Abstract
Because of their nuclear dimorphism, ciliates provide a unique opportunity to study the role of non-coding RNAs (ncRNAs) in the communication between germline and somatic lineages. In these unicellular eukaryotes, a new somatic nucleus develops at each sexual cycle from a copy of the zygotic (germline) nucleus, while the old somatic nucleus degenerates. In the ciliate Paramecium tetraurelia, the genome is massively rearranged during this process through the reproducible elimination of repeated sequences and the precise excision of over 45,000 short, single-copy Internal Eliminated Sequences (IESs). Different types of ncRNAs resulting from genome-wide transcription were shown to be involved in the epigenetic regulation of genome rearrangements. To understand how ncRNAs are produced from the entire genome, we have focused on a homolog of the TFIIS elongation factor, which regulates RNA polymerase II transcriptional pausing. Six TFIIS-paralogs, representing four distinct families, can be found in P. tetraurelia genome. Using RNA interference, we showed that TFIIS4, which encodes a development-specific TFIIS protein, is essential for the formation of a functional somatic genome. Molecular analyses and high-throughput DNA sequencing upon TFIIS4 RNAi demonstrated that TFIIS4 is involved in all kinds of genome rearrangements, including excision of ~48% of IESs. Localization of a GFP-TFIIS4 fusion revealed that TFIIS4 appears specifically in the new somatic nucleus at an early developmental stage, before IES excision. RT-PCR experiments showed that TFIIS4 is necessary for the synthesis of IES-containing non-coding transcripts. We propose that these IES+ transcripts originate from the developing somatic nucleus and serve as pairing substrates for germline-specific short RNAs that target elimination of their homologous sequences. Our study, therefore, connects the onset of zygotic non coding transcription to the control of genome plasticity in Paramecium, and establishes for the first time a specific role of TFIIS in non-coding transcription in eukaryotes.
Highlights
Recent progress in high-throughput transcriptome analysis has led to a constantly growing catalog of non-coding transcripts
At each sexual cycle, during development of the somatic nucleus from the germline nucleus, the genome is massively rearranged through the reproducible elimination of germline-specific sequences including thousands of short, single copy, non-coding Internal Eliminated Sequences (IES)
We demonstrate, using RNA interference, that the TFIIS4 gene encoding a developmentspecific homolog of RNA polymerase II elongation factor TFIIS, is indispensable for non-coding RNAs (ncRNAs) synthesis in the new somatic nucleus
Summary
Recent progress in high-throughput transcriptome analysis has led to a constantly growing catalog of non-coding transcripts (ncRNAs, for review see [1,2]). The prevalence of a variety of short (~20–35 nt; sRNAs) and long ncRNAs (>~200 nt; lncRNAs) has been reported in numerous organisms. Genomic regulatory elements give rise to ncRNAs: transcription start site-associated short RNAs (TSSa-RNAs) or promoter upstream transcripts (PROMPTs) are produced from promoter regions, and eRNAs from enhancer regions. Regulatory short micro-RNAs (miRNAs) or longer ncRNAs can be transcribed from gene introns. Long ncRNAs may as well be produced from transcriptionally active pseudogenes, and can in turn yield endogenous short interfering RNAs (siRNAs) or miRNAs. piwi-interacting RNAs (piRNAs), in Drosophila as in mammals, are derived from heterochromatin domains containing mostly transposable elements and degenerate transposons, and are involved in transposon silencing. The function of numerous ncRNAs, including those attributed to promiscuous transcription of non-coding genomic regions, still needs to be determined
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