RNA Deep Sequencing Reveals Differential MicroRNA Expression during Development of Sea Urchin and Sea Star

Article (PDF Available)inPLoS ONE 6(12):e29217 · December 2011with34 Reads
DOI: 10.1371/journal.pone.0029217 · Source: PubMed
Abstract
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.

Figures

RNA Deep Sequencing Reveals Differential MicroRNA
Expression during Development of Sea Urchin and Sea
Star
Sabah Kadri
1
, Veronica F. Hinman
2
, Panayiotis V. Benos
3
*
1 Lane Center for Computational Biology, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America, 2 Department of Biological Sciences, Carnegie
Mellon University, Pittsburgh, Pennsylvania, United States of America, 3 Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh,
Pennsylvania, United States of America
Abstract
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.
Citation: Kadri S, Hinman VF, Benos PV (2011) RNA Deep Sequencing Reveals Differential MicroRNA Expression during Development of Sea Urchin and Sea
Star. PLoS ONE 6(12): e29217. doi:10.1 371/journal.pone.0029217
Editor: Denis Dupuy, Inserm U869, France
Received June 7, 2011; Accepted November 22, 2011; Published December 28, 2011
Copyright: ß 2011 Kadri 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.
Funding: This work was supported by National Institutes of Health grants R01LM007994, and R01LM009657 (PVB), and National Science Foundation grant IOS
1024811. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: benos@pitt.edu
Introduction
The developmental program, the process that creates a
multicellular organism from a single cell, involves gene regulation
at various levels transcriptional and post-transcriptional.
microRNAs (miRNAs) are one such class of small (,22 nts),
non-coding RNA molecules that regulate protein coding gene
expression post-transcriptionally. miRNAs typically target 39
UTRs of protein coding genes, and usually downregulate their
expression by affecting their protein levels [1], either by inhibiting
mRNA translation, or by increasing its degradation rate [2,3].
miRNA genes are generally transcribed by RNA polymerase II, by
their own promoters [4,5], or as parts of introns of protein coding
genes [6,7,8]. The primary transcripts are processed into
characteristic RNA stem-loop structures, which are further
processed into ,22 nt long duplexes in the cytoplasm by the
RNAse III enzyme, Dicer [9,10,11]. The mature miRNAs typically
have relatively higher steady-state levels than their corresponding
miRNA*. However some miRNA* reach substantial levels and are
known to have regulatory roles [12].
The first miRNAs, lin-4 and let-7 were discovered in C. elegans,as
regulators of developmental timing [13,14], and since then,
miRNAs have been implicated in many developmental and tissue
differentiation processes [15,16]. miRNAs have been found in all
animal lineages, although specific miRNAs have been lost and
gained during evolution [17,18]. Some orthologous miRNAs are
associated with conserved expression in similar tissues, which may
suggest conservation of function [19].
The sea urchin, Strongylocentrotus purpuratus and the sea star,
Patiria miniata are used as model organisms for developmental and
evolutionary studies, due to their phylogenetic position (inverte-
brate deuterostomes), and their well-characterized transcription
factor gene networks. Despite the intense research that has been
devoted to their developmental transcriptional pathways
[20,21,22,23], little is known about miRNA expression in these
two organisms, especially during their early developmental stages.
In early work, Pasquinelli et al. [24] examined the expression of the
highly conserved let-7 miRNA in 14 species from 8 phyla, and
found that only sea urchin embryos lacked mature transcripts for
the miRNA. More recently, Song et al. [25] showed that the main
genes involved in the RNAi pathway are expressed in sea urchin
embryos, and Wheeler et al. [26] found 45 miRNAs to be
expressed in the adult sea urchin using 454 sequencing. They also
sequenced a species of sea star, H. sanguinolenta and found
42 miRNAs in this sea star adult. miRBase (v. 17, April 2011)
contains 64 entries for S. purpuratus miRNAs (including miRNA*
PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e29217
species) [26,27]. Since developmental transcription factor gene
networks are very detailed in these organisms (more than in any
other echinoderm species), a systematic overlay of miRNA level
regulation will provide invaluable insight into the cumulative
effects of transcriptional and post-transcriptional regulation on
developmental wiring.
In this paper, we present for the first time, concrete evidence that
many small non-coding RNA genes (including miRNAs) are
expressed in high-numbers in the early developmental stages of
two distantly related species, S. purpuratus and P. miniata, which last
shared a common ancestor almost 500 million years ago (MYA) [28].
The goal of this study is to determine the pool of miRNAs involved in
development of these two echinoderm species. We sequenced small
RNA libraries of mixed population embryos from each of these
echinoderms using Illumina Genome Analyzer (Illumina, Inc.),
which provides a better depth of sequencing compared to 454. In the
future, it will be extremely interesting to study stage-specific
expression of these miRNAs. Comparison of the two sequenced
datasets showed that a large number of miRNAs are expressed
during development in the two species. Most of the identified
miRNAs have homologs in other species, but a number of novel
(echinoderm-specific) miRNAs were also identified. The data
reported here will provide a valuable resource for evolutionary
comparisons across a broader distance in the phylogenetic branch of
deuterostomes, and this can help complete the puzzle of develop-
mental gene regulatory networks in these two model organisms.
Results and Discussion
A rich population of non-c oding RNAs is expressed in sea
urchin and sea star embryos
High-throughput sequencing data (Illumina Genome Analyzer,
Illumina, Inc.) corresponding to small RNAs were collected from a
mixed embryonic population, individually from S. purpuratus (sea
urchin) and P. miniata (sea star) as described in Methods. According
to the Illumina protocol, the method specifically targets small
RNAs with 39 hydroxyl group, so the RNAs processed by Dicer and
other RNA processing enzymes are preferentially sequenced with
this method. A collection of publicly available programs and in-
house made scripts were used to parse the Illumina reads, and
quantify known and novel miRNA gene expression (see Methods).
Illumina sequencing of the small RNA libraries returned ,13
million reads for sea urchin and ,9.8 million reads for sea star
embryos (Table 1). After removal of low quality 39 ends and
linker sequences, the remaining reads (,11.6 and ,9.01 million
reads from sea urchin and sea star, respectively) were collapsed
into ‘‘tags’’ based on sequence identity (see Methods). This process
resulted in a total of ,2.5 million tags from each species (Table 1).
We focuse d on seque nces of leng th 17–26 nts, since this is the
typical size class expected for miRNAs. The histograms of the
corresponding length d istributions of reads and tags show
similar trends between the two species (Fig. 1). In the sea
urchin reads, there is a peak of relatively highly expressed
sequences at 22–23 nts (corresponding to the typical length of a
miRNA) (Fig. 1). The quality of the RNA was checked using a
Bioanalyzer (Figure S1), before and after adapter l igat ion, and
Table 1. Summary statistics of sea urchin and sea star deep
sequencing data, and annotations.
S. purpuratus P. miniata
Genome 800 Mb 500 Mb
Total number of reads 12,907,171 9,760,097
Reads mapped to genome 9,401,944 N/A
Tags (collapsed reads)’’ 2,486,028 2,513,198
Reads mapped to:
tRNAs 7,550 33,551
rRNAs 288,036 319,035
snRNAs & snoRNAs 6,217 1,805
miRNAs (conserved) 376,007 48,320
miRNAs (potentially novel) 5,834 281
Number of conserved miRNAs 47 38
Potentially novel miRNAs (miRDeep) 11 3
Note that the number of reads for non-coding RNAs, such as tRNAs, rRNAs,
snRNAs, snoRNAs and miRNAs, are for the length range 17–26 nts. For
discovery of conserved miRNAs in the libraries, only tags with more than 2
reads were used, whereas, for potential novel predictions, tags with more than
5 reads were used.
doi:10.1371/journal.pone.0029217.t001
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.
doi:10.1371/journal.pone.0029217.g001
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indicated that the RNA was preserved (Fo r mo re details, check
Methods).
Presently, S. purpuratus is the only echinoderm with a sequenced
genome [29]. About 62% of the 17–26 nt sea urchin reads
mappe d to the genome (Fig. 2a) (81%, if reads of all lengths are
considered). The reads that do not map to the genome could be
the result of sequencing errors or genome quality. Since the sea
star genome is unavailable, we assign all unmapped re ads to the
‘‘unknown’’ category (Fig. 2d). Similarity searches against
miRNAs and other known RNAs (co ding and non-coding genes)
were performed (see Meth ods). Approxima tely one quarter of the
17–26 nt long reads map to non-coding RN As (14% to miRNAs
and 10% other non- coding RNAs), another one quarter are
mRNA degradation products, while 13% of reads map to the
genome, but do not map to any annotated re gions (Fig. 2a).
Fig. 2c & 2d sh ow the RNA composition of individual lengths in
this size range in the sea urchin and sea star respectively. The
22 nt lo ng sea urchin reads were most enriched for miRNAs,
while this trend was not seen in the sea star library. All the size
classes show an almost uniform distr ibution of mRNA an d rR NA
partial reads. The un-annotated reads could be attributed to the
relatively poor annotation quality of the sea urchin genome, or to
large -scale transcription as it has been observed in other species
[30,31,32]. For example, a re cent report showed that most
intergenic reads are found near transcrip tion start or termination
sites [33].
The relative abundance of the reads and tags that map to
various non-coding RNAs varies substantially between sea urchin
and sea star (Fig. 2b). This is particularly true for miRNAs, where
61.4% of the sea urchin reads (17–26 nts) map to miRNA
sequences compared to 12.6% of sea star reads. For sea urchin
embryos, the miRNA reads collapse to ,1,000 tags (that
correspond to 42 miRNA genes), indicating a high expression of
the miRNA genes (reads/gene average: 3,800; median: 413; 14
genes have .1,000 reads). By contrast, we found that a relatively
higher number of sea star embryonic reads are mapped to (parts
of) tRNA and rRNA genes (1.5% compared to 0.001%) (7.7% and
77.9% compared to 0.8% and 37% respectively) (Fig. 2b). This
may reflect a sampling bias, or may indicate that fewer miRNAs
are expressed in sea star embryos compared to sea urchin
embryos. We found miRNA* species for most miRNAs, and in
some cases, the miRNA* was more abundant than the miRNA
itself (for example, miR-200, miR-2008, miR-219, miR-2011)
(Figure S2).
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.
doi:10.1371/journal.pone.0029217.g002
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In summary, the sea urchin and sea star samples showed
differences in the distribution of annotated small RNA classes,
with the most striking difference being the relative higher
enrichment of miRNAs in sea urchin embryos.
Conservation of developmental miRNA gene expression
in echinoderms
We used sequence homology as well as information about the
secondary stem-loop structure of precursor sequence to search for
conserved and novel miRNAs in sea urchin and sea star
embryonic libraries (see Methods). We found a total of 47 sea
urchin and 38 sea star miRNAs mapping to known sequences in
the miRBase registry (v. 17, April 2011) [34] (Table 1). Fig. 3a
shows the overlap between miRNAs found expressed in the two
embryonic libraries as well as adult sea urchins [26]. Overall,
53 miRNAs are expressed in one or both embryonic samples,
whereas, 31 are expressed in sea urchin adults as well as in the
embryonic stages of both species (Fig. 3a). This figure does not
include the miRNA* species. When comparing miRNA expression
between the two species, 25 are present in sea urchin only, 4 in sea
star only (miR-92d, miR-1692, miR-100, miR-4171) and 34 in both
species (Fig. 3a). The common hits are considered as putative
candidates for phylum specific miRNAs. miR-100 is considered a
sea star specific miRNA in Fig. 3 as it was absent in our sea urchin
embryonic library and Wheeler et al. did not find this miRNA in
the sea urchin adult by 454 sequencing [26]. Additionally, the
current version of the sea urchin genome (version 2.1, UCSC
Genome Browser [35]) lacks miR-100 sequence as well. However,
northern blot analysis previously showed that miR-100 is present in
sea urchin adult (coelomycytes and mesenchyme) [18]. It will be
interesting to verify whether the adult tissue in sea urchin expresses
it or not, thus, deciding its position as a species specific or phylum-
conserved miRNA.
Novel miRNAs. We used miRDeep to identify potentially
novel miRNAs in sea urchin [36] (we were not able to use miRDeep
on the sea star dataset, because of the lack of the genomic sequence
in this species.) Of the 11 novel predictions, 8 genes (5,183 reads)
have seed sequences (positions 2–8) similar to known miRNAs in the
registry (Figure S3a), while 3 are novel sequences with a total
,400 reads. Each of the potentially novel sea urchin predictions is
part of stem-loop genomic hairpins, characteristic of Dicer processing
(Figure S3). The novel sea urchin predictions were also matched to
sea star reads. Three out of the 11 predictions were found in sea star
(Table 1). These three tags may therefore, represent echinoderm
specific miRNAs. The other 10 tags may represent genes that have
evolved after the divergence of the sea star and sea urchin lineages,
although the sea star genome sequence is required before we make a
definite assessment of this fact.
miRBase Release 17 (April 2011) [34] currently contains 64 sea
urchin gene entries, all obtained from adult tissue by 454
sequencing [26,27], including miRNA* species. No sea star
miRNA genes are present in miRBase. Our embryonic libraries
add 16 new sea urchin miRNA genes to this pool (2 conserved, 11
potentially novel and 3 miRNA*s); and 41 sea star miRNA genes
(38 conserved, 3 potentially novel).
Comparison of miRNA genes expressed in embryos and
adults.
Most of the sea urchin miRNAs (45 out of 59) are
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 (log
2
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.
doi:10.1371/journal.pone.0029217.g003
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expressed both in embryos (ou r dataset) and adults (miRBas e
registry) (Fig. 3a). However, twel ve miRNAs are present in the
adult sea urchin only, but not in the embryonic stages
considered. These may correspond to adult-specific miRNAs
with no role in development, or might have developmental roles
outside of the embryonic stages considered for this study. On the
other hand, miR-31b and miR-1b were found to be early
development specific for the sea urchin, with no expression in
the adult (Fig. 3). The most surprising result was let-7 reads in
the s ea urchin embryos. Pasquinelli et al. [24], using northern
blots, had shown that S. purpuratus embryos contain the let-7
prec ursors, but not the mature let-7 miRNA. We found 16 high-
quality reads corresponding to this miRNA in our sample. We
suspect that the relatively low abundance of this gen e made it
undetectable to northern blots. Figure S4 shows the differences
in sequence of S. purpuratus mature miRNAs between embryonic
(Illumina sequencing) data and the adult 454 seq uencing data.
Most sequences are the same and few differences are seen at the
59 or 39 end. However, miR-31b shows a difference of one base at
position 11.
There is no adult miRNA data for the P. miniata (PMI).
However, Wheeler et al. [26] sequenced a species of sea star, H.
sanguinolenta (HSN). On comparison of the PMI embryo data with
the HSN adult data, 34 miRNAs were found in both species, 13
were found in HSN only and 8 were found in PMI only (Figure
S5). Some changes are seen between the sequences of the same
miRNA (indicated by bold in Figure S5) but most of these are at
the 39 end of the miRNA and could be due to different sequencing
platforms or due to sequencing errors. The presence or absence of
miRNAs between the two datasets might be due to different
developmental stages, and might not represent species level
changes.
In summary, we find that the pool of miRNAs is more or less
conserved between embryonic and adult sea urchin. When we
compared the developmentally expressed miRNAs between the
two species we found that majority of them were conserved,
although some relatively highly abundant miRNAs in sea urchin
embryos did not have any reads in sea star embryos (for example,
miR-2008)(Fig. 3b). The overall conservation of miRNA genes
may imply that possible differences in miRNA function may be
due to differences in their spatial expression or their expression
levels.
miRNA gene expression shows similar trends between
the two echinoderm embryos
Fig. 3b shows a heat map corresponding to relative abundance
of overlapping miRNAs between the sea urchin and sea star
embryos. The miRNAs can be classified into 4 main groups based
on their expression trends, (1) relatively high abundance in both
species, (2) relatively high abundance in sea star embryos, but
lower abundance in sea urchin embryos, (3) relatively high
abundance in sea urchin embryos, but low abundance in sea star
embryos, and (4) medium to low abundance in both species.
Overall, we found that most miRNAs show similar patterns of
expression in the two species. This indicates that the two
echinoderms may share many features of their regulatory
programs. However, some differences are also become apparent.
Out of the 14 highly expressed sea urchin miRNAs, 11 are also
relatively highly expressed in sea star, which may indicate possible
overlap in the post-transcriptional gene regulatory mechanisms.
From the remaining three, two (miR-183 and miR-1a) are of
medium abundance in sea star, while miR-2008 has a single read in
sea star library (Fig. 3b). On the other hand, three highly
expressed and one moderately expressed miRNA in sea star (miR-
1692, miR-100, and miR-92d; and miR-4171, respectively) have no
reads in the sea urchin library (Fig. 3b). These differentially
expressed miRNAs are probably indicative of the differences
between the two developmental programs. We note, however, that
this is the first attempt to map the developmental post-
transcriptional regulome in echinoderms, and spatial as well as
temporal expression may vary even between the miRNAs that
appear to be abundant in both species.
Since the embryonic libraries were a mixed population sample,
northern blots of a few miRNAs in various early developmental
stages of sea urchin and sea star embryos were used to confirm the
presence of some conserved miRNAs (Figure S6). miR-2009 was
found in 1day, 2day and 3day old embryos in both species. miR-31
and miR-10 was found in all stages considered in sea urchin and
sea star respectively. miR-184 was only barely visible on the 3day
old embryos of sea urchin with undetectable levels in 1day and
2day old embryos, and might be more development specific than
the other miRNAs. However, the signal levels for sea star were
undetectable. This might be due to the low sensitivity of the
protocol (See Methods). It will be interesting to use whole mount
in situ hybridization to compare the spatial and temporal patterns
of these miRNAs.
Evolution of miRNA sequences in the echinoderm animal
lineage
miRNA families have been found in all analyzed animal
lineages. It has been shown that evolutionary trends across
metazoans show rare substitutions in mature miRNA sequence
[26]. We found that about half of the miRNAs in sea urchin and
sea star are identical in sequence, and the rest have acquired single
or multiple mutations. All alignments between the three species
are listed in Figure S7. Many of these differences are at the 39 end
of the miRNA, and represent the addition or loss of two or more
bases. A mutation at the last base of the miRNA between two
species is not treated as a change, as this might be a sequencing
error and in any case it is not expected to affect the function of the
mature miRNA. Differences at the 39 end may be due to
differences in the processing of the miRNA precursors between the
two species. Striking differences are seen in abundant miRNAs
such as, miR-2001, miR-182, miR-183, miR-2007 and miR-92b,
where the mutation(s) occurs in the middle of the sequence
(Figure S7). Fig. 4 shows the comparative analysis of mutations
in miRNAs between the two echinoderms, using the hemichor-
date, acorn worm, Saccoglossus kowalevskii as an outgroup. The
miRNAs can be grouped in several clusters based on the mutations
across evolutionarily divergent species (Fig. 4). Only ten of the 28
miRNAs that are present in all three species (Fig. 4, categories A,
B, and C) are identical in all of them; seven seem to have acquired
mutations in the S. kowalevskii lineage (or in the echinoderm
ancestor), five in the sea urchin lineage and only two in the sea star
lineage. The remaining four miRNAs have differences in all three
species (Fig. 4, category B). It will be very interesting to further
investigate the effects of these mutations on the loss or gain of
target sequences between the two echinoderms.
A very interesting observation was seen with miR-2008, which
seemed to present in S. purpuratus and S. kowalevskii, but not in P.
miniata based on our library data. Whole mount in situ
hybridization on late stage sea star embryos showed that miR-
2008 is indeed present in sea star, but is not expressed in the early
stage embryos considered for our library preparation (Figure S8).
We, thus, anticipate that our dataset will provide a rich source
for future evolutionary studies, as both the miRNA and target sites
may have evolved quite rapidly to facilitate new regulatory
interactions.
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Methods
Small RNA library preparation
Sea urchins and sea stars were collected by Marinus Scientific
LLC in Southern California (http://www.marinusscientific.com/)
and purchased by us. Total RNA was extracted from embryos at
24 h, 48 h and 72 h after fertilization using miRVana RNA
isolation kit (Ambion). Embryo populations were combined,
separately for each species, and the mixed population samples
were sent for small RNA library preparation and sequencing to the
Genomics & Microarray Facility at Wistar Institute, Philadelphia.
Prior to library preparation, RNA quality was checked using the
Bioanalyzer and was found to be very good with very little
degradation (see File S1 and Figure S1).
Illumina adapters were ligated to the 59 and 39 ends of RNA,
as described in the Illumina v1.5 protocol for small RNA
sequencing samples. Small RNA molecules were size selected
(Figure S1), and RT-PCR amplification was used to generate
the cDNA libraries for both species. The 36 bp run on the
Illumina Genome Analyzer (Illumina, Inc.) was used for
sequencing these cDNAs.
Computational analysis procedure and pipeline
Base calling was performed by the Bioinformatics facility at
Wistar Institute. The resulting sequences were subjected to our
computational pipeline (Fig. 5), which consists of a number of in-
house made scripts. First, we perform quality filtering by
converting the Illumina quality codes for each base to its Phred
quality score, and trimming the low quality 39 ends of the reads. A
cut-off of 20 was selected based on the histogram of qualities for all
reads (data not shown). 39 adapters were trimmed using the
novoalign program (www.novocraft.com). This program uses
ungapped semi-global alignment of adapter sequence against the
read using a weight matrix from read and base qualities, and
trimming is performed from start of the optimum alignment. 59
adapter sequence was trimmed based on perfect sequence match
of more than or equal to 10 nts at the 59 end. All reads shorter
than 17 nts or longer than 26 nts were excluded from further
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
#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
).
doi:10.1371/journal.pone.0029217.g004
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analysis except when it is noted otherwise. The remaining reads
with 100% sequence identity and length difference of 2 nts or less
were collapsed to produce ‘‘tags’’ of genes and calculate their
expression as number of independent reads each tag has. At this
stage, tRNAs, rRNAs, snRNAs and snoRNAs are removed based
on sequence identity to known genes. Also, similarity to known
miRNAs is used to identify evolutionary conserved miRNAs. If a
genome is available (i.e., sea urchin, in our case) the reads are
mapped to the genome and novel miRNA genes are discovered
using miRDeep [36].
Sea urchin tRNA sequences were obtained from UCSC (http://
gtrnadb.ucsc.edu/) and snRNA and snoRNA sequences from
GenBank [37]. rRNA sequences were gathered from a variety of
sources for three sea urchin species (S. purpuratus, P. lividus, L.
variegatus), including UCSC genome browser [35] and EBI
databases (http://www.ebi.ac.uk/Databases/). Since there is no
tRNA, snoRNA or snRNA data publicly available for the sea star,
the sequences from sea urchin were used for the search in sea star.
Due to the highly diverse nature of piRNAs and the fact that a
large number of them represent lowly expressed genes, we decided
to exclude piRNAs from our analysis. For sequence similarity
match we used BLAST [38]. The parameters used to map
miRNAs to Illumina reads were ‘‘-e 0.01 -p 100 -W 8’’. For
mapping reads to the genome and other conserved sequences,
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.
doi:10.1371/journal.pone.0029217.g005
MicroRNA Populations in Echinoderms
PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e29217
parameters used were ‘‘-W 12 -p 80’’. All hits with length less than
85% of the length of the query sequence were ignored. mRNA
sequences for the sea urchin and sea star were compiled using
NCBI predicted genes [37] and the SpBase (http://spbase.org)
database [39] was also used for S. purpuratus .
The computational pipeline to analyze Illumina reads is
available at http://www.benoslab.pitt.edu/services.html.
Hierarchical clustering of gene expression values
The relative abundance of each miRNA in each sample was
log2 transformed for better visualization of the data. Average
linkage hierarchical clustering was performed using Euclidean
distance as the distance metric. The distance between two clusters
X and Y is given by:
D(X,Y)~
P
n
X
i~1
P
n
Y
j~1
ed x
i
,y
j

n
X
n
Y
, x
i
[X,y
j
[Y,
where x
i
is the vector of log2 transformed relative abundances of
miRNA i, y
j
is the vector of log2 transformed relative abundances
of miRNA j, ed x
i
,y
j

is the Euclidean distance between x
i
[X and
y
j
[Y, n
X
is the number of samples in cluster X , n
Y
is the number
of samples in cluster Y .
Whole mount in situ hybridization
We followed our lab protocol [40] except we used an antisense
39 DIG labeled locked nucleic acid (LNA) probe (Exiqon Inc.) at
concentrations of between 2pmol to 4pmol per 100 ul of
hybridization solution and at 47uC as recommended by the
supplier.
Northern Blots
Total RNA was extracted from sea urchin and starfish embryos
using the miRVana kit by Ambion. Standard northern blot
protocols were performed using 10–15
mg of total RNA and
antisense miRNAs, starfireTM (IDT) a-P32 oligonucleotide
labeled probe. A 10 nt to 100 nt size ladder was used (Decade,
Ambion) to estimate size.
Supporting Information
File S1 RNA quality.
(DOC)
Figure S1 The RNA quality was checked using the
BioAnalyzer before (a,b) and after (c) adapter ligation.
(a) Distribution of lengths of the RNA sample from sea urchin
before adapters were ligated. The first peak (,20–25 nt)
corresponds to the small RNA population. (b) Length distribution
of sea star RNA sample before adapter ligation. (c) The adapter-
ligated RNA was run on a gel and size-selected for small RNAs.
(PNG)
Figure S2 Reads for mature miRNA and miRNA* in
UCSC genome browser for the sea urchin. Reads
(logarithm scale) for miRNA and miRNA* for cases in which
the miRNA* is more abundant than miRNA.
(PNG)
Figure S3 Stem-loop structures of the novel miRNA
miRDeep (1) predictions in sea urchin. (a) miRNAs that
share their seeds with known miRNAs. The temporary labels are
the names of miRNA (b) Precursors of novel miRNAs without any
seed conservation.
(PNG)
Figure S4 Comparison of mature miRNA sequences
between S. purpuratus adult (2) and embryonic data.
Differences are highlighted in bold. E: Embryonic data from
Illumina platform; A: Adult data from 454 sequencing platform.
(PDF)
Figure S5 Comparison of mature miRNA sequences
between H.sanguinolenta adult data (2) and P.miniata
embryonic data. Differences are highlighted in bold. Pmi: P.
miniata; Hsn: H. sanguinolenta.
(PDF)
Figure S6 Northern Blot showing the expression of a
few conserved miRNAs in S. purpuratus (sea urchin) and
P. miniata (sea star) embryos. 5S rRNA is used as the
loading control while miR-124 is used as the negative control.
(TIF)
Figure S7 Alignment of mature miRNA sequences in two
echinoderms and a hemichordate reference species. spu -
S. purpuratus; pmi - P. miniata; sko - S. kowalevskii.
(PDF)
Figure S8 Whole mount in situ hybridization of P.
miniata embryos using LNA probes antisense to miR-
2008. Blastula and gastrula stages do not show any expression for
this miRNA, consistent with the embryonic small RNA library.
However, we see expression of miR-2008 in late stage larvae.
(PNG)
Acknowledgments
We would like to thank Calen Nichols (formerly at Wistar Institute), for her
indispensable help with the small RNA library preparation and sequencing,
and an anonymous reviewer for helpful comments. The constructive
comments of one reviewer helped improve our manuscript. All data are
available from GEO (acc. no.: TBN) and from the authors’ web site
(http://www.benoslab.pitt.edu/services.html).
Author Contributions
Conceived and designed the experiments: SK VFH PVB. Performed the
experiments: SK. Analyzed the data: SK VFH PVB . Contributed
reagents/materials/analysis tools: SK VFH PVB. Wrote the paper: SK
VFH PVB.
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    • "We further compared the small RNA sequences with M. nipponense mRNAs, Rfam and Repbase, to identify rRNAs, tRNAs, snRNAs, snoRNAs, and mRNAs. The reads and type distribution of small RNAs revealed a large number of various rRNAs consistent with previous reports (Kadri et al., 2011; Mi et al., 2014). Analysis of 5′-and 3′-end nucleotides of these miRNAs revealed that uridine was the most common residue at both ends, a pattern that has been observed for miRNAs sequenced in other organisms (Chen et al., 2009; Legeai et al., 2010). "
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