Computational identification of Drosophila microRNA genes

BackgroundMicroRNAs (miRNAs) are a large family of 21-22 nucleotide non-coding RNAs with presumed post-transcriptional regulatory activity. Most miRNAs were identified by direct cloning of small RNAs, an approach that favors detection of abundant miRNAs. Three observations suggested that miRNA genes might be identified using a computational approach. First, miRNAs generally derive from precursor transcripts of 70-100 nucleotides with extended stem-loop structure. Second, miRNAs are usually highly conserved between the genomes of related species. Third, miRNAs display a characteristic pattern of evolutionary divergence.ResultsWe developed an informatic procedure called 'miRseeker', which analyzed the completed euchromatic sequences of Drosophila melanogaster and D. pseudoobscura for conserved sequences that adopt an extended stem-loop structure and display a pattern of nucleotide divergence characteristic of known miRNAs. The sensitivity of this computational procedure was demonstrated by the presence of 75% (18/24) of previously identified Drosophila miRNAs within the top 124 candidates. In total, we identified 48 novel miRNA candidates that were strongly conserved in more distant insect, nematode, or vertebrate genomes. We verified expression for a total of 24 novel miRNA genes, including 20 of 27 candidates conserved in a third species and 4 of 11 high-scoring, Drosophila-specific candidates. Our analyses lead us to estimate that drosophilid genomes contain around 110 miRNA genes.ConclusionsOur computational strategy succeeded in identifying bona fide miRNA genes and suggests that miRNAs constitute nearly 1% of predicted protein-coding genes in Drosophila, a percentage similar to the percentage of miRNAs recently attributed to other metazoan genomes.

[1]  V. Ambros,et al.  The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 , 1993, Cell.

[2]  G. Ruvkun,et al.  Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans , 1993, Cell.

[3]  G. Ruvkun,et al.  A bulged lin-4/lin-14 RNA duplex is sufficient for Caenorhabditis elegans lin-14 temporal gradient formation. , 1996, Genes & development.

[4]  E. Lai,et al.  The Bearded box, a novel 3' UTR sequence motif, mediates negative post-transcriptional regulation of Bearded and Enhancer of split Complex gene expression. , 1997, Development.

[5]  V. Ambros,et al.  The Cold Shock Domain Protein LIN-28 Controls Developmental Timing in C. elegans and Is Regulated by the lin-4 RNA , 1997, Cell.

[6]  E. Lai,et al.  Regulation of Drosophila Neurogenesis byRNA:RNA Duplexes? , 1998, Cell.

[7]  C Burks,et al.  The K box, a conserved 3' UTR sequence motif, negatively regulates accumulation of enhancer of split complex transcripts. , 1998, Development.

[8]  Michael Zuker,et al.  Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide , 1999 .

[9]  V. Ambros,et al.  The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation. , 1999, Developmental biology.

[10]  S. Hammond,et al.  An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells , 2000, Nature.

[11]  Phillip D. Zamore,et al.  RNA Interference , 2000, Science.

[12]  B. Reinhart,et al.  The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans , 2000, Nature.

[13]  B. Reinhart,et al.  Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA , 2000, Nature.

[14]  V. Ambros,et al.  An Extensive Class of Small RNAs in Caenorhabditis elegans , 2001, Science.

[15]  A. Pasquinelli,et al.  A Cellular Function for the RNA-Interference Enzyme Dicer in the Maturation of the let-7 Small Temporal RNA , 2001, Science.

[16]  B. Bass,et al.  A Role for the RNase III Enzyme DCR-1 in RNA Interference and Germ Line Development in Caenorhabditis elegans , 2001, Science.

[17]  G. Hannon,et al.  C . elegans involved in developmental timing in Dicer functions in RNA interference and in synthesis of small RNA , 2001 .

[18]  A. Caudy,et al.  Argonaute2, a Link Between Genetic and Biochemical Analyses of RNAi , 2001, Science.

[19]  L. Lim,et al.  An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans , 2001, Science.

[20]  A. Caudy,et al.  Role for a bidentate ribonuclease in the initiation step of RNA interference , 2001 .

[21]  T. Tuschl,et al.  Identification of Novel Genes Coding for Small Expressed RNAs , 2001, Science.

[22]  V. Ambros microRNAs Tiny Regulators with Great Potential , 2001, Cell.

[23]  E. Lai Micro RNAs are complementary to 3′ UTR sequence motifs that mediate negative post-transcriptional regulation , 2002, Nature Genetics.

[24]  T. Tuschl,et al.  Identification of Tissue-Specific MicroRNAs from Mouse , 2002, Current Biology.

[25]  V. Ambros,et al.  The expression of the let-7 small regulatory RNA is controlled by ecdysone during metamorphosis in Drosophila melanogaster. , 2002, Developmental biology.

[26]  S. Lewis,et al.  An integrated computational pipeline and database to support whole-genome sequence annotation , 2002, Genome Biology.

[27]  Jian Wang,et al.  The Genome Sequence of the Malaria Mosquito Anopheles gambiae , 2002, Science.

[28]  G. Hutvagner,et al.  A microRNA in a Multiple-Turnover RNAi Enzyme Complex , 2002, Science.

[29]  M. Mann,et al.  miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs. , 2002, Genes & development.

[30]  G. Hannon RNA interference : RNA , 2002 .

[31]  J. Messing,et al.  CARPEL FACTORY, a Dicer Homolog, and HEN1, a Novel Protein, Act in microRNA Metabolism in Arabidopsis thaliana , 2002, Current Biology.

[32]  B. Reinhart,et al.  Prediction of Plant MicroRNA Targets , 2002, Cell.

[33]  E. Myers,et al.  Finishing a whole-genome shotgun: Release 3 of the Drosophila melanogaster euchromatic genome sequence , 2002, Genome Biology.

[34]  A. Hüttenhofer,et al.  RNomics: identification and function of small, non-messenger RNAs. , 2002, Current opinion in chemical biology.

[35]  B. Reinhart,et al.  MicroRNAs in plants. , 2002, Genes & development.

[36]  M. A. Rector,et al.  Endogenous and Silencing-Associated Small RNAs in Plants Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.003210. , 2002, The Plant Cell Online.

[37]  Peer Bork,et al.  Comparative Genome and Proteome Analysis of Anopheles gambiae and Drosophila melanogaster , 2002, Science.

[38]  C. Llave,et al.  Cleavage of Scarecrow-like mRNA Targets Directed by a Class of Arabidopsis miRNA , 2002, Science.

[39]  V. Kim,et al.  MicroRNA maturation: stepwise processing and subcellular localization , 2002, The EMBO journal.

[40]  Michael Ashburner,et al.  Annotation of the Drosophila melanogaster euchromatic genome: a systematic review , 2002, Genome Biology.

[41]  B. Reinhart,et al.  A biochemical framework for RNA silencing in plants. , 2003, Genes & development.

[42]  L. Pachter,et al.  Strategies and tools for whole-genome alignments. , 2002, Genome research.

[43]  Nicholas L. Bray,et al.  AVID: A global alignment program. , 2003, Genome research.

[44]  T. Tuschl,et al.  New microRNAs from mouse and human. , 2003, RNA.

[45]  C. Burge,et al.  The microRNAs of Caenorhabditis elegans. , 2003, Genes & development.

[46]  C. Burge,et al.  Vertebrate MicroRNA Genes , 2003, Science.

[47]  R. Russell,et al.  bantam Encodes a Developmentally Regulated microRNA that Controls Cell Proliferation and Regulates the Proapoptotic Gene hid in Drosophila , 2003, Cell.

[48]  N. Perrimon,et al.  Coordinate regulation of small temporal RNAs at the onset of Drosophila metamorphosis. , 2003, Developmental biology.

[49]  G. Church,et al.  Computational and experimental identification of C. elegans microRNAs. , 2003, Molecular cell.

[50]  Phillip A Sharp,et al.  siRNAs can function as miRNAs , 2003 .

[51]  G. Ruvkun,et al.  A uniform system for microRNA annotation. , 2003, RNA.

[52]  V. Ambros,et al.  MicroRNAs and Other Tiny Endogenous RNAs in C. elegans , 2003, Current Biology.

[53]  V. Ambros,et al.  Temporal regulation of microRNA expression in Drosophila melanogaster mediated by hormonal signals and broad-Complex gene activity. , 2003, Developmental biology.