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. 2013 Oct 12;14:704.
doi: 10.1186/1471-2164-14-704.

Integrating microRNA and mRNA Expression Profiling in Symbiodinium Microadriaticum, a Dinoflagellate Symbiont of Reef-Building Corals

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Free PMC article

Integrating microRNA and mRNA Expression Profiling in Symbiodinium Microadriaticum, a Dinoflagellate Symbiont of Reef-Building Corals

Sebastian Baumgarten et al. BMC Genomics. .
Free PMC article

Abstract

Background: Animal and plant genomes produce numerous small RNAs (smRNAs) that regulate gene expression post-transcriptionally affecting metabolism, development, and epigenetic inheritance. In order to characterize the repertoire of endogenous smRNAs and potential gene targets in dinoflagellates, we conducted smRNA and mRNA expression profiling over 9 experimental treatments of cultures from Symbiodinium microadriaticum, a photosynthetic symbiont of scleractinian corals.

Results: We identified a set of 21 novel smRNAs that share stringent key features with functional microRNAs from other model organisms. smRNAs were predicted independently over all 9 treatments and their putative gene targets were identified. We found 1,720 animal-like target sites in the 3'UTRs of 12,858 mRNAs and 19 plant-like target sites in 51,917 genes. We assembled a transcriptome of 58,649 genes and determined differentially expressed genes (DEGs) between treatments. Heat stress was found to produce a much larger number of DEGs than other treatments that yielded only few DEGs. Analysis of DEGs also revealed that minicircle-encoded photosynthesis proteins seem to be common targets of transcriptional regulation. Furthermore, we identified the core RNAi protein machinery in Symbiodinium.

Conclusions: Integration of smRNA and mRNA expression profiling identified a variety of processes that could be under microRNA control, e.g. protein modification, signaling, gene expression, and response to DNA damage. Given that Symbiodinium seems to have a paucity of transcription factors and differentially expressed genes, identification and characterization of its smRNA repertoire establishes the possibility of a range of gene regulatory mechanisms in dinoflagellates acting post-transcriptionally.

Figures

Figure 1
Figure 1
Overview of smRNA and mRNA analysis workflow. Cultures of Symbiodinium microadriaticum were subjected to 9 experimental treatments (noon: 12 h/12 h day/night cycle, sampled at noon; 4°C: 4°C for 4 hours; 16°C: 16°C for 4 hours; 34°C: 34°C for 12 hours; 36°C: 36°C for 4 hours; 20 g: 20 g/L NaCl salt content for 4 hours; 60 g: 60 g/L NaCl salt content for 4 hours; DS (dark stress): 18 hour dark period; DC (dark cycle): 12 h/12 h day/night cycle, sampled at midnight). Noon was selected as the reference condition for differential expression analyses. A total of 137 million small RNA reads resulted in the prediction of 219 miRNAs in 9 experimental treatments with the software miRDeep2, yielding a set of 21 smRNAs after further quality filtering. miRNA target gene prediction yielded 1,720 animal- and 19 plant-like miRNA binding sites via bowtie software in the set of 12,858 3'UTRs and 51,917 genes, respectively. Annotated miRNA targets were subsequently tested for GO category enrichment. A total of 302 million paired-end (PE) reads were assembled to a final gene set of 58,649 genes ≥ 250 bp with the Oases software. smRNA and mRNA expression over 9 experimental treatments was quantified with the DESeq software. Expression estimates of 21 smRNAs and 19,893 GO-annotated genes were assessed for correlation over 9 experimental treatments, and smRNAs-mRNA expression pairs displaying a correlation > 0.8 or < −0.8 (Spearman Rank) were tested for GO category enrichment.
Figure 2
Figure 2
smRNA identification in Symbiodinium microadriaticum over 9 experimental treatments. (A) Lengths, read count distribution, and 5' identity of 5,125,940 distinct genome-matching small RNAs from the set of 137 million sequenced reads. All small RNA reads were mapped to a draft genome assembly via bowtie software. Genomic location is indicated in the pie chart. (B) miRDeep2 output for a miRNA precursor indicating the guide (red) and passenger (blue) strand as well as the hairpin loop (yellow). (C) miRDeep2 output for a siRNA precursor. Note the perfect complementarity between guide (red) and passenger (blue) strand as well as the hairpin loop (yellow).
Figure 3
Figure 3
smRNA target prediction for 21 smRNAs within the gene set of Symbiodinium microadriaticum. (A) 3 distinct animal-like target sites in the 3'UTR of genes exist that are characterized by seeds of lengths 6-8nt that display perfect complementary base pairing between the miRNA and mRNA sequence. Vertical dashes indicate Watson-Crick base pairing. The pie chart displays the relative frequency of these target sites in the 3'UTRs of 1,720 genes (from a set of 12,858 genes with available 3'UTRs). (B) 19 plant-like mRNA target sites were identified in the coding sequence and 3'UTRs of 51,917 genomic genes of Symbiodinium microadriaticum. Plant-like mRNA target sites are characterized by full-length complementary base pairing between a miRNA and its mRNA with only few mismatches (i.e. near-perfect). (C) Number of identified plant-like mRNA target sites (blue bars) in relation to number of mismatches allowed. Number of false positives in 1,000 randomly generated cohorts of small RNA sequences of length 22 nt (red bars) are displayed for comparison. A cutoff of 3 mismatches (mm) over the aligned smRNA and mRNA provides a False Positive Rate of about 1 in 5. (D) Enriched GO terms within the set of matching and annotated animal-like targets (n = 519, P < 0.05, 4,047 3' UTRs). (E) Enriched GO terms within the set of matching and annotated plant-like targets (n = 7, P < 0.05, 18,290 genes).

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