Academic journal article Genetics

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

Academic journal article Genetics

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

Article excerpt

RNA interference (RNAi) provides eukaryotic cells with a precise means of gene expression regulation through a variety of mechanisms (Kim 2005; Bartel 2009; Voinnet 2009). MicroRNAs, key components of the RNAi machinery, consist of endogenously encoded 20- to 24-nucleotide (nt) RNA molecules (Kim 2005). In plants and some green algae, these molecules are predominantly derived from RNA hairpins up to 300 bp in length that are processed by RNase III enzymes, the Dicer-like (DCL) family of proteins (Casas-Mollano et al. 2008; Voinnet 2009; Tarver et al. 2012). In Arabidopsis thaliana, most microRNAs (miRNAs) are generated by DCL1 in two processing steps, from the initial transcript [the primary miRNA (pri-miRNA)] to a smaller hairpin miRNA precursor (pre-miRNA) and then to a short duplex consisting of the miRNA and its complementary sequence (the miRNA* or passenger strand) (Voinnet 2009; Zhu et al. 2013). The short RNA duplex is loaded into the RNA-induced silencing complex (RISC), which contains an argonaute (AGO) protein as a central component and becomes active upon removal of the miRNA* strand. Active RISC can repress expression of target messenger RNAs (mRNAs) by preventing translation and/or by triggering transcript degradation (Bartel 2009; Voinnet 2009).

The extent of complementarity between miRNA and target mRNA has been proposed to play a major role in determining whether silencing occurs through transcript cleavage or translational repression/mRNA destabilization (Bartel 2009). The first nucleotide at the 59 end of a miRNA is secured within the middle (Mid) domain of argonaute, making this nucleotide inaccessible for binding with target transcripts, although it is important for determining miRNA loading into distinct AGOs (Mi et al. 2008; Wang et al. 2008a; Frank et al. 2010, 2012; Zha et al. 2012; Endo et al. 2013). Nucleotides 2-8 from the 59 end of a miRNA (the seed region, see Supporting Information, Figure S1) are often almost entirely complementary to a target mRNA, while the remaining nucleotides may contribute variably to the pairing interaction (Wang et al. 2008b; Bartel 2009; Sheng et al. 2014). In land plants, the complementarity of miRNAs and targets is commonly nearly perfect, resulting in cleavage of the target sequence between nucleotides 10 and 11 of the miRNA (Rogers and Chen 2013; Shen et al. 2013; Zhou and Luo 2013; Liu et al. 2014). In animals, by contrast, miRNAs frequently display extensive mismatching with their targets and regulate target expression through translational repression and/or transcript destabilization independent of AGO-mediated cleavage (Carthew and Sontheimer 2009; Djuranovic et al. 2012; Fujiwara and Yada 2013; Zheng et al. 2013). There is a growing body of evidence indicating that plants can perform this type of regulation as well; however, it still appears to be much less common than regulation through direct cleavage (Lanet et al. 2009; Beauclair et al. 2010; Yang et al. 2012; Axtell 2013; Li et al. 2013).

In the unicellular alga Chlamydomonas reinhardtii, miRNAs have been described relatively recently, based on deep sequencing of total cellular small RNA (sRNA) populations (Molnar et al. 2007; Zhao et al. 2007; Ibrahim et al. 2010; Shu and Hu 2012; Lv et al. 2013). However, the validity of many predicted Chlamydomonas miRNAs has been questioned of late because of their large hairpin precursor structures and the apparent imprecise processing of the sRNAs (Nozawa et al. 2012; Tarver et al. 2012). C. reinhardtii possesses core miRNA/sRNA processing and effector machinery similar to that in higher plants (Casas-Mollano et al. 2008). This machinery includes three DCL proteins along with three AGO proteins. DCL1 and AGO1 appear to be primarily involved in the silencing of transposable elements whereas AGO3 seems to be most extensively involved in miRNA functions (Casas-Mollano et al. 2008). MicroRNA quality control is at least partially directed by the MUT68 protein, which functions by placing untemplated nucleotides (generally uridyls) at the 39 end of the miRNA molecules, flagging them for degradation (Ibrahim et al. …

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