cloning and expression of new micrornas from …...31 new mirnas from zebrafish cloning and...

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Chapter 2

Cloning and Expression of New microRNAs from Zebrafish

reprinted and adapted from Nucleic Acids Research, 34:2558-69 (2006)

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New miRNAs from Zebrafish

Cloning and expression of new microRNAs fromzebrafishWigard P. Kloosterman, Florian A. Steiner, Eugene Berezikov, Ewart de Bruijn,

Jose van de Belt, Mark Verheul, Edwin Cuppen and Ronald H.A. Plasterk*

Hubrecht Laboratory, Centre for Biomedical Genetics, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

Received February 16, 2006; Revised March 21, 2006; Accepted April 4, 2006

ABSTRACT

MicroRNAs (miRNAs) play an important role in devel-opment and regulate the expression of many animalgenesbypost-transcriptional genesilencing.Herewedescribe the cloning and expression of new miRNAsfrom zebrafish. By high-throughput sequencing ofsmall-RNA cDNA libraries from 5-day-old zebrafishlarvae and adult zebrafish brain we found 139known miRNAs and 66 new miRNAs. For 65 knownmiRNAs and for 11 new miRNAs we also cloned themiRNA star sequence. We analyzed the temporal andspatialexpressionpatterns for35newmiRNAsandfor32 known miRNAs in the zebrafish by whole mountin situ hybridization and northern blotting. Overall,23 of the 35 new miRNAs and 30 of the 32 knownmiRNAs could be detected. We found that mostmiRNAs were expressed during later stages of devel-opment. Some were expressed ubiquitously, butmany of the miRNAs were expressed in a tissue-specific manner. Most newly discovered miRNAshave low expression levels and are less conservedin other vertebrate species. Our cloning and expres-sion analysis indicates that most abundant andconserved miRNAs in zebrafish are now known.

INTRODUCTION

Over the past few years it has become clear that the expres-sion of many genes is extensively controlled at the post-transcriptional level by microRNAs (miRNAs) (1,2).Although miRNAs were initially recognized as an oddityspecific to developmental switches in Caenorhabditis elegans(3,4), the cloning and computational prediction of hundredsof miRNAs in both animals and plants uncovered a wholenew layer of gene regulation (5). It appears now that a

mammalian genome may contain >500 genes encodingmiRNAs (6,7).

miRNAs are transcribed as long RNA polymerase II tran-scripts (8,9) which fold into characteristic stem–loop struc-tures. Once cleaved by the nuclear enzyme Drosha (10), asmaller precursor miRNA is transported to the cytoplasm(11,12), where the Dicer protein mediates maturation of themiRNA into a 20–23 nt species (13–16), a process which iscoupled to loading into a miRNP complex. These small-RNAmolecules bind to the 30-untranslated region of mRNAsby partial basepairing, which primarily results in inhibitionof mRNA translation (17). mRNAs that are repressed bymiRNAs are localized in cytoplasmic foci called P-bodies(18–20).

While plant miRNAs usually bind with perfect comple-mentarity to their target mRNA and induce mRNA degrada-tion (21), animal miRNAs in most cases regulate a mRNAcontaining a sequence complementary to the 7 nt seedof the miRNA (nt 1–7 or 2–8) (22–24). Computational pre-dictions indicate that thousands of genes might be regulatedby miRNAs and that the average number of genes that istargeted by a miRNA is �200 (25,26). Many miRNAtarget sites are evolutionarily conserved and the mRNAsthat bear conserved target sites are expressed at lower levelsin the tissue where the miRNA is expressed compared withthe tissues where the miRNA is not expressed (27,28).In addition, the mRNAs with conserved target sites areoften expressed in developmental stages prior to miRNAexpression (27).

The important role of miRNAs in animal development hasbeen shown by several approaches. Removal of all miRNAsresults in developmental arrest in mouse and fish (29–31).Several other studies have revealed the details of processeswhere miRNAs act. For example, the miR-1 knockout inDrosophila results in aberrant muscle growth (32), while inmouse, miR-1 regulates the transcription factor Hand2 duringheart development (33). In addition, miRNAs may regulatemajor signaling pathways like the notch signaling pathway

*To whom correspondence should be addressed. Tel: +31 30 2121963; Fax: +31 30 2516554; Email: [email protected]

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

� The Author 2006. Published by Oxford University Press. All rights reserved.

The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open accessversion of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Pressare attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety butonly in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected]

2558–2569 Nucleic Acids Research, 2006, Vol. 34, No. 9doi:10.1093/nar/gkl278

Published online May 12, 2006

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in Drosophila (34). In mouse and chick, miR-196 expressionregulates the expression of Sonic hedgehog through targetingthe transcription factor Hoxb8 (35).

In zebrafish there are currently 369 miRNA genes expres-sing 168 different miRNAs (5). Many of the conservedmiRNAs have a striking organ-specific expression patternin zebrafish and are mostly expressed at later stages of devel-opment (36). Also in Drosophila, miRNAs exhibit diversespatial expression patterns during embryonic developmentas indicated by the analysis of the expression of primarymiRNA transcripts (37). Strikingly, the expression patternsof some highly conserved miRNAs like miR-1 andmiR-124 are similar in flies, fish and mouse, suggestingancient roles in tissue development (37,38).

Encouraged by recent studies which indicate that there aremany more miRNAs than currently known (6,7), weattempted to find new miRNAs in the zebrafish by sequencingsmall-RNA cDNA libraries made from 5-day-old zebrafishlarvae and adult zebrafish brain. We found 139 knownmiRNAs and 66 new miRNAs in the zebrafish. In addition,we studied the temporal and spatial expression of miRNAswith unknown expression from three sources: 32 miRNAspredicted or cloned previously from zebrafish (7,39),34 miRNAs cloned in this study and one miRNA clonedfrom human (E. Berezikov, R. H. A. Plasterk and E. Cuppen,unpublished data). We used locked nucleic acid (LNA)probes to detect these 67 miRNAs in situ in the zebrafishembryo and on northern blots with total RNA from differentdevelopmental stages and adult tissues. In contrast to our pre-vious in situ hybridization screen for conserved vertebratemiRNAs (36), we could only detect miRNA expression insitu for a subset of 28 miRNAs. For 53 miRNAs we coulddetect a �22 nt species on northern blots. The remainder of14 miRNAs could not be detected by in situ hybridizationor northern blotting, although 13 of these were cloned inthis study and passed our computational analysis. Thesemight thus represent low abundant miRNAs or miRNAsexpressed only in a few cells.

Our data show that there are many more miRNAs inzebrafish than so far described. They also demonstrate thatnext to the highly abundant and tissue-specific miRNAs,most of which are conserved (36), there is a large set ofmiRNAs expressed at much lower levels, many of whichare less conserved.

MATERIALS AND METHODS

Small-RNA library construction

Two small-RNA cDNA libraries were prepared by VertisBiotechnologie AG (Freising-Weihenstephan, Germany).RNAs smaller than 200 bases were isolated from 5-day-oldzebrafish larvae and dissected adult zebrafish brain usingthe mirVana miRNA isolation kit (Ambion). Subsequently,the population of small-RNAs ranging in size from 15 to30 bp were purified from 12.5% polyacrylamide gel. Thissmall-RNA fraction was poly(A)-tailed followed by ligationof an RNA linker to the 50 end of the RNA. First strandcDNA synthesis was performed using oligo(dT)-linkerprimers and M-MLV-RNase H� reverse transcriptase. Theresulting cDNA was then PCR-amplified in 15 (larvae

library) or 23 (brain library) cycles. After limited exonu-clease treatment to generate 50 overhangs, the gel purifiedfraction of the cDNA in the range of 95–110 bp was direc-tionally ligated in the EcoRI and BamHI sites of pBSIISK+. Ligations were electroporated into T1 Phage resistantTransforMax� EC100� electrocompetent cells (Epicentre)resulting in 1.25 (larvae library) and 1.3 (brain library) · 106

recombinant clones.

Sequencing of small-RNA cDNA libraries

Both libraries were plated on Luria–Bertani (LB) amp platesand 12 288 individual colonies were automatically pickedand put into 384-well plates (Genetix QPix2; New MiltonHampshire, UK) containing 75 ml LB-Amp and grown over-night at 37�C with continuous shaking. All following pipet-ting steps were performed using liquid handling robots(Tecan Genesis RSP200 with integrated TeMo96 and Velo-city11 Vprep with BenchCell 4·). 5 ml of culture were trans-ferred to a 384-well PCR plate (Greiner) containing 20 mlwater. Cells were lysed by heating for 15 min at 95�C ina PCR machine. Lysate (1 ml) was transferred to a fresh384-well plate containing 4 ml PCR mix (final concentrations:0.2 mM M13forward, TGTAAAACGACGGCCAGT; 0.2 mMM13reverse, AGGAAACAGCTATGACCAT, 400 mM ofeach dNTP, 25 mM Tricine, 7.0% Glycerol (w/v), 1.6%dimethyl sulfoxide (w/v), 2 mM MgCl2, 85 mM Ammoniumacetate, pH 8.7 and 0.2 U Taq Polymerase in a total volumeof 10 ml) and the insert was amplified by 35 cycles of 20 minat 94�C, 10 min at 58�C, 30 min at 72�C. After adding 30 mlwater, 1 ml of PCR product was directly used for dideoxysequencing by transferring to a new 384-well PCR plate con-taining 4 ml sequencing mix (0.027 ml BigDye terminatormix v3.1 (Applied Biosystems, Foster City, CA), 1.96 ml of2.5· dilution buffer (Applied Biosystems), 0.01 ml sequen-cing oligo (100 mM stock T7, GTAATACGACTCACT-ATAGGGC) and 2 ml water). Thermocycling was performedfor 35 cycles of 10 min at 94�C, 10 min at 50�C, 20 min at60�C and final products were purified by ethanol precipitationin 384-well plates as recommended by the manufacturer(Applied Biosystems) and analyzed on ABI3730XL sequen-cers with a modified protocol for generating �100 nt sequen-cing reads.

Sequence analysis

Base calling and quality trimming of sequence chromato-grams was done by phred software (40). After masking ofvector and adapter sequences and removing redundancy,inserts of length 18 bases and longer were mapped to thezebrafish genome using megablast software (ftp://ftp.ncbi.nlm.nih.gov/blast/). Not all inserts matched perfectly to agenome, and detailed analysis of non-matching sequencesindicated that many of them represented known miRNAswith several additional nucleotides added to one of theends. These non-genomic sequences may be artifacts of thecloning procedure or a result of non-templated modificationof mature miRNAs (41). Such sequences were correctedaccording to the best blast hit to a genome. Next, for everygenomic locus matching to an insert, repeat annotationswere retrieved from the Ensembl database (http://www.ensembl.org) and repetitive regions were discarded from

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further analysis, with the exception of the followingrepeats: MIR, MER, L2, MARNA, MON, Arthur and trf,since these repeat annotations overlap with some knownmiRNAs. Genomic regions containing inserts with 100 ntflanks were retrieved from Ensembl and a sliding windowof 100 nt was used to calculate RNA secondary structuresby RNAfold (42). Only regions that folded into hairpinsand contained an insert in one of the hairpin arms wereused in further analysis. Since every non-redundant insertproduced independent hits at this stage, hairpins with overlap-ping genomic coordinates were merged into one region, tra-cing locations of matching inserts. In cases when severalinserts overlapped, the whole region covered by overlappinginserts was used in downstream calculations as a maturesequence. Next, gene and repeat annotations for hairpin geno-mic regions were retrieved from Ensembl, and repetitiveregions (with above mentioned exceptions) as well as riboso-mal RNAs, tRNAs and snoRNAs were discarded.

To find homologous hairpins in other genomes, matureregions were blasted against human, macaca, chimpanzee,mouse, rat, dog, cow, opossum, chicken, tetraodon, zebrafishand fugu genomes. Hits with length of at least 20 nt and iden-tity of at least 70% were extracted from genomes along withflanking sequences of length similar to that observed in origi-nal hairpins to which a certain mature query sequencebelonged. Extracted sequences were checked for hairpinstructures using RNAfold, and positive hairpins were alignedwith the original hairpin using clustalw (43). Only homologswith at least 70% overall identity and 90% identity within themature sequence were considered. In cases where severalhomologous hairpins in a species were identified, the bestclustalw-scoring hairpin was retained. Next, homologs fromdifferent organisms were aligned with the original hairpinby clustalw to produce a final multiple alignment of the hair-pin region. Chromosomal location of homologous sequenceswere used to retrieve gene and repeat annotations fromrespective species Ensembl databases. Hairpins that con-tained repeat/RNA annotations in one of the species, aswell as hairpins containing mature regions longer that 25 ntor with GC-content higher than 85% were discarded. Forremaining hairpins, randfold values were calculated forevery sequence in an alignment using mononucleotide shuf-fling and 1000 iterations. The cut-off of 0.01 was used forrandfold and only regions that contained a hairpin belowthis cut-off for at least one species in an alignment, wereconsidered as miRNA genes. Finally, positive hairpins weresplit into known and novel miRNAs according to annotations.To facilitate these annotations and also to track performanceof the pipeline, mature sequences of known miRNAs frommiRBase (5) were included into the analysis from the verybeginning.

In situ hybridization

Albino zebrafish embryos and larvae of 12, 16, 24, 48, 72 and120 hpf were fixed in 4% PFA in phosphate-buffered saline(PBS) overnight at 4�C. Proteinase K treatment was donefor 2, 5, 10, 30, 45 and 90 min, respectively. In situ hybridi-zation was performed as previously described (36,38). LNA-modified DNA probes (LNA probes) were designed andsynthesized by Exiqon (Denmark). The sequences of the

LNA probes complementary to the mature miRNAs are listedin Supplementary Table S1. The LNA probes were labeledwith digoxigenin (DIG) using the DIG 30 end labeling kit(Roche) and purified using Sephadex G25 MicroSpincolumns (Amersham).

Plastic sectioning

Embryos and larvae stained by whole-mount in situ hybridi-zation were transferred from benzyl benzoate/benzyl alcoholto 100% methanol and incubated for 10 min. Specimens werewashed twice with 100% ethanol for 10 min and incubatedovernight in 100% Technovit 8100 infiltration solution(Kulzer) at 4�C. Next, embryos were transferred to a moldand embedded overnight in Technovit 8100 embedding med-ium (Kulzer) deprived of air at 4�C. Sections of 7 mm thick-ness were cut with a microtome (Reichert-Jung 2050),stretched on water and mounted on glass slides. Sectionswere dried overnight. Counterstaining was done with 0.05%neutral red for 12 s, followed by extensive washing withwater. Sections were preserved with Pertex and mountedunder a coverslip.

Image acquisition

Embryos, larvae and sections were analyzed with Zeiss Axio-plan and Leica MZFLIII microscopes and subsequentlyphotographed with digital cameras. Images were adjustedwith Adobe Photoshop 7.0 software.

Northern blotting

Total RNA was isolated using trizol (Invitrogen). For eachsample �15 mg RNA was separated on 15% denaturing poly-acrylamide gels and blotted according to standard procedures.Blots were prehybridized for 30 min at 60�C in hybridizationbuffer (0.36 M Na2HPO4, 0.14 M NaH2PO4, 1 mM EDTAand 7% SDS) and hybridized overnight at 60�C in hybridiza-tion buffer containing 0.1 nM probe. After stringency washes(once for 30 min at 50�C in 2· SSC/0.1% SDS and once for30 min at 50�C in 0.5· SSC and 0.1% SDS) blots were rinsedin PBST (PBS with 0.1% Tween-20) and blocked for 30 minat room temperature in PBST with 5% milk powder. Subse-quently, blots were incubated for 1 h at room temperaturewith anti-DIG-AP antibody (Roche) in blocking buffer,washed six times for 15 min in PBST and twice for 5 minwith AP-buffer (0.1 M Tris–HCl, pH 9.5, 50 mM MgCl2,0.1 M NaCl and 0.1% Tween). Signal was detected byusing CDP-star chemiluminescent substrate (Roche) andexposing the blots to X-ray films. Films were scanned andpictures were processed using Adobe photoshop 7.0 software.To control for equal loading, blots were hybridized for 2 h at37�C with a radio-labeled probe against U6 snRNA. Afterwashing twice in 2· SSC/0.2% SDS, blots were exposed tophosphor-imager screens.

RESULTS

Cloning of new miRNAs from zebrafish

Recently, miRNAs were profiled in zebrafish by cloningsmall-RNAs from different developmental stages and zebra-fish cell lines (39). In addition to the miRNAs alreadyknown from other organisms, several new miRNAs were

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found. The total number of zebrafish miRNA genes in themiRNA registry is currently 369, corresponding to 168 differ-ent miRNAs (5). Computational identification, verificationand cloning of miRNAs by our group and others hasshown that the number of miRNAs in humans might bemuch higher and extend towards a thousand (E. Berezikov,R. H. A. Plasterk and E. Cuppen, unpublished data) (6,7).

In order to find more miRNAs in the zebrafish we per-formed sequencing of two newly generated small-RNAlibraries derived from 5-day-old zebrafish larvae (zf-larvae)and adult zebrafish brain (zf-brain). These two sample typeswere chosen based on our previous in situ expression analysisof conserved vertebrate miRNAs in the zebrafish embryo,which revealed that strongest expression for most miRNAsis observed in later stages of development and that one-thirdof this set of miRNAs was found to be expressed in thezebrafish embryonic brain (36).

For each library 12 288 individual clones were sequenced.Of these 12 288 sequence reads 2182 from the zf-larvaelibrary and 1231 reads from the zf-brain library were tooshort to be analyzed. The remaining sequence reads wereselected based on the presence of a 30 poly(A)-tail and a50 adapter sequence, both of which resulted from thecloning process. Clones containing inserts shorter than18 bp were also removed from the dataset. The clones thatfulfilled these criteria were analyzed using a computationalpipeline that includes RNA secondary structure. Based onthis analysis, 13 094 sequences could be annotated asmiRNAs, representing 205 distinct miRNAs (SupplementaryTables S2 and S3). From these small-RNA sequences,3,150 (zf-larvae) and 9,607 (zf-brain) could be annotated asknown miRNAs from zebrafish. In total, we found 139 outof 168 known zebrafish miRNAs and 126 of these werefound in both the zf-brain library and the zf-larvae library(Supplementary Figure S1 and Table S7). For 65 of theknown miRNAs, we sequenced clones from both the3p- and 5p-arm of the miRNA hairpin, i.e. both the miRNAand the miRNA star sequence were found (SupplementaryTable S5). All of the known miRNAs that we found wererepresented by multiple clones in the library (Figure 1 andSupplementary Table S2). In addition, 2.6% of the clonesrepresent 66 novel miRNAs (zf-larvae, 121 clones andzf-brain, 216 clones). These clones could be assigned to116 different hairpins in the zebrafish genome, thus repre-senting 116 potential new miRNA genes. For 11 of thenew miRNAs, clones were sequenced from both the5p- and the 3p-arm (Supplementary Table S6). Together,this limits the total number of unique miRNA hairpins to66 (Supplementary Table S4). Of the new miRNAs 37were found only once in one of both libraries. The overlapbetween the zf-larvae and zf-brain libraries for new miRNAswas 19 (Supplementary Figure S1 and Table S3). Althoughthe majority of the known miRNAs have a clear homologin other vertebrate species, many of the newly identifiedmiRNAs are less conserved (Figure 1, Supplementary TablesS2 and S3). The first nucleotide of the newly identifiedmiRNAs is most often a U, although this bias was lessstrong than for known miRNAs (25) (SupplementaryFigure S2).

In total, we found 66 novel miRNAs from zebrafish andthese represent a new set of less conserved miRNAs.

In situ hybridization analysis of the spatial expression ofknown and new miRNAs in the zebrafish

To determine the spatial and temporal expression of knownand new miRNAs during zebrafish development, we per-formed in situ hybridization and northern blotting usingLNA probes (Supplementary Table S1, Tables 1 and 2). Intotal we analyzed the expression of 67 miRNAs, derivedfrom three sources: 34 miRNAs cloned from the zf-larvaeand the zf-brain libraries, one miRNA cloned from human(E. Berezikov, R. H. A. Plasterk and E. Cuppen. unpublishedresults) and 32 miRNAs that were found previously (7,39). Ofthe latter set, six were already in our previous in situscreen (36) (130a, 187, 101b 135, 193b, 301b), but thesequences of these probes turned out to contain some mis-matches compared with the miRNA sequences cloned byChen et al. (39), so that we decided to test new and correctprobes.

First, we analyzed the expression of all 67 miRNAs byin situ hybridization on different zebrafish embryonic stages.Only a subset of 28 miRNAs could be detected in situ(Figure 2, Tables 1 and 2). For many of the miRNAs, theexpression was restricted to specific tissues or cell types,although overall we observed less tissue-restricted expressionas in our previous study for the conserved vertebrate miRNAset (Figure 2, Tables 1 and 2). Several miRNAs wereexpressed in (parts of) the brain (e.g. miR-92b, miR-500a/band miR-135) and these have unique patterns as revealedby sectioning of the embryos (Figure 2b). Other examplesof tissue-specific expression are miR-451, which is expressedin the blood cells, miR-455, which is expressed in thecartilage of the pharyngeal arches and head skeleton andmiR-459, which is only expressed in the anterior part ofthe gut.

The expression of several miRNAs was ubiquitous in thelater stages of development (miR-130a/b/c and miR-301b),whereas all members from the miR-430 family of miRNAswere expressed ubiquitously only in the earlier stages up to48 h of development, as described previously (29).

Of the 35 new miRNAs analyzed in this study, we couldonly detect four by in situ hybridization, which probablyreflects the low abundance of these new miRNA as alreadyindicated by the low cloning frequency. miR-34c-5p wasexpressed in the nose; miR-499 was expressed in the heart,the somitic muscles and the muscles of the head;miR-735-3p was expressed ubiquitously but primarily in thecentral nervous system; miR-733 was also expressed ubiqui-tously but more strongly in the gut and the cells surroundingthe yolk.

Northern blot analysis of known miRNAs

As outlined above, we could observe a clear in situ expres-sion pattern in the embryo for only 28 of the 67 miRNAs ana-lyzed. We next went on analyzing the expression of allmiRNAs by northern blot analysis, since this is a more sensi-tive method for detecting miRNAs, and it also determines thelength of the RNA species, providing further evidence for theexistence of a bonafide miRNA. Furthermore, the in situhybridization analysis is only restricted to embryonic stagesof development, whereas northern blot analysis enabled us


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Figure 1. Cloning frequency and conservation for all miRNAs cloned fromzebrafish small-RNA cDNA libraries. The upper panel depicts the cloning frequency for all small RNAs that were found in the two libraries and that passed our computational pipeline. All 139 known miRNAs (black data points) were cloned more than once, while 37 out of the 66 newmiRNAs (gray dots) were represented by a single sequenced clone. The lower panel shows a scatter plot of the conservation of known (black dots) and new (gray data dots) miRNAs in 12 vertebrate species (zebrafish, fugu, tetraodon, mouse, rat, human, dog, macaca, opossum, chicken, chimpanzee, cow). Forty-four of the new miRNAs were only found in zebrafish, while most of the known miRNAs were found in several species according to our conservation criteria.

to detect miRNAs in RNA derived from adult zebrafishtissues.

We first analyzed the temporal expression in five develop-mental stages ranging from 24 h to adult fish for the set of32 miRNAs that was already cloned or predicted previously(Figure 3a and Table 1). While some miRNAs could mainlybe detected in RNA from adult zebrafish (miR-202, miR-460), others were expressed at all time points analyzed(miR-130a/b/c) and some were also expressed mainly inembryonic stages, but at much lower levels in the adult fish(miR-363 and miR-301b). Again, all members of themiR-430 miRNA family were expressed abundantly in theearly embryonic stages up to �72 h, but were absent inRNA from 5-day-old larvae and adult fish.

Next, we compared the in situ patterns of miRNAs in theembryo with the spatial expression in the adult zebrafish(Figure 3b and Table 1). For the majority of miRNAs, theexpression in RNA derived from adult tissues is similar tothe expression in the embryo. For example, miR-135 is spe-cific for the embryonic and the adult brain. Similarly,miR-459 is expressed in the embryonic anterior gut and in

the adult fish it is expressed exclusively in the gut. However,for some miRNAs the expression in the embryo was differentcompared with the expression in the adult, e.g. miR-455 isexpressed in the cartilage of the embryo (Figure 2a and b),but the expression in the adult is in many tissues. Overall,on northern blots we could detect 30 out of the 32 knownmiRNAs analyzed.

Northern blot analysis of newly cloned miRNAs

Although we were able to obtain in situ expression patternsfor the majority of known miRNAs, we could only detect 4out of the 35 novel miRNAs that were cloned in this study(Figure 2 and Table 2). We next went on analyzing theexpression of these 35 newly identified miRNAs on northernblots using the same LNA probes as for in situ hybridization(Figure 4). First, we scanned the whole set of 35 miRNAs forpresence in large quantities of total RNA (�15 mg) from 24-h-old embryos and adult zebrafish (data not shown). For allmiRNAs that gave a positive signal, we performed new north-ern blots with total RNA from different developmental stages

Figure 1. Cloning frequency and conservation for all miRNAs cloned from zebrafish small-RNA cDNA libraries. The upper panel depicts the cloning frequency forall small RNAs thatwere found in the two libraries and that passed our computational pipeline.All 139 knownmiRNAs (blue data points)were clonedmore thanonce,while 37 out of the 66 newmiRNAs (pink dots) were represented by a single sequenced clone. The lower panel shows a scatter plot of the conservation of known (bluedots) and new (pink data dots) miRNAs in 12 vertebrate species (zebrafish, fugu, tetraodon, mouse, rat, human, dog, macaca, opossum, chicken, chimpanzee, cow).Forty-four of the newmiRNAswere only found in zebrafish, while most of the knownmiRNAswere found in several species according to our conservation criteria.


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and with total RNA from 10 dissected tissues from adultzebrafish. In total, we could detect 16 miRNAs on timeseries northern blots (Figure 4a). As for the known miRNAsanalyzed in Figure 3a, some miRNAs were expressedthroughout development (miR-15c and miR-736) and otherswere expressed only in the adult (miR-731). Another set ofthe new miRNAs was most abundant in 5-day-old larvae,while there was a strong drop in expression in the adultzebrafish (miR-726-3p, miR-729, miR-728 and miR-190b).Except for miR-726-3p, these have all been cloned primarilyfrom the 5-day-old larvae library. An additional three miR-NAs could be detected on northern blots containing RNAfrom dissected tissues (Figure 4b). For most miRNAs wecould only detect an RNA species corresponding to thelength of miRNAs, but for two we saw some additional bandscorresponding to larger RNA molecules (miR-735-3p andmiR-733), which is probably background.

There was a wide variety in expression in RNA from dis-sected tissues. For example, miR-499 is specific for the heartand the muscles. However, many newly cloned miRNAs wereexpressed in the adult brain (miR-739, miR-34c-5p, miR-728,miR-723-3p, miR-737-3p, miR-727-5p). Encouraged by this

finding, we analyzed the expression of the miRNAs, whichwe did not initially detect in total RNA samples from24-h-old embryos or adult fish, in RNA from adult fishbrain. By doing this, we could detect four more miRNAsthat were expressed in the adult brain sample, whereas hardlyany signal was detected in the total RNA sample (Figure 4c).Thus, by increasing the amount of RNA on the northern blotand by looking in a specific tissue, we enriched enough todetect some additional new miRNAs. In total, we could detect23 out of 35 newly cloned miRNAs by northern blotting.

To quantify the differential expression level of known andnew miRNAs, we performed parallel northern blots using adilution series of total adult fish RNA and an adult brainRNA sample. We probed these for two miRNAs that areeasily detectable, also in situ, and that are expressed in thebrain (miR-124 and miR-181b), with two new miRNAs thatwe found to be expressed at least in the adult fish brain(miR-489 and miR-34c-5p) (Figure 4d). Although miR-124and miR-181b were easily detected also in the more dilutedtotal RNA samples, miR-489 and miR-34c-5p could onlybe detected clearly in the adult brain samples. The expressionlevel of miR-34c-5p in the undiluted total RNA sample is

Table 1. Overview of expression data for known miRNAs

miRNA Expression timea Expression adult tissuea Expression in situ Temporal expression in situ12 h 16 h 24 h 48 h 72 h 5 days

dre-miR-101b Adult Ubiquitous No expression 0 0 0 0 0 0dre-miR-130a 24 h–adult Ubiquitous Head (gills, brain, jaw) 0 0 1 3 3 3dre-miR-130b 24 h–adult Ubiquitous Ubiquitous 0 0 1 3 3 3dre-miR-130c 24 h–adult Ubiquitous Head (gills, brain, jaw) 0 0 1 3 3 3dre-miR-135 48 h–adult Brain Structures in brain 0 0 0 3 3 3dre-miR-187 Not expressed ND No expression 0 0 0 0 0 0dre-miR-193b Adult Stronger in muscle, fins, skin Brain, jaw 0 0 0 0 3 3dre-miR-202 24 h–adult Not in any of the analyzed tissues No expression 0 0 0 0 0 0dre-miR-27c 48 h–adult Ubiquitous but not in liver Weak ubiquitous, pharyngeal arches, jaw 0 0 0 1 3 3dre-miR-301b 48 h–adult Ubiquitous but strongest in

brain and eyeUbiquitous 0 0 0 2 2 2

dre-miR-363 24 h–adult Strongest in muscle, liver, skin No expression 0 0 0 0 1 1dre-miR-365 48 h–adult Brain, eye, muscle, fins, skin Weak brain 0 0 0 0 3 3dre-miR-429 24 h–adult Gills, fins, skin Nose, neuromasts (hair and

supporting cells),taste buds, proctodeum

0 0 3 3 3 3

dre-miR-430a 24 h–5 days ND Ubiquitous in early stages 2 2 2 2 1 1dre-miR-430b 24 h–5 days ND Ubiquitous in early stages 2 2 2 2 1 0dre-miR-430c 24–48 h ND Ubiquitous in early stages 2 2 2 2 1 0dre-miR-430i 24–72 h ND Ubiquitous in early stages 2 2 2 2 1 1dre-miR-430j 24–5 days ND Ubiquitous in early stages 2 2 2 2 1 1dre-miR-451 48 h–adult Ubiquitous Blood 0 0 3 3 3 3dre-miR-454a 48 h–adult Ubiquitous, but stronger in

brain and eyeBrain and pharyngeal arches 0 0 0 3 3 3

dre-miR-454b 48 h–adult Ubiquitous, but stronger inbrain and eye

Brain and pharyngeal arches 0 0 0 3 3 3

dre-miR-455 72 h–5 days Eye, muscle, fins, skin Cartilage in head 0 0 0 3 3 3dre-miR-456 24 h–adult Weak brain Weak brain 0 0 1 1 3 3dre-miR-457a 24 h–adult Ubiquitous Brain 0 0 0 3 3 3dre-miR-457b 24 h–adult Ubiquitous Brain 0 0 0 3 3 3dre-miR-458 48 h–adult Stronger in brain and eye No expression 0 0 0 1 1 1dre-miR-459 72 h–adult Gut Gut 0 0 0 0 3 3dre-miR-460 72 h–adult Fins No expression 0 0 0 0 0 0dre-miR-461 Not expressed ND No expression 0 0 0 0 0 0dre-miR-462 Adult Gills, fins, skin Liver and head 0 0 0 0 3 3dre-miR-92b 24 h–adult Ubiquitous Outline of tectum and telencephalon 0 0 0 3 3 3dre-miR-740 Weak 5 days–adult ND No expression 0 0 0 0 0 0

ND ¼ not determined; 0 ¼ no expression; 1 ¼ weak ubiquitous or background expression; 2 ¼ strong ubiquitous expression; 3 ¼ specific expression.aDetermined by northern blot analysis.


37


comparable to the expression level of miR-124 in the mostdiluted (27·) total RNA sample, showing that there is a�30 fold difference in abundance between these two miR-NAs. These examples indicate that indeed many of the miR-NAs that we cloned in addition to the already knownmiRNAs are much less abundant and therefore more difficultto detect.

Cloning and expression of miRNA star sequences

For 65 known and 11 new miRNAs we also cloned the otherarm of the hairpin. The 65 star sequences of known miRNAswere represented by 621 clones (Supplementary Table S5)and the 11 star sequences of new miRNAs were represented

by 45 clones (Supplementary Table S6). In most cases, themiRNA was cloned more often than the star sequence.

In our expression analysis, we included six star sequencesof new miRNAs. We were unable to detect any of these byin situ hybridization. However, we detected miR-723-5pand miR-727-5p by northern blot analysis (Figure 4). Asexpected, the expression of these two miRNA star sequencesoverlaps in both cases with the expression of the miRNA.

DISCUSSION

Regulation of gene expression by miRNAs is an importantprocess for development of multicellular organims, andorganisms without miRNAs cannot live (29,30,44). This isstrengthened further by recent insight into the abundance

Table 2. Overview expression data new miRNAs

New ID Expression timea Expression adult tissuea Expression in situ Temporal expression in situ12 h 16 h 24 h 48 h 72 h 5 days

dre-miR-489b No expression Brain, eye No expression 0 0 0 0 0 0ZF_nl_11 No expression No expression No expression 0 0 0 0 0 0dre-miR-724-3pc No expression No expression No expression 0 0 0 0 0 0dre-miR-724-5p No expression Weak braind No expression 0 0 0 0 0 0dre-miR-725 48 h–adult Gills, fins No expression 0 0 0 0 0 0dre-miR-726-3p 72 h–adult Eye No expression 0 0 0 0 0 0dre-miR-726-5pc No expression No expression No expression 0 0 0 0 0 0ZF_nl_139 No expression No expression No expression 0 0 0 0 0 0ZF_nl_149 No expression No expression No expression 0 0 0 0 0 0ZF_nl_157 No expression No expression No expression 0 0 0 0 0 0dre-miR-727-3p No expression Braind No expression 0 0 0 0 0 0dre-miR-727-5pc No expression Brain, eye No expression 0 0 0 0 0 0dre-miR-728 48 h–adult Brain, eye No expression 0 0 0 0 0 0dre-miR-729 48 h–adult Eye No expression 0 0 0 0 0 0ZF_nl_21 No expression No expression No expression 0 0 0 0 0 0dre-miR-190b 48 h–adult Eye No expression 0 0 0 0 0 0ZF_nl_236 No expression No expression No expression 0 0 0 0 0 0dre-miR-34c-3pc No expression No expression No expression 0 0 0 0 0 0dre-miR-34c-5p 48 h–adult Brain Nose 0 0 0 0 3 3dre-miR-722 24 h–adult Brain, eye, gills, skin, liver No expression 0 0 0 0 0 0dre-miR-730-3pc No expression No expression No expression 0 0 0 0 0 0dre-miR-730-5p 24 h–adult Brain, eye, fins, skin No expression 0 0 0 0 0 0ZF_nl_263 No expression No expression No expression 0 0 0 0 0 0ZF_nl_264 no expression No expression No expression 0 0 0 0 0 0dre-miR-731 Adult Gills, fins, skin, gut, heart No expression 0 0 0 0 0 0ZF_nl_286 No expression No expression No expression 0 0 0 0 0 0dre-miR-732 No expression Weak braind No expression 0 0 0 0 0 0dre-miR-733 24 h–adult Eye, brain, muscle gills, fins, skin, gut Ubiquitous, but more in

yolk and gut2 2 3 3 3 3

dre-miR-15c 24 h–adult Ubiquitous, not in liver No expression 0 0 0 0 0 0ZF_nl_33 No expression No expression No expression 0 0 0 0 0 0dre-miR-734 No expression Braind No expression 0 0 0 0 0 0dre-miR-735-3p 24 h–adult Ubiquitous but not in liver and gut Ubiquitous (brain, neural tube,

outline neuromasts)2 2 2 3 3 3

ZF_nl_384 No expression No expression No expression 0 0 0 0 0 0dre-miR-736 24 h–adult Eye, gut No expression 0 0 0 0 0 0dre-miR-737-3p No expression Brain, eye No expression 0 0 0 0 0 0dre-miR-738 48 h–adult Gills No expression 0 0 0 0 0 0dre-miR-739 No expression Brain No expression 0 0 0 0 0 0ZF_nl_51 No expression No expression No expression 0 0 0 0 0 0dre-miR-499 24 h–adult Heart and muscles and fins Heart and muscles in head 0 0 0 3 3 3dre-miR-723-3p 5 days–adult Brain No expression 0 0 0 0 0 0dre-miR-723-5pc No expression Weak braind No expression 0 0 0 0 0 0

aDetermined by northern blot analysis.bPredicted based on verified mammalian sequence.cRegarded as miRNA star sequence.dOnly checked for expression in brain.0 ¼ no expression; 1 ¼ weak ubiquitous or background expression; 2 ¼ strong ubiquitous expression; 3 ¼ specific expression.


38

Chapter 2

and conservation of miRNAs and the high number of miRNAtargets found in animals (5,25,26).

Here we describe the cloning and expression of newmiRNAs from the zebrafish. By deep sequencing twosmall-RNA cDNA libraries we found 66 new miRNAs and11 star sequences corresponding to 116 potential miRNAhairpins in the zebrafish genome. The majority (97.4%) of

small-RNAs that were found in the libraries correspondedto known miRNAs and 56% of the new miRNAs wererepresented by a single sequenced clone. However, only13% of the new miRNAs that we detected on northernblots were cloned only once, indicating that these singlecloned miRNAs are more difficult to detect and also thatmiRNA cloning frequency reflects miRNA abundance. In

A

B

Figure 2. Examples of expression patterns of miRNAs in the zebrafish embryo as revealed by whole mount in situ hybridization. (A) Whole mount pictures of72-h-old and 5-day-old larvae and (B) pictures of sections from5-day-old larvae. (A andM)miR-454a is expressed in the brain, the pharyngeal arches and the jaw andthe eye; (B,Q andT) miR-429 is expressed in the hair and supporting cells of the lateral line organ (T), the taste buds (Q), the nose and the epithelium of the lips; (Cand S) miR-459 is expressed in the anterior gut; (D and U) miR-451 is expressed in the blood cells; (E and R) miR-92b is expressed in the proliferating zones ofthe preoptic region, optic tectum, tegmentum, telencephalon and octaval area; (F andP) miR-499 is expressed in the ventricle and atrium, themuscles of the head andthe somitic muscles; (G and L) miR-733 is expressed ubiquitously but primarily in the intestine; (H) miR-735-3p is expressed ubiquitously, but primarilyin the central nervous system; (I and N) miR-455 is expressed in the cartilage of the pharyngeal arches and head skeleton; (J) miR-34c-5p is expressed in thenose; (K and O) miR-135 is expressed in the pallium, optic ganglion, optic tectum, ventral telencephalon and at the beginning of the medulla oblongata.


39


addition, 67% of the newly cloned miRNAs are, out of 12 dif-ferent species, conserved only in zebrafish according to ourconservation criteria (>90% identity for the mature miRNAand >70% identity for the precursor). Thus, our cloning andexpression data show that although many miRNAs are abun-dantly expressed, there are also miRNAs that have muchlower expression levels or that are expressed in only a fewcells. For example, miR-34c-5p has a very restricted expres-sion pattern in the nose of the embryo. Of the new miRNAs37 were picked up only once, indicating that our sequencingdid not reach saturation yet. Furthermore, we screened only

two libraries derived from a limited amount of tissues. Ifsome miRNAs are only expressed in specific adult tissues,and some of them are (Figures 3 and 4), these will be missedby our libraries, which do not contain all adult tissues. Inorder to determine the complete set of miRNAs that governsgene expression in an organism, one should perform saturatedsequencing of small-RNA cDNA libraries from severaltissues.

Current estimates, which are based on cloning and compu-tational predictions of miRNAs from human, suggest thatthere might be up to a thousand miRNAs (6,7). Our data

Figure 3.Northern blot analysis of the expression of knownmiRNAs fromzebrafish. (A) Expression ofmiRNAs in five developmental stages: 24 h, 48 h, 72 h, 5 daysand adult zebrafish. (B) Expression of miRNAs in RNA derived from 10 adult zebrafish tissues: total, brain, eye, muscle, gills, fins, skin, liver, gut and heart. Forsome miRNAs we did not analyze the expression in the heart, because we could not obtain enough heart tissue for analyzing all the miRNAs by northern blotting.U6 snRNA serves as a loading control.


40

Chapter 2

show that there are more miRNAs to be found, but predictthat these will be lowly expressed and less conserved. Theset of abundantly expressed miRNAs in zebrafish is, basedon this study and previous work (36,39), limited to �150 dif-ferent miRNAs and mostly contains conserved miRNAs.

Analysis of the spatial and temporal expression of miRNAsmay shed light on their role in tissue specific processes. Manyof the new miRNAs analyzed in this study have a cell type ortissue specific expression, providing a basis for understandingspecific aspects of development that are under miRNA con-trol. The differences in temporal expression of many miRNAssuggests that some play a role in early development duringgastrulation and segmentation, whereas others may be

required for organ morphogenesis in later stages of embryo-nic development and a few miRNAs are exclusivelyexpressed in the adult fish. For some miRNAs there is a dif-ference in expression in the embryo compared to the adult,suggesting differences in function. For example, miR-92b isexpressed in the embryonic brain, but it is detected in manytissues in the adult fish.

In many cases, miRNA expression patterns correlate withmiRNA function in the Drosophila embryo, where, for exam-ple, knockdown of miRNAs expressed in the peripheral orcentral nervous system induced nervous system defects(37,45). Careful examination of the phenotypes caused bydepletion of specific miRNAs in the vertebrate embryo

Figure 4. Northern blot analysis of newly clonedmiRNAs from zebrafish. (A) Expression of miRNAs detected in five developmental stages: 24 h, 48 h, 72 h, 5 daysand adult zebrafish. (B) Expression ofmiRNAs in 10 different tissues from adult zebrafish: total, brain, eye, muscle, gills, fins, skin, liver, gut and heart. (C) miRNAsdetected by specifically probing RNA from adult fish brain. (D) Dilution series of adult fish RNA and one adult brain RNA sample. Blots were probed for twoabundant and brain-specific miRNAs (miR-124 and miR-181b) and two new and brain specific miRNAs cloned in this study (miR-489 and miR-34c-5p).U6 snRNA serves as a loading control. 1, regarded as the miRNA star sequence.


41


together with computational prediction of miRNA targetsmay further help in understanding their role in development.

ACKNOWLEDGEMENTS

We thankBrandonAson andReneKetting for critically readingof the manuscript. This work was supported by the NetherlandsGenomics Initiative and the European Union. Funding to paythe Open Access publication charges for this article wasprovided by the Council for Earth and Life Sciences of theNetherlands Organization for Scientific Research.

Conflict of interest statement. None declared.

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Supplementary information

Figure S1. Venn-diagram of known zebrafish miRNAs from the miRNA registry, miRNAs cloned from the zf-brain library and miRNAs cloned from the zf-embryo library.

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Figure S2. Bar diagram, showing the frequency of the base identity of the first base of known miRNAs (from the miRNA registry) and novel miRNAs cloned in this study.

new ID probe sequence new ID probe sequence

dre-miR-499 AAACATCACTGCAAGTCTTAAC dre-miR-726_5p1 AGTTCAGAACTAGCGGAATTCC

ZF_nl_384 CCCAATAAGGTTCCCAGGGA dre-miR-738 GAGGTCCCGACGCGGGCCGTAGC

dre-miR-731 CGATCCGGGAGAAAACGTGTCATT ZF_nl_33 AAACTCTGCTAAAACAGGACTCA

ZF_nl_21 CAGTGTCGTTACTCCCAATCGTG dre-miR-724_3p1 AAACAGTCGCAAATTCCCTTTAA

dre-miR-723_5p1 AAAGTAACATCATTTAAAACTGTC dre-miR-130c ATGCCCTTTTAATATTGCACTG

dre-miR-726_3p CCGAGTTCTGCTAGTAGTGAA dre-mir-451 AACTCAGTAATGGTAACGGTTT

dre-mir-489 AGCAGCCGTACATATGATGTCAC dre-mir-455 TGTATATGCCCATGGACTGCAT

ZF_nl_149 GTACCCGAGTGATGTCTGAAACCA dre-mir-92b GGAGGCCGGGACGAGTGCAATAT

ZF_nl_11 GAATCAGCGGAGTGGGGAGA dre-mir-429 ACGGCATTACCAGACAGTATTA

dre-miR-15c GAAAACCATGACGCGCTGCTT dre-mir-430a CTACCCCAACAAATAGCACTTA

dre-miR-730_5p ACACACAGCATGCACAATGAGGA dre-miR-130a ATGCCCTTTTAACATTGCACTG

dre-miR-733 AACCACTGAGCTAAACCAACGCA dre-mir-202 CAAAGAGGTATATGCATAGGAA

dre-miR-34c_5p GTAATCAACTAACTGCACTGCCT dre-mir-365 ATAAGGATTTTTAGGGGCATTA

dre-miR-739 AACCCTTCTCCACTTCGGCCT dre-mir-101b CTTCAGTTATCATAGTACTGTA

ZF_nl_236 ACCAATCCACCAGCTGTGCCCA dre-miR-130b ATGCCCTTTCATTATTGCACTG

ZF_nl_157 TGCCAGGGATTGTAGGTGTGA dre-miR-135 CACATAGGAATAGAAAGCCATA

ZF_nl_286 CGCAGGCAACTTAAAACAAGCC dre-miR-187 GTTCCACTGGCTGCAACACAAG

dre-miR-727_3p TAAGTCTTCAACTCGCCTCAAC dre-mir-193b AGCGGGACTTTGCGGGCCAGTT

dre-miR-725 ACTACTAGAAACAATGACTGAA dre-mir-27c GCAGAACTTAACCACTGTGAA

dre-miR-735_3p GTCAAGTTTAGCGGTGGGAGAG dre-mir-301b CAATGACAATACTATTGCACTG

dre-miR-737_3p TATTTTCTTTAGGTTTTGATT dre-mir-363 TACAGATGGATACCGTGCAATT

dre-miR-734 CGGTACGATTCTGCAGCATTTAC dre-mir-454a CCCTATTAGCAATATTGCACTA

Table S1. LNA probe sequences

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Chapter 2

dre-miR-727_5p1 GCTGGGAGGAATTGAAGACTGA dre-mir-454b CCCTATAAGCAATATTGCACTA

dre-miR-723_3p AGCACAGATTTAATTGATGTCTT dre-mir-430c CTACCCCAAAGAGAAGCACTTA

dre-miR-730_3p1 CCTCCACATTGCAGGCGCTGTG dre-mir-462 AGCTGCATTATGGGTTCCGTTA

dre-miR-34c_3p1 CCTGGTAGTGAGGTTAGTGATT dre-mir-460 CGCACAGTGTGTACAATGCAGG

dre-miR-732 ACCGAGAGTTCTCTGCTTTGAG dre-mir-461 TTGGCATTTAGCCCATTCCTGA

dre-miR-729 AACCCAGGTCGTATCATACCCATG dre-mir-430i CTACGCCAACAAATAGCACTTA

ZF_nl_263 GACGTCATTAGCGACCCGA dre-mir-430b CTACCCCAACTTGATAGCACTTT

dre-miR-724_5p CTTAAAAGGAAGGTGTGGCTG dre-mir-430j TACCCCAATTTGATAGCACTTT

ZF_nl_51 ACTGACAGAATCAGCGGAGCCG dre-mir-456 TGACAACCATCTAACCAGCCTG

ZF_nl_264 GAATGTACACTGGATGCAGACA dre-mir-457a TGCCAATATTGATGTGCTGCTT

dre-miR-728 GAAAACGTAGTGTACTTAGTAT dre-mir-457b CTCCAGTATTTATGTGCTGCTT

ZF_nl_139 AATGGCAATATGGAGGCATAGCA dre-mir-458 GCAGTACCATTCAAAGAGCTAT

dre-miR-722 AATCTGAAACGTTTCTGCAAAAAA dre-mir-459 CAGGATGAATCCTTGTTACTGA

dre-miR-736 AAAACTTTTTGTTCGTCTTAC dre-miR-740 ACTGTACCATACCACTTTTTAT

dre-miR-190b CAACCGAATATCAAACATATCA

1probes for miRNA star sequences

Table S2. Cloning frequency and conservation of known miRNAs from zebrafish

mir ID brain larvae total conservation

dre-let-7a 155 13 168 zfish,fugu,todon,chicken,opossum,chimp,human,macaca,cow,dog,mouse,rat

dre-let-7b 107 4 111 zfish,fugu,todon,chicken,cow,dog,chimp,macaca,human,opossum,mouse,rat

dre-let-7c 39 4 43 zfish,chicken,chimp,cow,dog,human,macaca,mouse,rat

dre-let-7d 44 10 54 zfish,fugu,todon,chicken,chimp,cow,dog,human,macaca,mouse,rat

dre-let-7e 7 1 8 zfish,fugu,chicken,todon,cow

dre-let-7f 6 0 6 zfish,mouse,rat,chimp,cow,dog,human,macaca,opossum,chicken,todon

dre-let-7g 6 4 10 zfish,todon,fugu,chimp,dog,human,mouse,rat,macaca,opossum,cow,chicken

dre-let-7h 2 0 2 zfish,todon,fugu,cow,chimp,human,macaca,rat,mouse,dog

dre-let-7j 2 0 2 zfish,chimp,dog,macaca,mouse

dre-miR-1 18 22 40 zfish,todon,fugu,macaca,chicken,chimp,human,mouse,opossum,cow,dog,rat

dre-miR-100 298 41 339 zfish,fugu,todon,mouse,rat,chimp,human,macaca,opossum,dog,cow

dre-miR-101a 24 5 29 zfish,fugu,chimp,cow,dog,macaca,mouse,rat,todon,opossum,human

dre-miR-103 17 2 19 zfish,fugu,todon,chicken,opossum

dre-miR-107 6 6 12 zfish,fugu,todon,mouse,chimp,cow,human,macaca,rat,dog,chicken

dre-miR-10a 38 43 81 zfish,chimp,human,macaca,mouse,rat,cow,dog,opossum,chicken

dre-miR-10b 18 25 43 zfish,fugu,todon

dre-miR-10d 15 22 37 zfish,mouse,rat,chimp,human,macaca,opossum,chicken,todon,fugu,cow,dog

dre-miR-122 14 18 32 zfish,todon,fugu,chimp,human,dog,chicken

dre-miR-124 92 40 132 zfish,fugu,todon,mouse,opossum,rat,chimp,human,dog,macaca,chicken,cow

dre-miR-125a 776 73 849 zfish,fugu

dre-miR-125b 2357 201 2558 zfish,todon,fugu,opossum,dog,chimp,human,macaca,rat,mouse

dre-miR-125c 163 17 180 zfish

dre-miR-126 39 5 44 zfish,todon,fugu,mouse,dog

dre-miR-128 450 9 459 zfish

dre-miR-129 255 12 267 zfish,todon,fugu,chimp,human,dog,mouse,macaca,rat,opossum

45


dre-miR-130a 4 3 7 zfish,chicken

dre-miR-130b 10 8 18 zfish,dog,chimp,macaca,rat,cow,mouse

dre-miR-132 165 6 171 zfish,todon,fugu

dre-miR-133a 64 73 137 zfish,mouse,opossum,chicken,dog,cow,fugu,chimp,human,macaca,rat,todon

dre-miR-133b 33 38 71 zfish,todon,chimp,human,macaca,opossum,rat,chicken,mouse

dre-miR-133c 13 18 31 zfish,todon

dre-miR-135 61 31 92 zfish,fugu,todon

dre-miR-135b 16 17 33 zfish

dre-miR-137 48 28 76 zfish,fugu,opossum,chicken,chimp,cow,dog,human,macaca,mouse,rat,todon

dre-miR-138 365 41 406 zfish,fugu,todon,chicken,rat,dog,chimp,cow,human,macaca,mouse,opossum

dre-miR-140 14 19 33 zfish,dog,chicken,mouse,chimp,human,macaca,rat,opossum,todon,fugu


dre-miR-142a 6 2 8 zfish,todon,fugu,chimp,dog,human,macaca,rat,chicken,mouse,opossum

dre-miR-142b 5 2 7 zfish,chicken,chimp,dog,human,macaca,rat,fugu,todon,mouse,opossum

dre-miR-143 9 5 14 zfish,rat,mouse,fugu,dog,human

dre-miR-144 2 4 6 zfish,fugu,todon,chicken

dre-miR-145 21 7 28 zfish,fugu

dre-miR-146a 3 0 3 zfish,todon




dre-miR-152 15 13 28 zfish,cow,dog,mouse,rat,human,macaca,chimp

dre-miR-153a 4 0 4 zfish,chicken,opossum,chimp,cow,human,macaca,dog,fugu,rat,todon,mouse

dre-miR-153b 12 8 20 zfish,todon,fugu,opossum,cow,chimp,human,macaca,rat



dre-miR-16a 15 4 19 zfish,fugu,todon,opossum,rat,cow,dog,chimp,chicken

dre-miR-16b 15 4 19 zfish,todon,fugu


dre-miR-17a 18 10 28 zfish,chicken,opossum

dre-miR-181a 264 131 395 zfish,fugu,todon,chicken,cow,mouse,rat,opossum,chimp,human,macaca,dog

dre-miR-181b 91 59 150 zfish,mouse,rat,chicken,macaca,chimp,dog,human,opossum,cow,fugu,todon


dre-miR-182 18 15 33 zfish,todon,fugu,opossum,mouse,macaca,dog,chimp,human

dre-miR-183 13 23 36 zfish,fugu,opossum,todon,chicken,chimp,human,cow,dog,macaca,rat

dre-miR-184 14 17 31 zfish,fugu,todon,opossum,macaca,chimp,human,mouse


dre-miR-18a 5 13 18 zfish,fugu,todon,chicken,cow,mouse,rat,chimp,dog,human,macaca,opossum


dre-miR-190 2 1 3 zfish,fugu,opossum,todon,chicken,chimp,cow,dog,human,macaca,mouse,rat





dre-miR-196a 2 3 5 zfish,todon,fugu


46

Chapter 2

dre-miR-199 117 122 239 zfish,fugu,todon,chicken,cow,mouse,chimp,human,dog,opossum,macaca

dre-miR-19a 58 63 121 zfish,fugu,todon,chimp,cow,dog,human,macaca,opossum,rat,chicken,mouse

dre-miR-19b 153 130 283 zfish,fugu,chimp,dog,human,cow

dre-miR-19c 19 32 51 zfish,chicken,opossum

dre-miR-19d 21 26 47 zfish,todon

dre-miR-200a 39 46 85 zfish,fugu,todon,macaca,cow,mouse,rat,chicken

dre-miR-200b 11 7 18 zfish,fugu,todon,chicken,mouse,rat

dre-miR-200c 14 7 21 zfish,fugu,cow,chicken

dre-miR-203b 88 125 213 zfish,todon

dre-miR-204 14 9 23 zfish,chicken,opossum,todon,fugu

dre-miR-205 35 60 95 zfish,fugu,todon,opossum

dre-miR-20a 28 20 48 zfish,fugu,todon,opossum


dre-miR-21 95 10 105 zfish,fugu,todon,chicken,mouse,opossum



dre-miR-214 4 2 6 zfish,fugu,todon,opossum,chimp,cow,dog,human,macaca,mouse,rat

dre-miR-216a 10 18 28 zfish


dre-miR-217 11 17 28 zfish,chicken,todon,chimp,human,macaca,fugu,dog,mouse,opossum,rat

dre-miR-218a 29 39 68 zfish,fugu,todon


dre-miR-219 206 129 335 zfish,todon,fugu,cow,rat

dre-miR-221 250 39 289 zfish,todon,fugu,cow,mouse,rat,chimp,human,macaca,dog,opossum,chicken

dre-miR-222 160 22 182 zfish,fugu,todon,chicken,rat,human,macaca,dog,mouse,opossum,cow


dre-miR-22a 102 58 160 zfish,fugu,todon,chicken,dog,opossum,chimp,cow,human,mouse,rat


dre-miR-23a 6 7 13 zfish,fugu,todon

dre-miR-24 31 10 41 zfish,cow,chimp,dog,human,macaca,mouse,rat

dre-miR-25 9 12 21 zfish,fugu,todon,dog,opossum,human,macaca,mouse

dre-miR-26a 51 22 73 zfish,todon,fugu

dre-miR-27a 2 1 3 zfish,fugu,todon,opossum

dre-miR-27b 17 2 19 zfish,chicken,chimp,human,macaca,mouse,rat,cow,dog,fugu,todon,opossum


dre-miR-27d 3 1 4 zfish,opossum,cow,chimp,human,macaca,mouse,rat,dog,chicken,fugu,todon

dre-miR-27e 4 1 5 zfish,todon,fugu

dre-miR-29a 19 4 23 zfish,todon,cow,fugu,chicken,opossum,dog,rat


dre-miR-301a 13 4 17 zfish,rat,mouse,chicken,dog,chimp,human,macaca,opossum


dre-miR-301c 6 9 15 zfish,todon,fugu



dre-miR-30d 38 6 44 zfish

dre-miR-30e 23 22 45 zfish,todon,fugu,chicken

47



dre-miR-34 28 6 34 zfish,todon,chicken,macaca,chimp,human,opossum,dog,cow,mouse,rat

dre-miR-363 5 5 10 zfish,dog,human,macaca,rat,mouse,opossum

dre-miR-365 7 2 9 zfish,fugu,todon,chicken,cow,dog,human,macaca,mouse,rat,opossum,chimp


dre-miR-429 0 4 4 zfish,fugu,todon,cow,mouse,rat,dog,human,chicken,chimp,opossum,macaca

dre-miR-454a 2 2 4 zfish,macaca,chicken,chimp,human,cow,dog,fugu


dre-miR-455 5 7 12 zfish,todon,fugu,chicken,opossum,chimp,dog,human,macaca,rat,mouse


dre-miR-457a 2 0 2 zfish


dre-miR-458 2 4 6 zfish,chicken,fugu

dre-miR-460 2 1 3 zfish,fugu


dre-miR-7b 35 9 44 zfish,fugu,macaca,mouse,opossum,chimp,human,rat,cow,dog,chicken

dre-miR-9 811 467 1278 zfish,fugu,todon,opossum,chimp,cow,dog,human,macaca,mouse,rat,chicken

dre-miR-92a 20 18 38 zfish,chimp,human,chicken,opossum,cow,dog,rat,fugu,todon



dre-miR-96 6 11 17 zfish,todon,fugu,opossum,cow,chimp,human,mouse,dog,rat

dre-miR-99 166 34 200 zfish,dog,chimp,cow,human,macaca,rat,chicken,mouse

total 9607 3150 12757

Table S3. Sequences, cloning frequency and conservation of new miRNAs from zebrafishmiRNA sequence brain larvae cloned total conservation

ZF_nl_107 CTTCAGAGCTGAGGTGAG 1 0 1 zfish

ZF_nl_108 ACAGTCTCTCGGAGCGCTCG 1 0 1 zfish

ZF_nl_11 TCTCCCCACTCCGCTGATTC 1 0 1 zfish

dre-miR-724 TTAAAGGGAATTTGCGACTGTT 21 0 21 zfish,fugu,todon

dre-miR-725 TTCAGTCATTGTTTCTAGTAGT 5 7 12 zfish

dre-miR-726 TTCACTACTAGCAGAACTCGG 4 1 5 zfish,todon,fugu

ZF_nl_125 TAATACTGCCTGGTAATGCCAT 1 0 1 zfish

ZF_nl_135 TTGCAGGTTCAATTCCTGTC 0 1 1 zfish

ZF_nl_138 TAGCACCATTTGAAATCGGTCG 1 1 2 zfish,opossum

ZF_nl_139 TGCTATGCCTCCATATTGCCATT 2 0 2 zfish

ZF_nl_149 TGGTTTCAGACATCACTCGGGTAC 0 1 1 zfish

ZF_nl_157 TCACACCTACAATCCCTGGCA 5 0 5 zfish

ZF_nl_164 GAACCTGATGGATCTTCTCT 0 1 1 zfish

dre-miR-727 GTTGAGGCGAGTTGAAGACTTA 13 4 17 zfish,fugu,todon

dre-miR-728 ATACTAAGTACACTACGTTTTC 0 2 2 zfish,todon

ZF_nl_176 TATGGCTTTTTATTCCTATGTG 5 2 7 zfish,todon

dre-miR-729 CATGGGTATGATACGACCTGGGTT 1 3 4 zfish

ZF_nl_21 CACGATTGGGAGTAACGACACTG 0 1 1 zfish

48

Chapter 2

ZF_nl_210 TGAGCGGCTCAGTGAGGTCCTCGGATC 2 0 2 zfish

ZF_nl_224 ACAGGCATGTGTGTGGGTAGAG 1 0 1 zfish

dre-miR-190b TGATATGTTTGATATTCGGTTG 17 24 41 zfish,fugu,todon

ZF_nl_230 CTGTGCCGAAAGACCTGGAACAAAC 1 0 1 zfish

ZF_nl_231 ACTGAGCTGCTGATCCGC 0 1 1 zfish,cow,dog,chimp,human,mouse,macaca

ZF_nl_236 TGGGCACAGCTGGTGGATTGGT 1 0 1 zfish

ZF_nl_240 TATTGTTATTCCTGCTGGTT 1 0 1 zfish

dre-miR-34c AGGCAGTGCAGTTAGTTGATTAC 0 2 2 zfish

ZF_nl_249 TCAGTGCATTACAGAACTTTG 8 7 15 zfish

dre-miR-722 TTTTTTGCAGAAACGTTTCAGATT 1 1 2 zfish,fugu,todon

ZF_nl_254 TGAATCTGGTCCTTCTGG 0 1 1 zfish

dre-miR-730 TCCTCATTGTGCATGCTGTGTGT 10 3 13 zfish,todon

ZF_nl_263 TCGGGTCGCTAATGACGTC 1 0 1 zfish

ZF_nl_264 TGTCTGCATCCAGTGTACATTC 1 0 1 zfish

dre-miR-731 AATGACACGTTTTCTCCCGGATCG 3 1 4 zfish

ZF_nl_286 GGCTTGTTTTAAGTTGCCTGCG 0 1 1 zfish,fugu,todon,chicken

ZF_nl_29 CTCTGCACTTCATTAGTG 1 0 1 zfish

ZF_nl_290 CGGTCGGGGGCGTCAGTGCC 0 1 1 zfish

dre-miR-732 CTCAAAGCAGAGAACTCTCGGT 0 1 1 zfish

ZF_nl_298 TGAGGTGGTTTTCTGTCAC 0 1 1 zfish

ZF_nl_300 GCCAAATGCCTCGTCATCTAAT 2 0 2 zfish,cow

ZF_nl_302 CTCGTACCGTGAGTAATAGTGC 10 10 20 zfish,todon,fugu

ZF_nl_304 CCTGAGACCCGGGTTTGTT 1 0 1 zfish

dre-miR-733 TGCGTTGGTTTAGCTCAGTGGTT 1 1 2 zfish

dre-miR-15c AAGCAGCGCGTCATGGTTTTC 1 0 1 zfish

ZF_nl_325 TCTACAGTGCATGTGTCTCCAGT 31 2 33 zfish,opossum,fugu,todon

ZF_nl_33 TGAGTCCTGTTTTAGCAGAGTTT 1 0 1 zfish

dre-miR-734 GTAAATGCTGCAGAATCGTACCG 3 1 4 zfish

ZF_nl_345 AAAATATCCTGAGCTGTTTTCT 1 0 1 zfish

ZF_nl_350 ATGCAGTCCATGGGCATATACACT 9 7 16 zfish,fugu,chicken,dog,todon,opossum,chimp,human,macaca,rat,mouse

ZF_nl_372 TCCCTCCGTCATTGAATTCCTG 1 0 1 zfish

ZF_nl_376 ACGAGAGCTGGGGTCTTGCTGG 0 1 1 zfish

dre-miR-735 CTCTCCCACCGCTAAACTTGAC 19 0 19 zfish

ZF_nl_384 TCCCTGGGAACCTTATTGGG 1 0 1 zfish

ZF_nl_39 TTTACAGGCTATGCTAATCTAT 1 0 1 zfish,fugu,todon

dre-miR-7361 GTAAGACGAACAAAAAGTTTT N.D. N.D. N.D. zfish,fugu

dre-miR-737 AATCAAAACCTAAAGAAAATA 0 7 7 zfish

dre-miR-738 GCTACGGCCCGCGTCGGGACCTC 4 0 4 zfish

dre-miR-739 AGGCCGAAGTGGAGAAGGGTT 0 1 1 zfish,mouse,chicken

ZF_nl_51 CGGCTCCGCTGATTCTGTCAGT 1 0 1 zfish

ZF_nl_58 TCAGAAAAGTTACCACAGGGAT 0 1 1 zfish,rat,cow

ZF_nl_63 CCGCAACACGAAACTGTCTT 0 1 1 zfish,fugu

dre-miR-499 TTAAGACTTGCAGTGATGTTTA 6 15 21 zfish,todon,fugu,chicken

ZF_nl_68 AGACACTGAGGGGTGTAG 0 1 1 zfish

dre-miR-723 AAGACATCAATTAAATCTGTGCT 3 3 6 zfish

ZF_nl_9 TGAACACGGTCTCTTTTT 1 0 1 zfish

49


ZF_nl_91 TGTCTGTCTCCCCGCTTTCT 1 0 1 zfish

ZF_nl_94 AGGCGATGGGCATCATGAACTCGTT 0 1 1 zfish,dog,fugu,opossum

dre-miR-4892 TGACATCATATGTACGGCTGCT 9 1 10 zfish,todon,fugu,chicken

total 216 121 337

N.D.=no data1cloned from human and conserved in zebrafish2predicted in zebrafish based on hsa-miR-489

Table S4. microRNA hairpins assigned for cloned sequences

miRNA hairpin IDs miRNA sequence

ZF_nl_107 ZF_nl_110 ZF_nl_114 ZF_nl_116 ZF_nl_117 ZF_nl_119 CTTCAGAGCTGAGGTGAG

ZF_nl_108 ACAGTCTCTCGGAGCGCTCG

ZF_nl_11 ZF_nl_34 ZF_nl_55 ZF_nl_56 ZF_nl_65 ZF_nl_66 ZF_nl_235 ZF_nl_269 ZF_nl_292 ZF_nl_317 ZF_nl_324

TCTCCCCACTCCGCTGATTC

dre-miR-724_5p ZF_nl_272 ZF_nl_273 TTAAAGGGAATTTGCGACTGTT

dre-miR-724_3p ZF_nl_272 ZF_nl_273 CAGCCACACCTTCCTTTTAAG

der-miR-725 ZF_nl_378 TTCAGTCATTGTTTCTAGTAGT

dre-miR-726_5p ZF_nl_134 GGAATTCCGCTAGTTCTGAACT

dre-miR-726_3p ZF_nl_134 TTCACTACTAGCAGAACTCGG

ZF_nl_125 TAATACTGCCTGGTAATGCCAT

ZF_nl_135 TTGCAGGTTCAATTCCTGTC

ZF_nl_138 TAGCACCATTTGAAATCGGTCG

ZF_nl_139 TGCTATGCCTCCATATTGCCATT

ZF_nl_149 TGGTTTCAGACATCACTCGGGTAC

ZF_nl_157 TCACACCTACAATCCCTGGCA

ZF_nl_164 ZF_nl_207 ZF_nl_348 ZF_nl_349 ZF_nl_360 ZF_nl_368 ZF_nl_369 GAACCTGATGGATCTTCTCT

dre-miR-727_5p TCAGTCTTCAATTCCTCCCAGC

dre-miR-727_3p GTTGAGGCGAGTTGAAGACTTA

dre-miR-728 ATACTAAGTACACTACGTTTTC

ZF_nl_176 TATGGCTTTTTATTCCTATGTG

dre-miR-729 CATGGGTATGATACGACCTGGGTT

ZF_nl_21 CACGATTGGGAGTAACGACACTG

ZF_nl_210_5p ZF_nl_212 ZF_nl_215 ZF_nl_217 TGAGCGGCTCAGTGAGGTCCTCGGATC

ZF_nl_210_3p ZF_nl_211 ZF_nl_212 ZF_nl_213 ZF_nl_214 ZF_nl_215 ZF_nl_216 ZF_nl_217 ZF_nl_342

TCGTAACAAGGTTTCCGT

ZF_nl_224 ACAGGCATGTGTGTGGGTAGAG

dre-miR-190b TGATATGTTTGATATTCGGTTG

ZF_nl_230 CTGTGCCGAAAGACCTGGAACAAAC

ZF_nl_231 ZF_nl_234 ZF_nl_362 ACTGAGCTGCTGATCCGC

ZF_nl_236 ZF_nl_379 TGGGCACAGCTGGTGGATTGGT

ZF_nl_240 TATTGTTATTCCTGCTGGTT

dre-miR-34c_5p ZF_nl_243 AGGCAGTGCAGTTAGTTGATTAC

dre-miR-34c_3p ZF_nl_243 AATCACTAACCTCACTACCAGG

ZF_nl_249 TCAGTGCATTACAGAACTTTG

dre-miR-722 ZF_nl_35 ZF_nl_185 TTTTTTGCAGAAACGTTTCAGATT

50

Chapter 2

ZF_nl_254 ZF_nl_344 TGAATCTGGTCCTTCTGG

dre-miR-730_5p TCCTCATTGTGCATGCTGTGTGT

dre-miR-730_3p CACAGCGCCTGCAATGTGGAGG

ZF_nl_263 TCGGGTCGCTAATGACGTC

ZF_nl_264 TGTCTGCATCCAGTGTACATTC

dre-miR-731 ZF_nl_330 AATGACACGTTTTCTCCCGGATCG

ZF_nl_286 GGCTTGTTTTAAGTTGCCTGCG

ZF_nl_29 CTCTGCACTTCATTAGTG

ZF_nl_290 ZF_nl_291 CGGTCGGGGGCGTCAGTGCC

dre-miR-732_nl_299 CTCAAAGCAGAGAACTCTCGGT

ZF_nl_298 TGAGGTGGTTTTCTGTCAC

ZF_nl_300 ZF_nl_336 GCCAAATGCCTCGTCATCTAAT

ZF_nl_302_5p CATTATTACTTTTGGTACGCG

ZF_nl_302_3p CTCGTACCGTGAGTAATAGTGC

ZF_nl_304 CCTGAGACCCGGGTTTGTT

dre-miR-733 TGCGTTGGTTTAGCTCAGTGGTT

dre-miR-15c AAGCAGCGCGTCATGGTTTTC

ZF_nl_325 TCTACAGTGCATGTGTCTCCAGT

ZF_nl_33 TGAGTCCTGTTTTAGCAGAGTTT

dre-miR-734 GTAAATGCTGCAGAATCGTACCG

ZF_nl_345 AAAATATCCTGAGCTGTTTTCT

ZF_nl_350_5p TATGTGCCCTTGGACTACATTGT

ZF_nl_350_3p ATGCAGTCCATGGGCATATACACT

ZF_nl_372 TCCCTCCGTCATTGAATTCCTG

ZF_nl_376 ACGAGAGCTGGGGTCTTGCTGG

dre-miR-735_5p GGCTGGTCCGAAGGCGGTGGGTTAGTC

dre-miR-735_3p CTCTCCCACCGCTAAACTTGAC

ZF_nl_384 TCCCTGGGAACCTTATTGGG

ZF_nl_39 TTTACAGGCTATGCTAATCTAT

dre-miR-7361 GTAAGACGAACAAAAAGTTTT

dre-miR-737_5p GTTTTTTTAGGTTTTGATTTT

dre-miR-737_3p AATCAAAACCTAAAGAAAATA

dre-miR-738 GCTACGGCCCGCGTCGGGACCTC

dre-miR-739 AGGCCGAAGTGGAGAAGGGTT

ZF_nl_51 ZF_nl_89 ZF_nl_90 ZF_nl_343 CGGCTCCGCTGATTCTGTCAGT

ZF_nl_58 TCAGAAAAGTTACCACAGGGAT

ZF_nl_63 CCGCAACACGAAACTGTCTT

dre-miR-499 ZF_nl_233 TTAAGACTTGCAGTGATGTTTA

ZF_nl_68 AGACACTGAGGGGTGTAG

dre-miR-723_5p GACAGTTTTAAATGATGTTACTTT

dre-miR-723_3p AAGACATCAATTAAATCTGTGCT

ZF_nl_9 TGAACACGGTCTCTTTTT

ZF_nl_91 TGTCTGTCTCCCCGCTTTCT

ZF_nl_94 ZF_nl_363 AGGCGATGGGCATCATGAACTCGTT

miR-4892 TGACATCATATGTACGGCTGCT

1cloned from human and conserved in zebrafish2predicted in zebrafish based on hsa-miR-489

51


Table S5. miRNA and star sequences of known miRNAs

miRNA sequence number of clones

let-7b TGAGGTAGTAGGTTGTGTGGT 109

let-7b* CTATACAACCTACTGCCTTCCC 2

miR-1 TGGAATGTAAAGAAGTATGTATT 39

miR-1* ACATACTTCTTTATGTGCCCAT 1

miR-100* CAAGCTCGTGTCTATAGGTATG 2

miR-100 AACCCGTAGATCCGAACTTGT 337

miR-103 AGCAGCATTGTACAGGGCTATGA 17

miR-103* AGCCTCTTTACGGTGCTGCCTTG 2

miR-107 AGCAGCATTGTACAGGGCTATCA 10

miR-107* AGCTTCTTTACAGTGTTGTCTTG 2

miR-10b TACCCTGTAGAACCGAATTTGTG 42

miR-10b* CAAATACGTCTCTACAGGAAT 1

miR-124 TAAGGCACGCGGTGAATGCC 123

miR-124* TGTGTTCACAGTGGACCTTGATT 9

miR-125a TCCCTGAGACCCTTAACCTGTGAG 848

miR-125a* ACAGGTGAGGTCCTCAGGAAC 1

miR-125b TCCCTGAGACCCTAACTTGTGA 2553

miR-125b* ACGGGTTAGGTTCTTGGGAGCT 5

miR-126* CATTATTACTTTTGGTACGCG 2

miR-126 TCGTACCGTGAGTAATAATGC 42

miR-128 TCACAGTGAACCGGTCTCTTTTT 458

miR-128* CGGGGCCGTGGCACTGTATGAG 1

miR-129* AAGCCCTTACCCCAAAAAGTAT 205

miR-129 CTTTTTGCGGTCTGGGCTTGC 62

miR-130b* ACTCTTTCCCTGTTGCACTACTG 10

miR-130b CAGTGCAATAATGAAAGGGCAT 8

miR-132 TAACAGTCTACAGCCATGGTCG 169

miR-132* ACCGTGGCTTTAGATTGTTACT 2

miR-133a* AGCTGGTAAAATGGAACCAAAT 1

miR-133a TTTGGTCCCCTTCAACCAGCTGT 136

miR-135 TATGGCTTTCTATTCCTATGTG 91

miR-135* ACATAGGGTTCAAAGCCATTGG 1

miR-135b* TATAGGGATGGAAGCCATGCAG 5

miR-135b TATGGCTTTTTATTCCTATCTGA 28

miR-137 TTATTGCTTAAGAATACGCGTAG 74

miR-137* ACGGGTATTCTTGGGTGGATAAT 2

miR-138 AGCTGGTGTTGTGAATCAGGCCG 401

miR-138* GCTATTTCACAACACCAGGGT 5

miR-140 CAGTGGTTTTACCCTATGGT 26

miR-140* TACCACAGGGTAGAACCACGG 7

miR-142a-3p TGTAGTGTTTCCTACTTTATGG 6

miR-142a-5p CATAAAGTAGAAAGCACTACT 2

miR-142b-3p TGTAGTGTTTCCTACTTTATGG 6

miR-142b-5p CATAAAGTAGACAGCACTACTA 1

miRNA sequence number of clones

miR-143 TGAGATGAAGCACTGTAGCTC 12

miR-144* GGATATCATCGTATACTGTAAGTT 1

miR-144 TACAGTATAGATGATGTACT 5

miR-145 GTCCAGTTTTCCCAGGAATCCCTT 24

miR-145* GGATTCCTGGAAATACTGTTCT 4

miR-148 TCAGTGCATTACAGAACTTTGT 18

miR-148* AAGTTCTGTGATACACTCCGAC 1

miR-153b TTGCATAGTCACAAAAATGAGC 18

miR-153b* GTCATTTTTGTGGT TTGCAGCT 2

miR-16c TAGCAGCATGTAAATATTGGAGT 7

miR-16c* GCCTCCAATATTGCTCGTGCTG 3

miR-17a CAAAGTGCTTACAGTGCAGGT 27

miR-17a* ACTGCAGTGGAGGCACTTCAAG 1

miR-181a AACATTCAACGCTGTCGGTGAGTTT 391

miR-181a* ACCATCGACCGTTGACTGTACC 4

miR-181b* ACTCACTGATCAATGAATGC 2

miR-181b AACATTCATTGCTGTCGGTGGGTT 148

miR-181c CACATTCATTGCTGTCGGTGGG 26

miR-181c* CTCGCCGGACAATGAATGAG 1

miR-182 TTTGGCAATGGTAGAACTCAC 30

miR-182* TGGTTCTAGACTTGCCAACTA 3

miR-183 TATGGCACTGGTAGAATTCACT 35

miR-183* TGAATTACCAAAGGGCCAT 1

miR-184 TGGACGGAGAACTGATAAGGG 30

miR-184* TCCTTATCACTTTTCCAGCCCAG 1

miR-187 TCGTGTCTTGTGTTGCAGCCAG 48

miR-187* GGCTGCAACACAGGACATGG 2

miR-18a* ACTGCCCTAAGTGCTCCTTCTG 1

miR-18a TAAGGTGCATCTAGTGCAGATAG 17

miR-18c TAAGGTGCATCTTGTGTAGTTAG 5

miR-18c* TACTGCGCTAGATGTTCCTTTTG 2

miR-192 ATGACCTATGAATTGACAGCCA 49

miR-192* CCTGTCAGTTCTGTAGGCCACTG 4

miR-193b* GACTTTGGGGGCGAGATG 1

miR-193b AACTGGCCCGCAAAGTCCCGCT 6

miR-194b* TGGAGAAGCTGTTACCTG 1

miR-194b TGTAACAGCCGCTCCATGTGGA 1

miR-196a TAGGTAGTTTCATGTTGTTGGG 4

miR-196a* CTGCAACGTGAAACTGTCTTAA 1

miR-199 CCCAGTGTTCAGACTACCTGTTCA 197

miR-199* ACAGTAGTCTGCACATTGGTT 42

miR-19d TGTGCAAACCCATGCAAAACTGA 46

miR-19d* AGCTTTGCGGGGTGGGCAGTCAGC 1

miR-203b TGAAATGTTCAGGACCACTTG 194

52

Chapter 2

miR-203b* AGTGGTTCTCAACAGTTCAACAGT 19

miR-20a TAAAGTGCTTATAGTGCAGGTAG 43

miR-20a* ACTGCAGTGTGAGCACTTGAAG 5

miR-210 CTGTGCGTGTGACAGCGGCT 74

miR-210* AGCCACTGACTAACGCACATTG 6

miR-212* ACCTTGGCTCTAGACTGCTTACT 35

miR-212 TAACAGTCTACAGTCATGGCTAC 8

miR-214 ACAGCAGGCACAGACAGGCAG 3

miR-214* GCCTGTCTACACTTGCTGTGC 3

miR-216a TAATCTCAGCTGGCAACTGTGA 27

miR-216a* CACAGGCGCTGCTGGGGTTCTG 1

miR-218a* ATGGTTCCGTCAAGCACCAGG 1

miR-218a TTGTGCTTGATCTAACCATGTG 67

miR-219 TGATTGTCCAAACGCAATTCTTG 333

miR-219* GGAGTTGTGGATGGACATCATG 2

miR-222 AGCTACATCTGGCTACTGGGTCTC 181

miR-222* TGCTCAGTAGTCAGTGTAGAT 1

miR-22a* AGTTCTTCACTGGCAAGCTTT 9

miR-22a AAAGCTGCCAGCTGAAGAACTGT 151

miR-22b* CGTTCTTCACTGGCTAGCTTT 1

miR-22b AAGCTGCCAGTTGAAGAGCTG 4

miR-24 TGGCTCAGTTCAGCAGGAACAGG 40

miR-24* GTGCCTTCTGAGCTGATATCAG 1

miR-26a* CCTATTCATGATTACTTGCACT 1

miR-26a TTCAAGTAATCCAGGATAGGCT 72

miR-27d TTCACAGTGGCTAAGTTCTTC 3

miR-27d* CAGAGCTTGGCTGATTGGTG 1

miR-29b TAGCACCATTTGAAATCAGTGT 3

miR-29b* GCTGAATTCAGATGGTGCCATAG 1

miR-30c TGTAAACATCCTACACTCTCAGCT 85

miR-30c* CCGGGAGTGGGATGTTTG 1

miR-30e* CTTTCAGTCGGATGTTTGCAGC 16

miR-30e TGTAAACATCCTTGACTGGAAGCT 29

miR-455* ATGCAGTCCATGGGCATATACAC 8

miR-455 TATGTGCCCTTGGACTACATCG 4

miR-460-3p CACAGCGCATACAATGTGGATG 1

miR-460-5p CCTGCATTGTACACACTGTGCG 2

miR-9 TCTTTGGTTATCTAGCTGTATG 1121

miR-9* TAAAGCTAGATAACCGAAAGT 157

miR-92b* AGGTGTGGGATGTTGTGCAGTGTT 1

miR-92b TATTGCACTCGTCCCGGCCTCC 6

Table S6. miRNA and star sequences of new miRNAs

miRNA sequence brain larvae total clones tested detection miRNA

dre-miR-724_3p CAGCCACACCTTCCTTTTAAG 1 0 1 yes no

dre-miR-724_5p TTAAAGGGAATTTGCGACTGTT 20 0 20 yes northern 5p

dre-miR-726_3p TTCACTACTAGCAGAACTCGG 3 0 3 yes northern 3p

dre-miR-726_5p GGAATTCCGCTAGTTCTGAACT 1 1 2 yes no

dre-miR-727_3p GTTGAGGCGAGTTGAAGACTTA 8 4 12 yes northern 3p

dre-miR-727_5p TCAGTCTTCAATTCCTCCCAGC 5 0 5 yes northern

ZF_nl_210_3p TCGTAACAAGGTTTCCGT 1 0 1 no

ZF_nl_210_5p TGAGCGGCTCAGTGAGGTCCTCGGATC 1 0 1 no 5p

dre-miR-34c_3p AATCACTAACCTCACTACCAGG 0 1 1 yes no

dre-miR-34c_5p AGGCAGTGCAGTTAGTTGATTAC 0 1 1 yes northern, in situ 5p

dre-miR-730_3p CACAGCGCCTGCAATGTGGAGG 1 0 1 yes no

dre-miR-730_5p TCCTCATTGTGCATGCTGTGTGT 9 3 12 yes northern 5p

ZF_nl_302_3p CTCGTACCGTGAGTAATAGTGC 8 10 18 no 3p

ZF_nl_302_5p CATTATTACTTTTGGTACGCG 2 0 2 no

ZF_nl_350_3p ATGCAGTCCATGGGCATATACACT 3 6 9 no 3p

ZF_nl_350_5p TATGTGCCCTTGGACTACATTGT 6 1 7 no

dre-miR-735_3p CTCTCCCACCGCTAAACTTGAC 1 0 1 yes northern,in situ 3p

dre-miR-735_5p GGCTGGTCCGAAGGCGGTGGGTTAGTC 18 0 18 no

dre-miR-723_3p AAGACATCAATTAAATCTGTGCT 3 2 5 yes northern 3p

53


Table S7. Overview of miRNA cloing and expression

dre-miR-723_5p GACAGTTTTAAATGATGTTACTTT 0 1 1 yes northern

dre-miR-737_3p AATCAAAACCTAAAGAAAATA 0 1 1 yes norhern 3p

dre-miR-737_5p GTTTTTTTAGGTTTTGATTTT 0 6 6 no

known new

miRNAs in registry 168

miRNA genes in registry 369

miRNAs cloned 139 66

star sequences cloned 65 11

miRNA genes 255 116

analyzed miRNAs 32 35

analyzed star sequences 0 6

miRNAs detected in situ 24 4

star sequences detected in situ 0 0

miRNAs detected on Northern 30 23

star sequences detected on Northern

0 2

brain larvae total

known miRNAs cloned 136 129 139

of which found in both libraries 126

new miRNAs cloned 47 38 66

of which found in both libraries 19

cloning and expression of new micrornas from …...31 new mirnas from zebrafish cloning and...

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