Two classes of endogenous small RNAs in Tetrahymena thermophila

In diverse eukaryotes from parasitic protozoa to humans, RNA interference (RNAi) pathways regulate gene expression, establish heterochromatin, and/or protect the genome from viruses and mobile DNA elements (Matzke and Birchler 2005; Sontheimer and Carthew 2005). Although the biological function of RNAi varies, central to all pathways are ∼21–30-nucleotide (nt) small noncoding RNAs (sRNAs) that provide specificity for RNA or DNA targets. In multicellular organisms, three major classes of endogenous sRNAs have been characterized in detail: micro RNAs (miRNAs), repeat-associated small interfering RNAs (rasiRNAs), and trans-acting small interfering RNAs (ta-siRNAs) (Bartel 2005; Sontheimer and Carthew 2005). The miRNAs and tasiRNAs direct translational repression and/or degradation of messenger RNAs. The rasiRNAs, derived from repetitive DNA elements such as transposons and centromeres, function to promote heterochromatin formation, DNA methylation, and/or RNA degradation. Less-well-characterized sRNAs include those with precise complementarity to protein-coding genes, pseudogenes, and intergenic regions (e.g., see Ambros et al. 2003). The biogenesis of diverse sRNAs depends on an RNaseIII family nuclease called Dicer (Tomari and Zamore 2005). The Dicer substrates for miRNA production are single-stranded RNAs with stem-loop structures, while precursors to ta-siRNAs and most rasiRNAs are double-stranded RNAs (dsRNAs) resulting from bidirectional transcription or RNA-dependent RNA polymerase activity. Dicer processing of precursors yields short sRNA duplexes of homogeneous length. One strand of each sRNA duplex is stabilized by assembly into an effector ribonucleoprotein (RNP) containing a Piwi/PAZ domain (PPD) protein of the Argonaute family. Multicellular eukaryotes express multiple paralogs of RNAi pathway components that are specialized in function. In contrast to the diversity of sRNAs in multicellular organisms, unicellular eukaryotes are only known to express rasiRNA-like sRNAs (Djikeng et al. 2001; Reinhart and Bartel 2002; Chicas et al. 2004; Ullu et al. 2005). In the free-living ciliated protozoan Tetrahymena thermophila, RNAs ∼26–31 nt in length direct developmentally programmed DNA elimination (Mochizuki and Gorovsky 2004b). T. thermophila, like other ciliates, has nuclear dualism, with a diploid, germline micronucleus (MIC) that remains phenotypically silent and a polyploid, transcriptionally active, somatic macronucleus (MAC). When starved for nutrients, T. thermophila ceases to divide vegetatively and becomes competent to reproduce sexually by conjugation. In conjugating cells, new MACs are developed from mitotic siblings of the zygotic MIC in a process involving site-specific chromosome fragmentation and deletion of ∼6000 internally eliminated sequences (IESs). The IESs are single-copy elements or moderately repetitive, transposon-like sequences that together account for ∼15% of the MIC genome (Yao and Chao 2005). DNA elimination occurs under epigenetic regulation: Sequences in the parental MAC can protect corresponding sequences in the developing MAC from elimination. Normal MAC development and the conjugation-induced accumulation of ∼26–31-nt sRNAs require the PPD-containing TWI1 and the Dicer-like DCL1 (Mochizuki et al. 2002; Malone et al. 2005; Mochizuki and Gorovsky 2005). Bidirectional nongenic transcription in the MIC during conjugation (Chalker and Yao 2001) is proposed to provide dsRNA precursors that are processed by Dcl1p into sRNAs (Yao et al. 2003; Mochizuki and Gorovsky 2004b). Northern blot assays have confirmed that a known MIC-limited IES is represented in the conjugation-induced sRNA population (Chalker et al. 2005). In addition, DNA hybridization studies using sRNAs isolated from conjugating cells have suggested that as conjugation progresses, the sRNA population becomes enriched for MIC-limited sequence (Mochizuki and Gorovsky 2004a). To account for this finding and provide a mechanism for the epigenetic influence of the parental MAC, the ∼26–31-nt sRNAs, termed the scan (scn)RNAs, are proposed to enter the parental MAC in association with Twi1p and scan for homologous sequence in a manner that results in degradation of MAC-cognate sRNAs. The sRNAs remaining after parental MAC subtraction are thought to then transit to the developing MAC where they guide the histone H3 Lys 9 (H3K9) methylation of MIC-limited chromatin, which likely marks IESs for subsequent elimination (Taverna et al. 2002; Liu et al. 2004). In this manner, sRNA-guided DNA elimination in T. thermophila is similar to rasiRNA-guided heterochromatin formation in Schizosaccharomyces pombe (Matzke and Birchler 2005). The recently sequenced MAC genome of T. thermophila encodes multiple Dicer and PPD family members, implying the existence of additional RNAi pathways with roles other than DNA elimination. RasiRNA-like sRNAs derived from MIC centromeres may function in MIC maintenance in a manner dependent on DCL1 during vegetative growth (Mochizuki and Gorovsky 2005), although conflicting results have been reported (Malone et al. 2005). However, the full complexity of sRNAs in T. thermophila has not been examined. Here we present our analysis of sRNAs expressed in vegetatively growing, starving, and conjugating cells. We describe a second class of T. thermophila sRNAs with ubiquitous accumulation throughout the life cycle. These ∼23–24-nt sRNAs have features characteristic of sRNAs from other organisms but with interesting differences that suggest a novel biogenesis pathway distinct from those previously described for miRNAs, rasiRNAs, and ta-siRNAs. Analogous to the diversity of sRNAs found in multicellular organisms, the ∼27–30-nt sRNAs and the ∼23–24-nt sRNAs in T. thermophila represent coexisting yet genetically separable RNAi pathways.