Identification of AGO3-Associated miRNAs and Computational Prediction of Their Targets in the Green Alga Chlamydomonas reinhardtii

Abstract

The unicellular green alga Chlamydomonas reinhardtii harbors many types of small RNAs (sRNAs) but little is known about their role(s) in the regulation of endogenous genes and cellular processes. To define functional microRNAs (miRNAs) in Chlamydomonas, we characterized sRNAs associated with an argonaute protein, AGO3, by affinity purification and deep sequencing. Using a stringent set of criteria for canonical miRNA annotation, we identified 39 precursor miRNAs, which produce 45 unique, AGO3-associated miRNA sequences including 13 previously reported miRNAs and 32 novel ones. Potential miRNA targets were identified based on the complementarity of miRNAs with candidate binding sites on transcripts and classified, depending on the extent of complementarity, as being likely to be regulated through cleavage or translational repression. The search for cleavage targets identified 74 transcripts. However, only 6 of them showed an increase in messenger RNA (mRNA) levels in a mutant strain almost devoid of sRNAs. The search for translational repression targets, which used complementarity criteria more stringent than those empirically required for a reduction in target protein levels, identified 488 transcripts. However, unlike observations in metazoans, most predicted translation repression targets did not show appreciable changes in transcript abundance in the absence of sRNAs. Additionally, of three candidate targets examined at the protein level, only one showed a moderate variation in polypeptide amount in the mutant strain. Our results emphasize the difficulty in identifying genuine miRNA targets in Chlamydomonas and suggest that miRNAs, under standard laboratory conditions, might have mainly a modulatory role in endogenous gene regulation in this alga.

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).…”

“…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).…”

“…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). Interestingly, there appears to be little conservation of miRNA genes among algal species or with those encoded in the genomes of land plants or animals (Cerutti et al, 2011;Li et al, 2014;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. Interestingly, most characterized land plant miRNAs appear to regulate transcripts with highly complementary binding sites and preferentially trigger their endonucleolytic cleavage by Argonaute proteins (Bartel, 2009;Axtell, 2013;Rogers and Chen, 2013).…”

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

“…It is likely, therefore, that there are at least 43 mRNAs in C. reinhardtii that may be subject to direct cleavage by DCL3. 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).…”