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Cloning and expression of new microRNAs from zebrafish.

ABSTRACT: MicroRNAs (miRNAs) play an important role in development and regulate the expression of many animal genes by post-transcriptional gene silencing. Here we describe the cloning and expression of new miRNAs from zebrafish. By high-throughput sequencing of small-RNA cDNA libraries from 5-day-old zebrafish larvae and adult zebrafish brain we found 139 known miRNAs and 66 new miRNAs. For 65 known miRNAs and for 11 new miRNAs we also cloned the miRNA star sequence. We analyzed the temporal and spatial expression patterns for 35 new miRNAs and for 32 known miRNAs in the zebrafish by whole mount in situ hybridization and northern blotting. Overall, 23 of the 35 new miRNAs and 30 of the 32 known miRNAs could be detected. We found that most miRNAs were expressed during later stages of development. Some were expressed ubiquitously, but many of the miRNAs were expressed in a tissue-specific manner. Most newly discovered miRNAs have low expression levels and are less conserved in other vertebrate species. Our cloning and expression analysis indicates that most abundant and conserved miRNAs in zebrafish are now known.

SUBMITTER: Kloosterman WP

PROVIDER: S-EPMC3303176 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Cloning and expression of new microRNAs from zebrafish.

Kloosterman Wigard P WP Steiner Florian A FA Berezikov Eugene E de Bruijn Ewart E van de Belt Jose J Verheul Mark M Cuppen Edwin E Plasterk Ronald H A RH

Nucleic acids research 20060512 9

MicroRNAs (miRNAs) play an important role in development and regulate the expression of many animal genes by post-transcriptional gene silencing. Here we describe the cloning and expression of new miRNAs from zebrafish. By high-throughput sequencing of small-RNA cDNA libraries from 5-day-old zebrafish larvae and adult zebrafish brain we found 139 known miRNAs and 66 new miRNAs. For 65 known miRNAs and for 11 new miRNAs we also cloned the miRNA star sequence. We analyzed the temporal and spatial ...[more]

PMID: 16698962

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Project description:BackgroundMicroRNAs (miRNAs) are a new class of small RNAs of approximately 22 nucleotides in length that control eukaryotic gene expression by fine tuning mRNA translation. They regulate a wide variety of biological processes, namely developmental timing, cell differentiation, cell proliferation, immune response and infection. For this reason, their identification is essential to understand eukaryotic biology. Their small size, low abundance and high instability complicated early identification, however cloning/Sanger sequencing and new generation genome sequencing approaches overcame most technical hurdles and are being used for rapid miRNA identification in many eukaryotes.ResultsWe have applied 454 DNA pyrosequencing technology to miRNA discovery in zebrafish (Danio rerio). For this, a series of cDNA libraries were prepared from miRNAs isolated at different embryonic time points and from fully developed organs. Each cDNA library was tagged with specific sequences and was sequenced using the Roche FLX genome sequencer. This approach retrieved 90% of the 192 miRNAs previously identified by cloning/Sanger sequencing and bioinformatics. Twenty five novel miRNAs were predicted, 107 miRNA star sequences and also 41 candidate miRNA targets were identified. A miRNA expression profile built on the basis of pyrosequencing read numbers showed high expression of most miRNAs throughout zebrafish development and identified tissue specific miRNAs.ConclusionThis study increases the number of zebrafish miRNAs from 192 to 217 and demonstrates that a single DNA mini-chip pyrosequencing run is effective in miRNA identification in zebrafish. This methodology also produced sufficient information to elucidate miRNA expression patterns during development and in differentiated organs. Moreover, some zebrafish miRNA star sequences were more abundant than their corresponding miRNAs, suggesting a functional role for the former in gene expression control in this vertebrate model organism.

Project description:BackgroundmicroRNAs (miRNAs) are non-coding RNAs that are now recognized as a major class of gene-regulating molecules widely distributed in metozoans and plants. miRNAs have been found to play important roles in apoptosis, cancer, development, differentiation, inflammation, longevity, and viral infection. There are a few reports describing miRNAs in the African malaria mosquito, Anopheles gambiae, on the basis of similarity to known miRNAs from other species. An. stephensi is the most important malaria vector in Asia and it is becoming a model Anopheline species for physiological and genetics studies.ResultsWe report the cloning and characterization of 27 distinct miRNAs from 17-day old An. stephensi female mosquitoes. Seventeen of the 27 miRNAs matched previously predicted An. gambiae miRNAs, offering the first experimental verification of miRNAs from mosquito species. Ten of the 27 are miRNAs previously unknown to mosquitoes, four of which did not match any known miRNAs in any organism. Twenty-five of the 27 Anopheles miRNAs had conserved sequences in the genome of a divergent relative, the yellow fever mosquito Aedes aegypti. Two clusters of miRNAs were found within introns of orthologous genes in An. gambiae, Ae. aegypti, and Drosophila melanogaster. Mature miRNAs were detected in An. stephensi for all of the nine selected miRNAs, including the four novel miRNAs (miR-x1- miR-x4), either by northern blot or by Ribonuclease Protection Assay. Expression profile analysis of eight of these miRNAs revealed distinct expression patterns from early embryo to adult stages in An. stephensi. In both An. stephensi and Ae. aegypti, the expression of miR-x2 was restricted to adult females and predominantly in the ovaries. A significant reduction of miR-x2 level was observed 72 hrs after a blood meal. Thus miR-x2 is likely involved in female reproduction and its function may be conserved among divergent mosquitoes. A mosquito homolog of miR-14, a regulator of longevity and apoptosis in D. melanogaster, represented 25% of all sequenced miRNA clones from 17-day old An. stephensi female mosquitoes. An. stephensi miR-14 displayed a relatively strong signal from late embryonic to adult stages. miR-14 expression is consistent during the adult lifespan regardless of age, sex, and blood feeding status. Thus miR-14 is likely important across all mosquito life stages.ConclusionThis study provides experimental evidence for 23 conserved and four new microRNAs in An. stephensi mosquitoes. Comparisons between miRNA gene clusters in Anopheles and Aedes mosquitoes, and in D. melanogaster suggest the loss or significant change of two miRNA genes in Ae. aegypti. Expression profile analysis of eight miRNAs, including the four new miRNAs, revealed distinct patterns from early embryo to adult stages in An. stephensi. Further analysis showed that miR-x2 is likely involved in female reproduction and its function may be conserved among divergent mosquitoes. Consistent expression of miR-14 suggests that it is likely important across all mosquito life stages from embryos to aged adults. Understanding the functions of mosquito miRNAs will undoubtedly contribute to a better understanding of mosquito biology including longevity, reproduction, and mosquito-pathogen interactions, which are important to disease transmission.

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Cloning and expression of new microRNAs from zebrafish.

Publications

Cloning and expression of new microRNAs from zebrafish.

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