Exogenous RNAi mechanisms contribute to transcriptome adaptation by phased siRNA clusters in Paramecium

Abstract

Extensive research has characterized distinct exogenous RNAi pathways interfering in gene expression during vegetative growth of the unicellular model ciliate Paramecium. However, role of RNAi in endogenous transcriptome regulation, and environmental adaptation is unknown. Here, we describe the first genome-wide profiling of endogenous sRNAs in context of different transcriptomic states (serotypes). We developed a pipeline to identify, and characterize 2602 siRNA producing clusters (SRCs). Our data show no evidence that SRCs produce miRNAs, and in contrast to other species, no preference for strand specificity of siRNAs. Interestingly, most SRCs overlap coding genes and a separate group show siRNA phasing along the entire open reading frame, suggesting that the mRNA transcript serves as a source for siRNAs. Integrative analysis of siRNA abundance and gene expression levels revealed surprisingly that mRNA and siRNA show negative as well as positive associations. Two RNA-dependent RNA Polymerase mutants, RDR1 and RDR2, show a drastic loss of siRNAs especially in phased SRCs accompanied with increased mRNA levels. Importantly, most SRCs depend on both RDRs, reminiscent to primary siRNAs in the RNAi against exogenous RNA, indicating mechanistic overlaps between exogenous and endogenous RNAi contributing to flexible transcriptome adaptation.

Characterisation of the RNA-interference pathway as a Tool for Genetics in the Nascent Phototrophic Endosymbiosis,Paramecium bursaria

“…The ciliate-specific scan (scn)RNA (25-nt) and internal eliminated sequence (ies)RNA (~28-nt) pathways are endogenous, and function primarily to eliminate the bulk of noncoding DNA present in the germline micronuclear genome during development of the somatic macronucleus 35,[41][42][43]46,47 . Paramecium also encodes a shortinterfering (si)RNA (23-nt) pathway capable of processing both exogenously [48][49][50][51] and endogenously 32,44 derived RNA precursors. Although siRNA is believed to have evolved to protect against foreign genetic elements (such as viruses, transposons and transgenes) 52 , some siRNA-based RNAi factors have also been implicated in the regulation of endogenous transcriptome expression in the non-photo-endosymbiotic model system, P. tetraurelia 32 .…”

Two Piwis with Ago-like functions silence somatic genes at the chromatin level

“…In addition, Ptiwi13 also enriches for fragments of rRNA and snRNA. In contrast, Ptiwi14 IPs show accumulation of transgene-associated sRNAs and other siRNA producing genes (SRCs) of the Paramecium genome [23]. Our data indicates that Ptiwi14 exclusively loads these two classes and interestingly both are also enriched in Ptiwi13.…”

“…We finally had a look for endogenous siRNAs. We have recently described siRNA producing loci in the Paramecium genome showing read length preference of 23nt [23]. Figure 7A shows also for these endogenous clusters a predominant overlap of 21nt indicating Dicer to be involved at least in the majority of them.…”

“…Ptiwi IPs show that both Ptiwis select for 23nt siRNAs which is the predominant length of somatic sRNAs in Paramecium [23,30], but this is not the case for all Ptiwis as developmental Ptiwi09 does not show a clear length preference for sRNAs [12]. In transgene-induced silencing, it seems likely that the 5'-U preference is due to Ptiwi selection rather than a Dicer1 preference because the preference differs between both Ptiwis.…”

The Obligate Symbiont “Candidatus Megaira polyxenophila” Has Variable Effects on the Growth of Different Host Species

“…Paramecium (Ciliophora, Oligohymenophorea) is a unicellular protist with a broad, nearly global distribution in fresh and brackish water bodies. This ciliate is studied, among other things ( Karunanithi et al, 2019 ; Kelz and Mashour, 2019 ; Mayne et al, 2019 ; Soares et al, 2019 ; Arnaiz et al, 2020 ) for the abundance and diversity of its endosymbionts ( Floriano et al, 2018 ; Garushyants et al, 2018 ; Grosser et al, 2018 ; Potekhin et al, 2018 ; Sabaneyeva et al, 2018 ; Schrallhammer et al, 2018 ; Castelli et al, 2019a , b ; Fokin et al, 2019 ; Koehler et al, 2019 ; Lanzoni et al, 2019 ; Plotnikov et al, 2019 ). Host-symbiont interactions and their outcome have been studied for example using Holospora ( Lohse et al, 2006 ; Hori et al, 2008 ; Fokin and Görtz, 2009 ; Nidelet et al, 2009 ; Duncan et al, 2013 , 2018 ; Banerji et al, 2015 ; Castelli et al, 2015 ; Dusi et al, 2015 ; Garushyants et al, 2018 ; Grosser et al, 2018 ) Caedibacter ( Kusch et al, 2002 ; Dusi et al, 2014 ; Grosser et al, 2018 ; Schu and Schrallhammer, 2018 ; Koehler et al, 2019 ) and Preeria ( Bella et al, 2016 ; Potekhin et al, 2018 ).…”