Diversification of the Core RNA Interference Machinery in Chlamydomonas reinhardtii and the Role of DCL1 in Transposon Silencing

Abstract

Small RNA-guided gene silencing is an evolutionarily conserved process that operates by a variety of molecular mechanisms. In multicellular eukaryotes, the core components of RNA-mediated silencing have significantly expanded and diversified, resulting in partly distinct pathways for the epigenetic control of gene expression and genomic parasites. In contrast, many unicellular organisms with small nuclear genomes seem to have lost entirely the RNA-silencing machinery or have retained only a basic set of components. We report here that Chlamydomonas reinhardtii, a unicellular eukaryote with a relatively large nuclear genome, has undergone extensive duplication of Dicer and Argonaute polypeptides after the divergence of the green algae and land plant lineages. Chlamydomonas encodes three Dicers and three Argonautes with DICER-LIKE1 (DCL1) and ARGONAUTE1 being more divergent than the other paralogs. Interestingly, DCL1 is uniquely involved in the post-transcriptional silencing of retrotransposons such as TOC1. Moreover, on the basis of the subcellular distribution of TOC1 small RNAs and target transcripts, this pathway most likely operates in the nucleus. However, Chlamydomonas also relies on a DCL1-independent, transcriptional silencing mechanism(s) for the maintenance of transposon repression. Our results suggest that multiple, partly redundant epigenetic processes are involved in preventing transposon mobilization in this green alga.

Most microRNAs in the single-cell alga Chlamydomonas reinhardtii are produced by Dicer-like 3-mediated cleavage of introns and untranslated regions of coding RNAs

“…A role under starvation of sulphate and/or phosphate is possible because these conditions affect sRNA profiles in Chlamydomonas (Shu and Hu 2012;Zheng et al 2015). The DCL3-dependent silencing might also act redundantly with other silencing systems as indicated by the loss of transposon silencing in C. reinhardtii that was dependent on loss of function at both DCL1 and of a histone methyltransferase (Casas-Mollano et al 2008). The availability of DCL3 mutants will now allow us to test these possibilities.…”

“…It has a complex RNA silencing machinery with three DCLs (DCL1-3) and three AGOs (AGO1-3) (Merchant et al 2007;Casas-Mollano et al 2008). These proteins are not encoded by orthologs of genes in higher plants, although it is well established that C. reinhardtii sRNAs, including miRNAs, are like those of higher plants in that they direct cleavage of their mRNA targets (Molnár et al 2007;Zhao et al 2007).…”

RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii

“…Although many studies have shed light on the mechanism(s) of RNA-mediated gene silencing, it remains poorly understood in the unicellular green alga Chlamydomonas reinhardtii (32)(33)(34)(35)(36)(37)(38)(39)(40)(41), particularly with respect to miRNA biogenesis. Here we show that the Chlamydomonas RNA-binding protein Dull slicer-16 (DUS16), which has an ssRNA-binding domain (RRM) and a dsRNA-binding domain (dsRBD), associates with nascent primiRNA transcripts cotranscriptionally and assists in processing pri-miRNAs into mature miRNAs.…”

Uridylation of mature miRNAs and siRNAs by the MUT68 nucleotidyltransferase promotes their degradation in Chlamydomonas

“…miRNAs originate from endogenous noncoding RNA transcripts or introns that fold into imperfect stem loop structures and often modulate the expression of genes with roles in development, physiological processes, or stress responses (1)(2)(3). siRNAs are produced from long, near-perfect complementarity doublestranded RNAs (dsRNAs) of diverse origins, including the transcripts of long inverted repeats, the products of convergent transcription or RNA-dependent RNA polymerase activity, viral RNAs, or dsRNAs experimentally introduced into cells (1)(2)(3)(4)(5). These siRNAs play various roles in posttranscriptional regulation of gene expression, suppression of viruses and transposable elements, and/or heterochromatin formation (1)(2)(3)(4)(5).…”

“…siRNAs are produced from long, near-perfect complementarity doublestranded RNAs (dsRNAs) of diverse origins, including the transcripts of long inverted repeats, the products of convergent transcription or RNA-dependent RNA polymerase activity, viral RNAs, or dsRNAs experimentally introduced into cells (1)(2)(3)(4)(5). These siRNAs play various roles in posttranscriptional regulation of gene expression, suppression of viruses and transposable elements, and/or heterochromatin formation (1)(2)(3)(4)(5). Hairpin and long dsRNAs are processed into sRNAs by an RNaseIII-like endonuclease named Dicer (1, 2).…”