Kadri S et al. (2011), RNA deep sequencing reveals differential microR...

ECB-ART-42288

PLoS One 2011 Jan 01;612:e29217. doi: 10.1371/journal.pone.0029217.

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RNA deep sequencing reveals differential microRNA expression during development of sea urchin and sea star.

Kadri S , Hinman VF , Benos PV .

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microRNAs (miRNAs) are small (20-23 nt), non-coding single stranded RNA molecules that act as post-transcriptional regulators of mRNA gene expression. They have been implicated in regulation of developmental processes in diverse organisms. The echinoderms, Strongylocentrotus purpuratus (sea urchin) and Patiria miniata (sea star) are excellent model organisms for studying development with well-characterized transcriptional networks. However, to date, nothing is known about the role of miRNAs during development in these organisms, except that the genes that are involved in the miRNA biogenesis pathway are expressed during their developmental stages. In this paper, we used Illumina Genome Analyzer (Illumina, Inc.) to sequence small RNA libraries in mixed stage population of embryos from one to three days after fertilization of sea urchin and sea star (total of 22,670,000 reads). Analysis of these data revealed the miRNA populations in these two species. We found that 47 and 38 known miRNAs are expressed in sea urchin and sea star, respectively, during early development (32 in common). We also found 13 potentially novel miRNAs in the sea urchin embryonic library. miRNA expression is generally conserved between the two species during development, but 7 miRNAs are highly expressed in only one species. We expect that our two datasets will be a valuable resource for everyone working in the field of developmental biology and the regulatory networks that affect it. The computational pipeline to analyze Illumina reads is available at http://www.benoslab.pitt.edu/services.html.

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Genes referenced: LOC100887844 LOC590297 mpi

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	Figure 1. Length distributions of sea urchin and sea star reads.Histogram of length distribution of reads and tags in sea urchin and sea star small RNA Illumina libraries. The peak corresponding to the typical length of a miRNA is seen at 22 nts in sea urchin, but this peak is not as enhanced in the sea star library. Spu: Strongylocentrotus purpuratus; Pmi: Patiria miniata.
	Figure 2. Distribution of annotated reads in small RNA libraries.(a) Bar showing the distribution of annotated reads 17 to 26 nts in length, for sea urchin. (b) Fractional distribution of non-coding RNAs in sea urchin and sea star embryonic small RNA libraries. Mapping of the annotated classes to reads and tags, shows the relative abundance (frequency) of each class per tag. All classes of non-coding RNAs compared were mapped to reads of lengths 17 to 26 nts. Spu: Strongylocentrotus purpuratus; Pmi: Patiria miniata.
	Figure 3. Comparison between sea urchin and sea star miRNAs.(a) Venn Diagram showing overlap between conserved miRNAs in sea urchin and sea embryos, and sea urchin adult (miRBase [34]). Only Illumina tags >2 reads were treated as potential true miRNAs. This figure does not include the miRNA* species. (b) Heat map showing the relative miRNA expression between sea urchin and sea star embryos (log2 transformed relative expression values). Average linkage clustering using Euclidean distance as the distance metric was used to generate the heat map (Methods). Since the genome sequence for sea star is unavailable, absence of certain miRNAs from the small RNA library in sea star, but its presence in sea urchin is treated as missing values for sea star. Missing values for sea star are indicated by the background color. Only miRNAs with zero reads are treated as missing values, whereas miRNAs with 1 or 2 reads are shown in the heat map.
	Figure 4. Phylogenetic comparison of sequence similarities between sea urchin, S. purpuratus and sea star, P. miniata.The hemichordate, S. kowalevskii has been used as the outgroup and the sequences in that species are used as the reference sequences. miRNA sequences in S. purpuratus or P. miniata that differ from the reference sequence are colored. Same color represents identical sequences. Absence of a miRNA from a species (represented by a blank) indicates absence of that miRNA from the reads and the registry. The miRNAs can be classified into 6 groups: (A) identical sequence and present in all three species; (B) present in all three species, but the sequence differences in all miRNAs; (C) present in all three species, but one or more species show mutations; (D1) identical sequence and present in S. purpuratus and P. miniata; (D2) identical sequence and present in S. purpuratus and S. kowalevskii; (E) present in two species with difference(s) in sequence; (F) the gene gained in a single species or lost in other two species. Group F is represented by the blue miRNAs at the node for the specific species #: miRNA is in the registry but has â‰¤2 read frequency in the embryonic reads nb: miRNA was shown to be present in adult tissue by northern blot [18] but is not present in registry. **: miR-2008 was found in late sea star embryos by whole mount in situ hybridization but not in early embryos ( Figure S8 ).
	Figure 5. Computational pipeline for analysis of deep sequencing libraries for discovery of small non-coding RNAs.Illumina reads undergo numerous filtering steps based on quality and length. The pipeline has two branches: for a species with genome sequence, and for a species without a sequenced genome, but a closely related sequenced species. Spu: Strongylocentrotus purpuratus; Pmi: Patiria miniata. miRDeep [36]; BLAST [38]. Green color: Reads Orange: Tags.

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