The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena’s germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.DOI: http://dx.doi.org/10.7554/eLife.19090.001

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 ...

SummarySmall RNAs are used to silence transposable elements (TEs) in many eukaryotes, which use diverse evolutionary solutions to identify TEs. In ciliated protozoans, small-RNA-mediated comparison of the germline and somatic genomes underlies identification of TE-related sequences, which are then eliminated from the soma. Here, we describe an additional mechanism of small-RNA-mediated identification of TE-related sequences in the ciliate Tetrahymena. We show that a limited set of internal eliminated sequences (IESs) containing potentially active TEs produces a class of small RNAs that recognize not only the IESs from which they are derived, but also other IESs in trans. This trans recognition triggers the expression of yet another class of small RNAs that identify other IESs. Therefore, TE-related sequences in Tetrahymena are robustly targeted for elimination by a genome-wide trans-recognition network accompanied by a chain reaction of small RNA production.

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.