Identification and analysis of functional associations among natural eukaryotic genome editing components

Estienne C Swart,Pamela Y Sandoval,Mariusz Nowacki,Miroslav Arambasic,Michael Ignarski,Cristina Hoehener,Cyril Denby Wilkes,Dominique I Furrer,Aditi Singh

doi:10.12688/f1000research.12121.1

Estienne C Swart, Pamela Y Sandoval + Show 7 more

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https://doi.org/10.12688/f1000research.12121.1

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During development in the ciliate Paramecium, excess DNA interspersed throughout the germline genome is deleted to generate a new somatic genome. In this process, most of the intervening DNA is excised by a Piggybac-derived transposase, assisted by small RNAs (scnRNAs and iesRNAs) and chromatin remodelling. As the list of genes involved in DNA elimination has been growing, a need for a general approach to discover functional relationships among these genes now exists. We show that deep sequencing-based comparisons of experimentally-induced DNA retention provide a sensitive, quantitative approach to identify and analyze functional associations among genes involved in native genome editing. This reveals two functional molecular groups: (i) iesRNAs/scnRNAs, the putative Piwi- and RNA-binding Nowa1/2 proteins, and the transcription elongation factor TFIIS4; and (ii) PtCAF1 and Ezl1, two proteins involved in chromatin remodelling. Comparative analyses of silencing effects upon the largely unstudied regions comprising most developmentally eliminated DNA in Paramecium suggests a continuum between precise and imprecise DNA elimination. These findings show there is now a way forward to systematically elucidate the main components of natural eukaryotic genome editing systems.

Highlights

Paramecium, like other ciliates, contains both a large, somatic, polyploid macronucleus (MAC), and a relatively tiny, gametic, diploid micronucleus (MIC), within the same cell
Given the role of TFIIS4 and Dcl2/3/5 in producing and processing of RNAs, and the suggested role of Nowa[1] in RNA interactions, we classify these proteins as “RNA-associated” proteins involved in Paramecium MAC genome development
The strong similarity of internally eliminated sequences (IESs) retention following silencing of DCL2/3/5, NOWA1/2 and TFIIS4 suggests that the protein products of these genes may work in the same small RNAs (sRNAs)-dependent DNA elimination pathway during short IES excision, and that DCL2/3, NOWA1/2 and TFIIS4 work in the predominantly scnRNA-dependent DNA pathway responsible for other eliminated sequences (OESs) and long IES elimination

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Introduction

Paramecium, like other ciliates, contains both a large, somatic, polyploid macronucleus (MAC), and a relatively tiny, gametic, diploid micronucleus (MIC), within the same cell. During sexual or asexual “reproduction” (conjugation or autogamy), a copy of Paramecium’s micronucleus develops into a new macronucleus, and at the same time the micronuclear genome it contains is transformed into a new macronuclear genome by extensive, targeted DNA deletion. Characterized Paramecium IESs are typically short (> 90% are < 150 bp), TA-flanked, and unique (∼45,000 IESs per Paramecium tetraurelia MIC genome[1]). These IESs are recognized and generally precisely removed by the PiggyMac transposase complex[2]. For some IESs, development-specific small RNAs (sRNAs) are required for the recognition and efficient removal of all their copies[3]

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