Study of an RNA helicase implicates small RNA–noncoding RNA interactions in programmed DNA elimination in Tetrahymena

Abstract

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 ...

2′-O-methylation stabilizes Piwi-associated small RNAs and ensures DNA elimination in Tetrahymena

“…Although 38 of 56 progeny of the HEN1 KO strains showed complete or only reduced elimination of the four IESs, the removal of many of the other z6000 IESs was likely to be affected in these progeny. Since DNA elimination is believed to be essential for producing viable sexual progeny (Coyne et al 1999;Nikiforov et al 1999;Mochizuki et al 2002;Liu et al 2007;Aronica et al 2008), the reduced production of viable offspring in the HEN1 KO strains (Supplemental Fig. S5C) is likely due to this partial DNA elimination defect.…”

“…The samples were blocked for 1 h with 3% BSA (Sigma), 10% normal goat serum (Invitrogen), and 0.1% Tween 20 in PBS followed by overnight incubation at 4°C in blocking solution containing a 1:500 dilution of anti-Hen1p or a 1:200 dilution of anti-Twi1p (Aronica et al 2008). The anti-Hen1p antibody was obtained by immunizing a rabbit with recombinant Hen1p.…”

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

“…Interestingly, piRNAs direct specific genome rearrangements in ciliates and this precise genome editing results in either somatic elimination [72] or retention [73] , indicating the versatility of this particular RNAi pathway. PiRNAs can target mRNAs through a PTGS-like mechanism, however they may also induce TGS by directing heterochromatin formation at the target regions [74,75] .…”

Non-coding RNAs in Polycomb/Trithorax regulation

“…33 The targeting mechanism involves small (28 nt) RNAs carried by the Tetrahymena Argonaute protein Twi1p, which very probably act as guiding molecules by hybridising to a long nascent non-coding RNA. 34 Thus Tetrahymena targets its Polycomb proteins using RNAi components in a similar manner to that of fission yeast in heterochromatin formation. However in contrast to higher organisms, the Tetrahymena PcG and heterochromatin proteins are fused into a single system.…”