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

Sivarajan Karunanithi,Karl Nordström,Marcello Pirritano,Franziska Drews,Vidya Oruganti,Martin Simon,Simone Marker,Angela M Rodriguez-Viana,Marcel H Schulz

doi:10.1093/nar/gkz553

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.

Highlights

RNA interference (RNAi) is a conserved eukaryotic mechanism, which involves small non coding RNAs to regulate gene expression and genome integrity
Paramecium undergoes antigenic variation similar to pathogenic protists; exclusive expression of surface antigen genes are associated with environmental parameters such as cultivation temperature [17,40] and we have previously shown that individual serotypes are associated with massive transcriptome alterations, rather than switching of the surface antigen only [27]
We have described the same dependency for 1o short interfering RNAs (siRNAs) produced from exogenous double-stranded RNA (dsRNA), it is still not understood why RNA-dependent RNA polymerase (RDR) are necessary for dicing the dsRNA [15]

Summary

Introduction

RNA interference (RNAi) is a conserved eukaryotic mechanism, which involves small non coding RNAs to regulate gene expression and genome integrity. Among the broad variety of small RNA biogenesis pathways and functions, micro RNAs (miRNAs) and short interfering RNAs (siRNAs) are two of the best studied classes [1]. The latter are usually produced from double-stranded RNA (dsRNA) precursors arising from bidirectional transcription or activity of a RNA-dependent RNA polymerase (RDR). This dsRNA acts as a substrate for Dicer, a RNAse III domain containing enzyme, which cleaves dsRNA into discrete siRNA duplexes.

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Conclusion