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      Characterization of the Small RNA Transcriptome of the Marine Coccolithophorid, Emiliania huxleyi

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          Abstract

          Small RNAs (smRNAs) control a variety of cellular processes by silencing target genes at the transcriptional or post-transcription level. While extensively studied in plants, relatively little is known about smRNAs and their targets in marine phytoplankton, such as Emiliania huxleyi (E. huxleyi). Deep sequencing was performed of smRNAs extracted at different time points as E. huxleyi cells transition from logarithmic to stationary phase growth in batch culture. Computational analyses predicted 18 E. huxleyi specific miRNAs. The 18 miRNA candidates and their precursors vary in length (18–24 nt and 71–252 nt, respectively), genome copy number (3–1,459), and the number of genes targeted (2–107). Stem-loop real time reverse transcriptase (RT) PCR was used to validate miRNA expression which varied by nearly three orders of magnitude when growth slows and cells enter stationary phase. Stem-loop RT PCR was also used to examine the expression profiles of miRNA in calcifying and non-calcifying cultures, and a small subset was found to be differentially expressed when nutrients become limiting and calcification is enhanced. In addition to miRNAs, endogenous small RNAs such as ra-siRNAs, ta-siRNAs, nat-siRNAs, and piwiRNAs were predicted along with the machinery for the biogenesis and processing of si-RNAs. This study is the first genome-wide investigation smRNAs pathways in E. huxleyi. Results provide new insights into the importance of smRNAs in regulating aspects of physiological growth and adaptation in marine phytoplankton and further challenge the notion that smRNAs evolved with multicellularity, expanding our perspective of these ancient regulatory pathways.

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          Most cited references46

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          psRNATarget: a plant small RNA target analysis server

          Plant endogenous non-coding short small RNAs (20–24 nt), including microRNAs (miRNAs) and a subset of small interfering RNAs (ta-siRNAs), play important role in gene expression regulatory networks (GRNs). For example, many transcription factors and development-related genes have been reported as targets of these regulatory small RNAs. Although a number of miRNA target prediction algorithms and programs have been developed, most of them were designed for animal miRNAs which are significantly different from plant miRNAs in the target recognition process. These differences demand the development of separate plant miRNA (and ta-siRNA) target analysis tool(s). We present psRNATarget, a plant small RNA target analysis server, which features two important analysis functions: (i) reverse complementary matching between small RNA and target transcript using a proven scoring schema, and (ii) target-site accessibility evaluation by calculating unpaired energy (UPE) required to ‘open’ secondary structure around small RNA’s target site on mRNA. The psRNATarget incorporates recent discoveries in plant miRNA target recognition, e.g. it distinguishes translational and post-transcriptional inhibition, and it reports the number of small RNA/target site pairs that may affect small RNA binding activity to target transcript. The psRNATarget server is designed for high-throughput analysis of next-generation data with an efficient distributed computing back-end pipeline that runs on a Linux cluster. The server front-end integrates three simplified user-friendly interfaces to accept user-submitted or preloaded small RNAs and transcript sequences; and outputs a comprehensive list of small RNA/target pairs along with the online tools for batch downloading, key word searching and results sorting. The psRNATarget server is freely available at http://plantgrn.noble.org/psRNATarget/.
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            A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana.

            To better understand the diversity of small silencing RNAs expressed in plants, we employed high-throughput pyrosequencing to obtain 887,000 reads corresponding to Arabidopsis thaliana small RNAs. They represented 340,000 unique sequences, a substantially greater diversity than previously obtained in any species. Most of the small RNAs had the properties of heterochromatic small interfering RNAs (siRNAs) associated with DNA silencing in that they were preferentially 24 nucleotides long and mapped to intergenic regions. Their density was greatest in the proximal and distal pericentromeric regions, with only a slightly preferential propensity to match repetitive elements. Also present were 38 newly identified microRNAs (miRNAs) and dozens of other plausible candidates. One miRNA mapped within an intron of DICER-LIKE 1 (DCL1), suggesting a second homeostatic autoregulatory mechanism for DCL1 expression; another defined the phase for siRNAs deriving from a newly identified trans-acting siRNA gene (TAS4); and two depended on DCL4 rather than DCL1 for their accumulation, indicating a second pathway for miRNA biogenesis in plants. More generally, our results revealed the existence of a layer of miRNA-based control beyond that found previously that is evolutionarily much more fluid, employing many newly emergent and diverse miRNAs, each expressed in specialized tissues or at low levels under standard growth conditions.
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              GtRNAdb: a database of transfer RNA genes detected in genomic sequence

              Transfer RNAs (tRNAs) represent the single largest, best-understood class of non-protein coding RNA genes found in all living organisms. By far, the major source of new tRNAs is computational identification of genes within newly sequenced genomes. To organize the rapidly growing collection and enable systematic analyses, we created the Genomic tRNA Database (GtRNAdb), currently including over 74 000 tRNA genes predicted from 740 species. The web resource provides overview statistics of tRNA genes within each analyzed genome, including information by isotype and genetic locus, easily downloadable primary sequences, graphical secondary structures and multiple sequence alignments. Direct links for each gene to UCSC eukaryotic and microbial genome browsers provide graphical display of tRNA genes in the context of all other local genetic information. The database can be searched by primary sequence similarity, tRNA characteristics or phylogenetic group. The database is publicly available at http://gtrnadb.ucsc.edu.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                21 April 2016
                2016
                : 11
                : 4
                : e0154279
                Affiliations
                [1 ]Department of Computer Science and Information Systems, California State University, San Marcos, CA, 92096, United States of America
                [2 ]Department of Biological Sciences, California State University, San Marcos, CA, 92096, United States of America
                [3 ]School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich, Norfolk, NR4 7TJ, United Kingdom
                Mount Allison University, CANADA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: BR XZ. Performed the experiments: BR SC EC AH. Analyzed the data: XZ JG ARH. Contributed reagents/materials/analysis tools: TM. Wrote the paper: XZ BR.

                Article
                PONE-D-14-51739
                10.1371/journal.pone.0154279
                4839659
                27101007
                6044fa85-9ba2-4bb9-8439-9a4752b8803b
                © 2016 Zhang et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 17 November 2014
                : 11 April 2016
                Page count
                Figures: 7, Tables: 2, Pages: 26
                Funding
                Funded by: NIH SCORE
                Award ID: 5SC3GM092765
                Award Recipient :
                Support for the work of XZ and BR was provided by National Institutes of Health (NIH) Support of Competitive Research (SCORE) Grant 5SC3GM092765 “Characterization of Small Silencing RNAs in Emiliania Huxleyi”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and life sciences
                Genetics
                Gene expression
                Gene regulation
                MicroRNAs
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                MicroRNAs
                Biology and life sciences
                Genetics
                Gene expression
                Gene regulation
                Small interfering RNAs
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Non-coding RNA
                Small interfering RNAs
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Alignment
                Research and Analysis Methods
                Molecular Biology Techniques
                Sequencing Techniques
                Sequence Analysis
                Sequence Alignment
                Research and Analysis Methods
                Database and Informatics Methods
                Biological Databases
                Genomic Databases
                Biology and Life Sciences
                Computational Biology
                Genome Analysis
                Genomic Databases
                Biology and Life Sciences
                Genetics
                Genomics
                Genome Analysis
                Genomic Databases
                Biology and Life Sciences
                Computational Biology
                Genome Analysis
                Genomic Libraries
                Biology and Life Sciences
                Genetics
                Genomics
                Genome Analysis
                Genomic Libraries
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Double stranded RNA
                Biology and life sciences
                Genetics
                Epigenetics
                RNA interference
                Biology and life sciences
                Genetics
                Gene expression
                RNA interference
                Biology and life sciences
                Genetics
                Genetic interference
                RNA interference
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                RNA interference
                Research and analysis methods
                Extraction techniques
                RNA extraction
                Custom metadata
                All small RNA sequence data are available from the NCBI SRA database (accession number SRX756940).

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