Only recently it has been shown that animal and plant genomes produce numerous small, noncoding RNAs that act as a guide for the Argonaute effector protein regulating gene expression and affecting processes of metabolism, development, epigenetic inheritance, and others [1–4]. Three classes of small RNAs (smRNAs) have been described, microRNAs (miRNAs), small interfering RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs) . miRNAs are the most common and best understood class of non-coding RNAs, but with ongoing research in the field of RNAi, differences and similarities in biogenesis and functionality of the different smRNA classes are becoming clearer . miRNAs are ~22 nt small non-coding RNAs implicated in the regulation of gene expression in development and cell differentiation, the immune system, and homeostasis [7, 8]. Homologous binding of a miRNA to its target genes leads to mRNA degradation and translational inhibition but also induces DNA methylation [9–14].
miRNAs are assumed to occur at a frequency of approximately 1% - 2% of the total number of genes in the genome of an organism . Furthermore, it is estimated that about 20% to 30% of human genes are targeted by miRNAs as indicated by conserved seed pairing, often flanked by adenosines . After the discovery of the first miRNAs in Caenorhabditis elegans, sequencing surveys have identified miRNAs in more than 100 organisms including those at the base of the metazoan tree . Only recently, miRNAs have been shown to be expressed in unicellular eukaryotes and algae, e.g. Chlamydomonas reinhardtii and Ectocarpus siliculosus. Accordingly, it has been suggested that miRNAs have a long evolutionary history among eukaryotes . However, a recent study by Tarver et al.  that proposed a number of criteria to unambiguously identify miRNAs (e.g. presence of miRNA and miRNA*, non-repetitive match to the genome, miRNA and miRNA* form a 2 nt overhang on the 3′ ends of the duplex) showed that the majority of identified miRNA types from unicellular protists might be explained by alternative means. The authors consequently stated that while the RNAi core molecular pathway and genes are conserved among eukaryotes (e.g. Dicer and Argonaute proteins), the products they produce are not, and hence RNAi might be an example of molecular exaptation .
Dinoflagellates are typically unicellular, photosynthetic, free-swimming, biflagellate organisms. They are important primary producers and constitute an important component of freshwater and marine phytoplanktonic communities. There are currently ~2,000 living species of dinoflagellates known, which are classified in ~125 genera. Dinoflagellates form one of the three main phyla of the alveolates (together with the ciliates and apicomplexans) . About half of all dinoflagellates are autotrophic (photosynthetic), some are heterotrophic, saprophytic, symbiotic, or even parasitic. The autotrophic dinoflagellates are either free-living, or associated with a broad range of hosts as endosymbionts. Dinoflagellates possess unique molecular traits that differ from ‘classical’ model organisms. For instance, dinoflagellates have permanently condensed chromosomes [21–23] and DNA that contains some 5-hydroxymethyluracil in place of thymine . Furthermore, dinoflagellates seem to harbor unusual genes and gene arrangements, such as unidirectional orientation of genes in the genome , bacterial type II RUBISCO , and minicircular plastid DNA . Recent transcriptome studies in dinoflagellates show that dinoflagellates have a paucity of common transcription factors, and seem to only regulate few genes at the level of transcription [21, 22, 27–29].
One of the most successful mutualistic associations of dinoflagellates is found with scleractinian corals, which contain members of the genus Symbiodinium as endosymbiotic algae. This endosymbiotic relationship provides the foundation of coral reef ecosystems by providing the energy to construct the three-dimensional framework of coral reefs . Together with a specific assemblage of bacteria (among other organisms) the coral host and dinoflagellate symbiont constitute the so-called coral holobiont . While coral reefs form biodiversity hotspots in the oceans, their presence is declining because of local (e.g. overfishing, eutrophication, tourism) and global (e.g. ocean acidification and warming) impacts . In order to characterize the molecular mechanisms driving these processes, understanding the contribution of each of the holobiont members to coral functioning is crucial. So far, researchers have conducted gene expression analyses mainly in the coral host [33–39] and looked at changes in the microbial community [40, 41], while large scale gene expression studies in Symbiodinium are lacking. Given the apparent paucity of regulation of gene expression in Symbiodinium and dinoflagellates, a study investigating the integrated expression of smRNAs and mRNAs presents a compelling possibility to determine the presence of RNAi-related regulatory mechanisms that act post-transcriptionally, and provide an alternative means of regulating gene expression.
In this study, we conducted a comprehensive smRNA and mRNA expression-profiling screen in the dinoflagellate Symbiodinium microadriaticum (clade A1, strain CCMP2467, strain synonym 370, National Center for Marine Algae and Microbiota), which is a photosynthetic symbiont of scleractinian corals. We sequenced and analyzed 9 different experimental treatments of a cultured strain via Illumina single and paired-end sequencing. We were interested in 1) understanding presence, diversity, and expression of smRNAs and mRNAs, 2) identifying proteins of the RNAi machinery, and 3) integrating smRNA and mRNA expression in order to identify functional links between genes and potential smRNA regulators.