn1p and Rdf1p, or Rdn1p and Rdf2p, or Rdn2p) that govern cellular template selection (Lee and Collins 2007;Lee et al 2009;Talsky and Collins 2012). The sRNAs loaded into Twi2p exhibit extreme strand bias, arising only from the RDRCsynthesized strand antisense to cellular transcripts with degenerate or no open reading frame (Lee and Collins 2006;Couvillion et al 2009).…”