Biased transcription and selective degradation of small RNAs shape the pattern of DNA elimination in Tetrahymena

Abstract

The ciliated protozoan Tetrahymena undergoes extensive programmed DNA elimination when the germline micronucleus produces the new macronucleus during sexual reproduction. DNA elimination is epigenetically controlled by DNA sequences of the parental macronuclear genome, and this epigenetic regulation is mediated by small RNAs (scan RNAs [scnRNAs]) of~28-30 nucleotides that are produced and function by an RNAi-related mechanism. Here, we examine scnRNA production and turnover by deep sequencing. scnRNAs are produced exclusively from the micronucleus and nonhomogeneously from a variety of chromosomal locations. scnRNAs are preferentially derived from the eliminated sequences, and this preference is mainly determined at the level of transcription. Despite this bias, a significant fraction of scnRNAs is also derived from the macronuclear-destined sequences, and these scnRNAs are degraded during the course of sexual reproduction. These results indicate that the pattern of DNA elimination in the new macronucleus is shaped by the biased transcription in the micronucleus and the selective degradation of scnRNAs in the parental macronucleus.

Dynamic distributions of long double-stranded RNA in Tetrahymena during nuclear development and genome rearrangements

“…Dcl1p is known to be localized in meiotic MICs and to be absent from the parental MACs, whereas the localization in other stages is not clear. Recent deep-sequencing studies have shown that these small RNAs are enriched in sequences from IESs and some MDSs, and, at early stages, are derived exclusively from the MICs Schoeberl et al, 2012). We found that dsRNA staining in MICs was indeed highly elevated in dcl1Δ mutants, supporting the idea that it is processed by Dcl1p.…”

“…At least some of these transcripts are processed by the Dicer-family protein Dcl1p (Malone et al, 2005;Mochizuki and Gorovsky, 2005) to produce ∼29-nt small RNAs (also referred to as scanning RNAs or scnRNAs) between the stages of meiosis and new MAC development (Chalker and Yao, 2001;Mochizuki et al, 2002). Deep sequencing revealed their origins from both strands of IESs and some MAC-destined sequences (MDSs, sequences that are not deleted from new MACs) (Schoeberl et al, 2012). These small RNAs are bound by the piwi-related Tetrahymena argonaute proteins Twi1p and Twi11p (Mochizuki et al, 2002;Couvillion et al, 2009;Noto et al, 2015) and are thought to guide heterochromatin targeting through homology recognition, though the targeting mechanism is still unknown.…”

Intercepting noncoding messages between germline and soma: Figure 1.

“…With the recently available Tetrahymena micronuclear genome sequence as the reference, a detailed deepsequencing analysis of scnRNAs and their ncRNA precursors was performed by Mochizuki's laboratory (Schoeberl et al 2012). By comparing scnRNAs from different conjugation time points as well as the wild-type and RNAideficient strains, the study provides definitive support for the selective degradation of scnRNAs homologous to MDS, a key prediction by the scnRNA model.…”

“…The new study by Mochizuki and colleagues (Schoeberl et al 2012) has systematically addressed the turnover of scnRNAs in early to mid-conjugation, during which scnRNAs are first generated by Dcl1p-dependent processing of ncRNA transcripts from the meiotic micronucleus and then enriched for IES homologous sequences. The latter process is the cornerstone for the scnRNA model.…”

Targeted Gene Disruption by Ectopic Induction of DNA Elimination in Tetrahymena

“…During the development of the new MAC of Tetrahymena, .8000 DNA segments called internal eliminated sequences (IESs) were reproducibly eliminated. IESs are recognized by 26-to 32-nt small RNAs called scnRNAs (Schoeberl et al 2012). IESs can be classified into two types: type A IESs that are recognized by scnRNAs produced by themselves and type B IESs that are recognized by scnRNAs produced by type A IESs .…”