Identification of microRNAs in the Toxigenic Dinoflagellate Alexandrium catenella by High-Throughput Illumina Sequencing and Bioinformatic Analysis

Abstract

Micro-ribonucleic acids (miRNAs) are a large group of endogenous, tiny, non-coding RNAs consisting of 19–25 nucleotides that regulate gene expression at either the transcriptional or post-transcriptional level by mediating gene silencing in eukaryotes. They are considered to be important regulators that affect growth, development, and response to various stresses in plants. Alexandrium catenella is an important marine toxic phytoplankton species that can cause harmful algal blooms (HABs). To date, identification and function analysis of miRNAs in A. catenella remain largely unexamined. In this study, high-throughput sequencing was performed on A. catenella to identify and quantitatively profile the repertoire of small RNAs from two different growth phases. A total of 38,092,056 and 32,969,156 raw reads were obtained from the two small RNA libraries, respectively. In total, 88 mature miRNAs belonging to 32 miRNA families were identified. Significant differences were found in the member number, expression level of various families, and expression abundance of each member within a family. A total of 15 potentially novel miRNAs were identified. Comparative profiling showed that 12 known miRNAs exhibited differential expression between the lag phase and the logarithmic phase. Real-time quantitative RT-PCR (qPCR) was performed to confirm the expression of two differentially expressed miRNAs that were one up-regulated novel miRNA (aca-miR-3p-456915), and one down-regulated conserved miRNA (tae-miR159a). The expression trend of the qPCR assay was generally consistent with the deep sequencing result. Target predictions of the 12 differentially expressed miRNAs resulted in 1813target genes. Gene ontology (GO) analysis and the Kyoto Encyclopedia of Genes and Genomes pathway database (KEGG) annotations revealed that some miRNAs were associated with growth and developmental processes of the alga. These results provide insights into the roles that miRNAs play in the growth of A. catenella, and they provide the basis for further studies of the molecular mechanisms that underlie bloom growth in red tides species.

Biosynthesis of Saxitoxin in Marine Dinoflagellates: An Omics Perspective

“…Regulation of saxitoxin biosynthesis at the post-transcriptional level via trans-splicing is additionally bolstered by sequence analysis of sxtA4 and sxtG transcripts that demonstrated the presence of a dinoflagellate spliced leader sequence in these genes [43,44]. Additionally, apart from the coding RNA transcripts in dinoflagellates, there are many non-coding RNA transcripts in dinoflagellates, e.g., microRNA (miRNA) [76][77][78][79][80]. These miRNAs were observed to post-transcriptionally direct the expression of several genes associated with different biological processes in dinoflagellates [79,80].…”

“…These miRNAs were observed to post-transcriptionally direct the expression of several genes associated with different biological processes in dinoflagellates [79,80]. Given their potential role in gene regulation, the significance of miRNAs in regulating saxitoxin biosynthesis cannot be ignored, as several miRNAs were documented in saxitoxin-producing dinoflagellates [77,78]. In most eukaryotes, the protein translational mechanism begins with eukaryotic translation initiation factor 4E (eIF4E) through its interaction with the 5'-cap structure of mRNA [81].…”

“…Enzyme in the TCA cycle exhibited circadian changes in accordance with protein abundance, whereas its messenger RNA (mRNA) level remained constant throughout the cycle [84] Presence of unique splice leader at 5' of dinoflagellates mRNA might provide translational regulation in dinoflagellates via trans-splicing [72,73] Expression of conserved S-phase genes in Karenia brevis remains unchanged throughout cell cycle, but other protein expression level was observed [85] Presence of dinoflagellate spliced leader sequence at 5' of sxtA and sxtG genes might indicate that saxitoxin biosynthesis is regulated at the translational level [43,44] Daily circadian system in dinoflagellate Lingulodinium showed lack of regulation at the transcript level using RNA-sequencing approach, suggesting the involvement of translational or post-translational control of this system [86] Identification of microRNAs (miRNAs) in several species of dinoflagellates, including saxitoxin-producing dinoflagellates, indicates regulation of several genes in dinoflagellates at post-transcriptional level via a small RNA gene silencing mechanism [76][77][78][79][80] Characterization of extensive transcript encoding protein elF4E family in dinoflagellates [83] Genome sequence of Symbiodinium kawagutii revealed substantial translational control by miRNA in biological processes involving carbohydrate metabolism, transcription regulation, and biosynthesis of amino acids and antibiotics [79] Poor correlation between protein and mRNA level in dinoflagellate Lingulodinium [87]…”

Current Knowledge and Recent Advances in Marine Dinoflagellate Transcriptomic Research

“…Only recently, a number of studies have started looking into the diversity of miRNA in dinoflagellates using a transcriptomics approach [31][32][33][34]. Baumgarten et al [31] discovered that Symbiodinium microadriaticum not only produce miRNA, but also small (or short) interfering RNA (siRNA).…”

RNA-Seq as an Emerging Tool for Marine Dinoflagellate Transcriptome Analysis: Process and Challenges

“…To date, the transcriptomics approach using either microarray or RNA-seq only record the levels of RNA (cDNA) present and are thus not able to detect any post transcriptional events [79]. Although until now there is no detailed study regarding correlation between mRNA and protein abundance in dinoflagellates, this limitation needs to be considered as there is strong proof for post-transcriptional gene regulation such as regulation by miRNA in dinoflagellates [4,19,24,33,[80][81][82].…”