Computational and experimental identification of mirtrons in Drosophila melanogaster and Caenorhabditis elegans.

Mirtrons are intronic hairpin substrates of the dicing machinery that generate functional microRNAs. In this study, we describe experimental assays that defined the essential requirements for entry of introns into the mirtron pathway. These data informed a bioinformatic screen that effectively identified functional mirtrons from the Drosophila melanogaster transcriptome. These included 17 known and six confident novel mirtrons among the top 51 candidates, and additional candidates had limited read evidence in available small RNA data. Our computational model also proved effective on Caenorhabditis elegans, for which the identification of 14 cloned mirtrons among the top 22 candidates more than tripled the number of validated mirtrons in this species. A few low-scoring introns generated mirtron-like read patterns from atypical RNA structures, but their paucity suggests that relatively few such loci were not captured by our model. Unexpectedly, we uncovered examples of clustered mirtrons in both fly and worm genomes, including a <8-kb region in C. elegans harboring eight distinct mirtrons. Altogether, we demonstrate that discovery of functional mirtrons, unlike canonical miRNAs, is amenable to computational methods independent of evolutionary constraint.

[1]  Eric C Lai,et al.  microRNAs: Runts of the Genome Assert Themselves , 2003, Current Biology.

[2]  William Ritchie,et al.  Mireval: a web tool for simple microRNA prediction in genome sequences , 2008, Bioinform..

[3]  Satoshi Shibata,et al.  A High-Resolution Structure of the Pre-microRNA Nuclear Export Machinery , 2009, Science.

[4]  Eugene Berezikov,et al.  Repertoire and evolution of miRNA genes in four divergent nematode species. , 2009, Genome research.

[5]  V. Kim,et al.  Biogenesis of small RNAs in animals , 2009, Nature Reviews Molecular Cell Biology.

[6]  Ryan D. Morin,et al.  Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. , 2008, Genome research.

[7]  Colin N. Dewey,et al.  Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures , 2007, Nature.

[8]  B. Cullen,et al.  Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. , 2003, Genes & development.

[9]  Robert Blelloch,et al.  Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs. , 2008, Genes & development.

[10]  L. Goff,et al.  Ago2 Immunoprecipitation Identifies Predicted MicroRNAs in Human Embryonic Stem Cells and Neural Precursors , 2009, PloS one.

[11]  Pedro J. Batista,et al.  Argonautes ALG-3 and ALG-4 are required for spermatogenesis-specific 26G-RNAs and thermotolerant sperm in Caenorhabditis elegans , 2010, Proceedings of the National Academy of Sciences.

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

[13]  S. Armstrong,et al.  A novel mutation in the miR-128b gene reduces miRNA processing and leads to glucocorticoid resistance of MLL-AF4 Acute Lymphocytic Leukemia cells , 2010, Cell cycle.

[14]  Byoung-Tak Zhang,et al.  Human microRNA prediction through a probabilistic co-learning model of sequence and structure , 2005, Nucleic acids research.

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

[16]  Z. Weng,et al.  Endogenous siRNAs Derived from Transposons and mRNAs in Drosophila Somatic Cells , 2008, Science.

[17]  Panayiotis V. Benos,et al.  HHMMiR: efficient de novo prediction of microRNAs using hierarchical hidden Markov models , 2009, BMC Bioinformatics.

[18]  E. Lai,et al.  Endogenous RNA Interference Provides a Somatic Defense against Drosophila Transposons , 2008, Current Biology.

[19]  Rafael A. Irizarry,et al.  Evolutionary flux of canonical micrornAs and mirtrons in Drosophila , 2010 .

[20]  M. Zuker,et al.  Comparing RNA secondary structures using a relaxed base-pair score. , 2010, RNA.

[21]  Stijn van Dongen,et al.  miRBase: tools for microRNA genomics , 2007, Nucleic Acids Res..

[22]  Ayelet T. Lamm,et al.  Distinct phases of siRNA synthesis in an endogenous RNAi pathway in C. elegans soma. , 2010, Molecular cell.

[23]  Anastasia Khvorova,et al.  Functional siRNAs and miRNAs Exhibit Strand Bias , 2003, Cell.

[24]  B. Lenhard,et al.  Mammalian MicroRNA Prediction through a Support Vector Machine Model of Sequence and Structure , 2007, PloS one.

[25]  N. Perrimon,et al.  Hierarchical rules for Argonaute loading in Drosophila. , 2009, Molecular cell.

[26]  Peter F. Stadler,et al.  Hairpins in a Haystack: recognizing microRNA precursors in comparative genomics data , 2006, ISMB.

[27]  Fei Li,et al.  Classification of real and pseudo microRNA precursors using local structure-sequence features and support vector machine , 2005, BMC Bioinformatics.

[28]  William Ritchie,et al.  RNA stem-loops: to be or not to be cleaved by RNAse III. , 2007, RNA.

[29]  A. Villeneuve,et al.  A Caenorhabditis elegans RNA-Directed RNA Polymerase in Sperm Development and Endogenous RNA Interference , 2009, Genetics.

[30]  G. Rubin,et al.  Computational identification of Drosophila microRNA genes , 2003, Genome Biology.

[31]  Alexander J. Smola,et al.  Learning with kernels , 1998 .

[32]  U. Kutay,et al.  Nuclear Export of MicroRNA Precursors , 2004, Science.

[33]  William Ritchie,et al.  miREval 2.0: a web tool for simple microRNA prediction in genome sequences , 2008, Bioinform..

[34]  Michael Zuker,et al.  UNAFold: software for nucleic acid folding and hybridization. , 2008, Methods in molecular biology.

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

[36]  Pedro J. Batista,et al.  PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C. elegans. , 2008, Molecular cell.

[37]  Taishin Kin,et al.  Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells , 2008, Nature.

[38]  Hervé Seitz,et al.  Argonaute Loading Improves the 5′ Precision of Both MicroRNAs and Their miRNA∗ Strands in Flies , 2008, Current Biology.

[39]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[40]  Manolis Kellis,et al.  Systematic discovery and characterization of fly microRNAs using 12 Drosophila genomes. , 2007, Genome research.

[41]  Taishin Kin,et al.  miRRim: a novel system to find conserved miRNAs with high sensitivity and specificity. , 2007, RNA.

[42]  Liwei Liu,et al.  Comparing RNA Secondary Structures Based on LZ Complexity , 2012 .

[43]  N. Perrimon,et al.  An endogenous small interfering RNA pathway in Drosophila , 2008, Nature.

[44]  Carsten Wiuf,et al.  Ab Initio Identification of Human Micrornas Based on Structure Motifs Ab Initio Identification of Human Micrornas Based on Struc- Ture Motifs , 2007 .

[45]  Robert J. Moore,et al.  A microRNA catalog of the developing chicken embryo identified by a deep sequencing approach. , 2008, Genome research.

[46]  N. Perrimon,et al.  Processing of Drosophila endo-siRNAs depends on a specific Loquacious isoform. , 2009, RNA.

[47]  Jan-Peter Nap,et al.  In silico miRNA prediction in metazoan genomes: balancing between sensitivity and specificity , 2009, BMC Genomics.

[48]  Yvonne Tay,et al.  A Pattern-Based Method for the Identification of MicroRNA Binding Sites and Their Corresponding Heteroduplexes , 2006, Cell.

[49]  Zachary Pincus,et al.  Dynamic expression of small non-coding RNAs, including novel microRNAs and piRNAs/21U-RNAs, during Caenorhabditis elegans development , 2009, Genome Biology.

[50]  Pedro J. Batista,et al.  CDE-1 Affects Chromosome Segregation through Uridylation of CSR-1-Bound siRNAs , 2009, Cell.

[51]  Kuniaki Saito,et al.  Endo‐siRNAs depend on a new isoform of loquacious and target artificially introduced, high‐copy sequences , 2009, The EMBO journal.

[52]  F. Piano,et al.  Large scale sorting of C. elegans embryos reveals the dynamics of small RNA expression , 2009, Nature Methods.

[53]  D. Bartel,et al.  Intronic microRNA precursors that bypass Drosha processing , 2007, Nature.

[54]  Mihaela Zavolan,et al.  Identification of Clustered Micrornas Using an Ab Initio Prediction Method , 2022 .

[55]  E. Lai,et al.  MicroRNA biogenesis via splicing and exosome-mediated trimming in Drosophila. , 2010, Molecular cell.

[56]  Manolis Kellis,et al.  Evolution, biogenesis, expression, and target predictions of a substantially expanded set of Drosophila microRNAs. , 2007, Genome research.

[57]  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.

[58]  Pedro J. Batista,et al.  The Argonaute CSR-1 and Its 22G-RNA Cofactors Are Required for Holocentric Chromosome Segregation , 2009, Cell.

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

[60]  Eugene Berezikov,et al.  Mammalian mirtron genes. , 2007, Molecular cell.

[61]  Ola R. Snøve,et al.  Reliable prediction of Drosha processing sites improves microRNA gene prediction. , 2007, Bioinformatics.

[62]  Louise C. Showe,et al.  Bioinformatics Original Paper Combining Multi-species Genomic Data for Microrna Identification Using a Naı¨ve Bayes Classifier , 2022 .

[63]  T. Du,et al.  Asymmetry in the Assembly of the RNAi Enzyme Complex , 2003, Cell.

[64]  M. Gerstein,et al.  Unlocking the secrets of the genome , 2009, Nature.

[65]  Byoung-Tak Zhang,et al.  Molecular Basis for the Recognition of Primary microRNAs by the Drosha-DGCR8 Complex , 2006, Cell.

[66]  Peng Jin,et al.  Single nucleotide polymorphism associated with mature miR-125a alters the processing of pri-miRNA. , 2007, Human molecular genetics.

[67]  Bin Fan,et al.  MiRFinder: an improved approach and software implementation for genome-wide fast microRNA precursor scans , 2007, BMC Bioinformatics.

[68]  E. Lai,et al.  Distinct mechanisms for microRNA strand selection by Drosophila Argonautes. , 2009, Molecular cell.

[69]  Santosh K. Mishra,et al.  De novo SVM classification of precursor microRNAs from genomic pseudo hairpins using global and intrinsic folding measures , 2007, Bioinform..

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

[71]  N. Rajewsky,et al.  Discovering microRNAs from deep sequencing data using miRDeep , 2008, Nature Biotechnology.

[72]  David Haussler,et al.  The UCSC Genome Browser database: update 2010 , 2009, Nucleic Acids Res..

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

[74]  R. Sachidanandam,et al.  An Epigenetic Role for Maternally Inherited piRNAs in Transposon Silencing , 2008, Science.

[75]  R. Aharonov,et al.  Identification of hundreds of conserved and nonconserved human microRNAs , 2005, Nature Genetics.

[76]  C. Nusbaum,et al.  Mammalian microRNAs: experimental evaluation of novel and previously annotated genes. , 2010, Genes & development.

[77]  Julius Brennecke,et al.  Specialized piRNA Pathways Act in Germline and Somatic Tissues of the Drosophila Ovary , 2009, Cell.

[78]  Mary Goldman,et al.  The UCSC Genome Browser database: update 2011 , 2010, Nucleic Acids Res..

[79]  E. Lai,et al.  The Mirtron Pathway Generates microRNA-Class Regulatory RNAs in Drosophila , 2007, Cell.

[80]  Thomas Sandmann,et al.  Identification of Novel Drosophila melanogaster MicroRNAs , 2007, PloS one.

[81]  Peng Jiang,et al.  MiPred: classification of real and pseudo microRNA precursors using random forest prediction model with combined features , 2007, Nucleic Acids Res..

[82]  Yukio Kawahara,et al.  RNA editing of the microRNA‐151 precursor blocks cleavage by the Dicer–TRBP complex , 2007, EMBO reports.

[83]  Christopher M. Player,et al.  Large-Scale Sequencing Reveals 21U-RNAs and Additional MicroRNAs and Endogenous siRNAs in C. elegans , 2006, Cell.

[84]  Vasile Palade,et al.  microPred: effective classification of pre-miRNAs for human miRNA gene prediction , 2009, Bioinform..

[85]  Z. Weng,et al.  Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway. , 2010, RNA.

[86]  Eugene Berezikov,et al.  Approaches to microRNA discovery , 2006, Nature Genetics.

[87]  G. Hannon,et al.  Evolutionary flux of canonical microRNAs and mirtrons in Drosophila , 2010, Nature Genetics.

[88]  Bernhard Schölkopf,et al.  Learning with kernels , 2001 .

[89]  Katsutomo Okamura,et al.  The evolution and functional diversification of animal microRNA genes , 2008, Cell Research.