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

Abstract

We describe here a forward genetic screen to investigate the biogenesis, mode of action, and biological function of miRNAmediated RNA silencing in the model algal species, Chlamydomonas reinhardtii. Among the mutants from this screen, there were three at Dicer-like 3 that failed to produce both miRNAs and siRNAs and others affecting diverse post-biogenesis stages of miRNA-mediated silencing. The DCL3-dependent siRNAs fell into several classes including transposon-and repeat-derived siRNAs as in higher plants. The DCL3-dependent miRNAs differ from those of higher plants, however, in that many of them are derived from mRNAs or from the introns of pre-mRNAs. Transcriptome analysis of the wild-type and dcl3 mutant strains revealed a further difference from higher plants in that the sRNAs are rarely negative switches of mRNA accumulation. The few transcripts that were more abundant in dcl3 mutant strains than in wild-type cells were not due to sRNAtargeted RNA degradation but to direct DCL3 cleavage of miRNA and siRNA precursor structures embedded in the untranslated (and translated) regions of the mRNAs. Our analysis reveals that the miRNA-mediated RNA silencing in C. reinhardtii differs from that of higher plants and informs about the evolution and function of this pathway in eukaryotes.

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

“…Consistent with DUS16's being part of a microprocessor complex, affinity purification of tagged DUS16 and subsequent mass spectrometry analysis revealed that the protein associates with DICER-LIKE 3 (DCL3), which is the predominant DICER function in Chlamydomonas (Fig. 4F) (41). These results suggest that DUS16 recognizes and associates with nascent pri-miRNAs cotranscriptionally and assists DCL3-mediated processing of pri-miRNAs.…”

“…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.…”

Global characterization of the Dicer-like protein DrnB roles in miRNA biogenesis in the social amoeba Dictyostelium discoideum

“…Similar division of functionality between Dicer/Dicer-like proteins is also seen in other organisms such as plants and the two Dicers present in Drosophila where Dicer-1 and Dicer-2 are devoted to miRNA and siRNA maturation, respectively [6,57,58]. However, in the single cell alga C. reinhardtii , one of the three Dicer homologs, DCL-3 is required for both miRNA and siRNA maturation, a situation similar to mammals and worms where a single Dicer protein produce both classes of small RNAs [31,57]. It should be noted that a small proportion of the small RNAs, derived from the LTR retrotransposon TRE-3A [41], for unknown reasons accumulate in drnB - cells.…”

“…No obvious homolog to Drosha is present in D. discoideum eventhough the domain structure of DrnB is similar[30]. Interestingly, this Drosha-like domain arrangement is also present in in DCL-3, the Dicer protein responsible for miRNA maturation in the unicellular alga C. reinhardtii [31]. A feature of the miRNA machinery in D. discoideum shared with animals is the absence of 3ʹ methylation of miRNAs.…”

“…Interestingly, the domain composition of DrnB is more similar to Drosha than e.g. Dicer-like proteins in plant, a feature shared with the DCL-3 involved in miRNA maturation in C. reinhadtii [30,31]. RbdB together with DrnB has been proposed to constitute the microprocessor in D. discoideum and depletion of RbdB abolishes the maturation of some tested miRNAs, similar to the results shown for strains where drnB has been disrupted [28,29].…”