Complementarity to an miRNA seed region is sufficient to induce moderate repression of a target transcript in the unicellular green alga Chlamydomonas reinhardtii

Abstract

SUMMARYMicroRNAs (miRNAs) are 20-24 nt non-coding RNAs that play important regulatory roles in a broad range of eukaryotes by pairing with mRNAs to direct post-transcriptional repression. The mechanistic details of miRNA-mediated post-transcriptional regulation have been well documented in multicellular model organisms. However, this process remains poorly studied in algae such as Chlamydomonas reinhardtii, and specific features of miRNA biogenesis, target mRNA recognition and subsequent silencing are not well understood. In this study, we report on the characterization of a Chlamydomonas miRNA, cre-miR1174.2, , which is processed from a near-perfect hairpin RNA. Using Gaussia luciferase (gluc) reporter genes, we have demonstrated that cre-miR1174.2 is functional in Chlamydomonas and capable of triggering site-specific cleavage at the center of a perfectly complementary target sequence. A mismatch tolerance test assay, based on pools of transgenic strains, revealed that target hybridization to nucleotides of the seed region, at the 5′ end of an miRNA, was sufficient to induce moderate repression of expression. In contrast, pairing to the 3′ region of the miRNA was not critical for silencing. Our results suggest that the base-pairing requirements for small RNAmediated repression in C. reinhardtii are more similar to those of metazoans compared with the extensive complementarity that is typical of land plants. Individual Chlamydomonas miRNAs may potentially modulate the expression of numerous endogenous targets as a result of these relaxed base-pairing requirements.

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

“…Remarkably, six of 13 UTR miRNAs bind AGO3 (Voshall et al 2015), one of the three AGO proteins in C. reinhardtii. These observations prompt us to suggest that C. reinhardtii DCL3, like animal Drosha, has a dual role in mRNA regulation: It is first a ribonuclease that controls the levels of certain mRNAs by direct cleavage; and second, it is involved in biogenesis of sRNAs that act in trans to influence either mRNA accumulation or translation (Ma et al 2013;Yamasaki et al 2013;Voshall et al 2015).…”

“…Effective miRNA silencing in higher plants depends on near complete complementarity of the miRNA and its target (Liu et al 2014) whereas, in C. reinhardtii, pairing in the miRNA seed region is sufficient to induce down-regulation (Yamasaki et al 2013). …”

Gene silencing in microalgae: Mechanisms and biological roles

“…This also appears to be the case for some miRNAs in both C. reinhardtii and V. carteri, where expected target RNA cleavage products have been experimentally detected (Molnar et al, 2007;Zhao et al, 2007;Li et al, 2014;Voshall et al, 2014). Yet, very few of the reported Chlamydomonas or Volvox miRNAs have identifiable targets with near perfect complementarity (Molnar et al, 2007;Li et al, 2014;Voshall et al, 2014) and recent evidence suggests that miRNA regulation of transcript expression in Chlamydomonas may operate, at least for certain targets, by translation repression (Ma et al, 2013;Yamasaki et al, 2013;Voshall et al, 2014).…”

“…Within the examined microalgae, candidate miRNAs have been experimentally identified in C. reinhardtii (Molnar et al, 2007;Zhao et al, 2007;Yamasaki et al, 2013;Voshall et al, 2014) and in V. carteri (Li et al, 2014), and potential endogenous targets have been predicted by computational approaches (Molnar et al, 2007;Zhao et al, 2007;Li et al, 2014;Voshall et al, 2014). However, target gene identification is a challenging problem (Thomas et al, 2010;Voshall et al, 2014) and the false-positive prediction rate for miRNA targets in microalgae is currently unknown.…”

“…The occurrence of small RNAs, potentially generated by Dicer-mediated processing, has been examined in very few of the sampled algal species. C. reinhardtii has been shown to contain a complex set of endogenous sRNAs including miRNAs, phased siRNAs, as well as siRNAs originating from transposons and repeated DNA regions (Molnar et al, 2007;Zhao et al, 2007;Yamasaki et al, 2013;Voshall et al, 2014). Likewise, V. carteri has been demonstrated to possess miRNAs, many of them being preferentially enriched in either gonidia or somatic cells (Li et al, 2014).…”