Tetrahymena eliminates micronuclear-limited sequences from the developing macronucleus during sexual reproduction. Homology between the sequences to be eliminated and ∼28-nucleotide small RNAs (scnRNAs) associated with an Argonaute family protein Twi1p likely underlies this elimination process. However, the mechanism by which Twi1p-scnRNA complexes identify micronuclear-limited sequences is not well understood. We show that a Twi1p-associated putative RNA helicase Ema1p is required for the interaction between Twi1p and chromatin. This requirement explains the phenotypes of EMA1 KO strains, including loss of selective down-regulation of scnRNAs homologous to macronuclear-destined sequences, loss of H3K9 and K27 methylation in the developing new macronucleus, and failure to eliminate DNA. We further demonstrate that Twi1p interacts with noncoding transcripts derived from parental and developing macronuclei and this interaction is greatly reduced in the absence of Ema1p. We propose that Ema1p functions in DNA elimination by stimulating base-pairing interactions between scnRNAs and noncoding transcripts in both parental and developing new macronuclei.[Keywords: RNA; heterochromatin; small RNA; noncoding RNA; Tetrahymena] Supplemental material is available at http://www.genesdev.org. Received April 7, 2008; revised version accepted June 25, 2008. Heterochromatin functions in various chromosomal processes, including regulation of gene expression, chromosome segregation, and nuclear organization (for review, see Grewal and Jia 2007). In diverse eukaryotes, RNAirelated mechanisms involving small RNAs complexed with Argonaute family proteins mediate heterochromatin formation (for review, see Martienssen and Moazed 2006;Grewal and Jia 2007). However, the mechanism by which small RNAs target heterochromatin formation is not completely understood. In ciliated protozoans, heterochromatin formation is also induced by an RNAi-related mechanism, followed by programmed DNA elimination of germline-specific sequences from the developing somatic nucleus (for review, see Meyer and Chalker 2006). Thus, programmed DNA elimination in ciliates serves as a model to study small RNA-mediated heterochromatin formation.Like most ciliated protozoans, Tetrahymena thermophila exhibits nuclear dimorphism. Each cell contains a germline micronucleus (Mic) and a somatic macronucleus (Mac). It is likely that only the Mac contributes to gene expression. In vegetative growth, the Mic and Mac replicate/divide, and sister nuclei are segregated to daughter cells. In the sexual process of conjugation ( Fig. 1A; see also Supplemental Fig. S1), the Mic undergoes meiosis to form two haploid pronuclei, one of which is reciprocally exchanged between the two conjugating cells. The migratory and stationary pronuclei then fuse to create a zygotic nucleus that divides mitotically twice to produce the next generation of new Macs and Mics. Then, paired cells separate, one of the two new Mics and the parental Mac are destroyed and, if fed, they resume vegetative ...

Summary Emerging evidence suggests that RNAi-related processes act both in the cytoplasm and in the nucleus. However, the process by which the RNAi machinery is transported into the nucleus remains poorly understood. The Tetrahymena Argonaute protein Twi1p localizes to the nucleus and is crucial for small RNA-directed programmed DNA elimination. In this study, we identify Giw1p, which binds to Twi1p and is required for its nuclear localization. Further, the endoribonuclease (Slicer)-activity of Twi1p plays a vital role in the removal of one of the two strands of Twi1p-associated siRNAs, leading to a functionally mature Twi1p-siRNA complex. Slicer activity is also shown to be required for nuclear localization of Twi1p and for its association with Giw1p. These results suggest that Giw1p senses the state of Twi1p-associated siRNAs and selectively transports the mature Twi1p-siRNA complex into the nucleus.

The formation of RNA–DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.