TFIIS-Dependent Non-coding Transcription Regulates Developmental Genome Rearrangements

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.

Prolonged exposure to constant environmental conditions prompts nonrandom genetic variation

“…Finally, we asked how extensively IESs that accumulated in the MA lines’ somatic genome overlap with IESs that are epigenetically controlled in the control stock 51 (Lhuillier-Akakpo et al 2014; Sandoval et al 2014; Maliszewska-Olejniczak et al 2015) . We found that a significant excess of the IESs that are retained in the MA lines fall into a class of IESs that are under the control of small RNAs (epi-IESs) in stock 51 ( Figure S5 ).…”

A mating-type mutagenesis screen identifies a zinc-finger protein required for specific DNA excision events in Paramecium

“…The C2H2 zinc finger present in both mtGa and mtGb is predicted to bind GCTA or GCTT, suggesting that direct binding to IESs is required for correct excision. We currently cannot exclude the possibility that mtG proteins bind to the TFIIS4-dependent nascent transcripts found to play a role in IES excision ( 15 ), rather than to DNA itself. In both cases, the co-localization of mtGa-GFP and H3K9me3 marks in what appears to be DNA elimination structures would suggest that binding is maintained until excision.…”

“…After being loaded on the Piwi proteins Ptiwi01 and Ptiwi09 ( 12 , 13 ), scnRNAs are imported into the parental MAC and those able to pair with pervasive nascent transcripts are somehow inactivated ( 14 ). Thus, only those originating from MIC-specific sequences would be licensed to later target the same sequences in the developing zygotic MAC, by pairing with TFIIS4-dependent nascent transcripts ( 15 ). In both parental and zygotic MACs, pairing of scnRNAs with nascent transcripts is thought to involve the RNA-binding and Ago-hook-containing proteins Nowa1/2 ( 16 , 17 ).…”

Encoding activities of non-coding RNAs

“…A large, but as of yet undetermined, number of non-coding RNAs (ncRNAs) have recently been uncovered. We, along with other groups, have demonstrated that these ncRNAs can regulate normal development and disease occurrence through controlling RNA maturation and protein synthesis 6 - 12 . Notably, the number of ncRNAs is more than that of protein-coding genes within the genome 13 , 14 .…”