Abstract
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.
Highlights
MicroRNAs are a new class of small RNAs of approximately 22 nucleotides in length that control eukaryotic gene expression by fine tuning mRNA translation
Small RNAs ranging from 15 to 30 nt in size were isolated from gels and subjected to two successive ligations, i.e., a first ligation with a 3' adapter was followed by a second ligation with a 5' adapter
The search for novel and known miRNAs in our ZF samples was complemented by a megaBlast alignment between our dataset and mature sequences deposited in miRBase 12.0 and the novel miRNA transcripts predicted by Ensembl and by Thatcher et al [44]
Summary
MicroRNAs (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. MicroRNAs (miRNAs) are small RNAs that regulate eukaryotic gene expression at the post-transcriptional level [1] They are transcribed as long precursor RNA molecules (pri-miRNAs) and are successively processed by two key RNAses, namely Drosha and Dicer, into their mature forms of ~22 nucleotides [2]. These small RNAs regulate gene expression by binding to target sites in the 3' untranslated region of mRNAs (3'UTR) [3,4]. MiRNAs direct rapid deadenylation of target mRNAs, leading to decapping and rapid mRNA decay by the combined activities of the exosome (3' to 5' degradation) and the exoribonuclease Xrn (5' to 3'degradation) [7,8]
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