Resource AMammalianmicroRNAExpressionAtlas Based on Small RNA Library Sequencing Pablo Landgraf,1 Mirabela Rusu,2 Robert Sheridan,3 Alain Sewer,2,19,29 Nicola Iovino,1,27 Alexei Aravin,1,26 Sebastien �� Pfeffer,1,25 Amanda Rice,1 Alice O. Kamphorst,1 Markus Landthaler,1 Carolina Lin,1 Nicholas D. Socci,3 Leandro Hermida,2 Valerio Fulci,4 Sabina Chiaretti,4 Robin Foa,4 ` Julia Schliwka,5 Uta Fuchs,6 Astrid Novosel,6,28 Roman-Ulrich Muller,1,7 �� Bernhard Schermer,7 Ute Bissels,8 Jason Inman,9 Quang Phan,10 Minchen Chien,11 David B. Weir,11 Ruchi Choksi,11 Gabriella De Vita,12 Daniela Frezzetti,12 Hans-Ingo Trompeter,13 Veit Hornung,23,24 Grace Teng,14 Gunther Hartmann,18 Miklos Palkovits,15 Roberto Di Lauro,12,20 Peter Wernet,13 Giuseppe Macino,4 Charles E. Rogler,16 James W. Nagle,22 Jingyue Ju,11,21 F. Nina Papavasiliou,14 Thomas Benzing,7 Peter Lichter,5 Wayne Tam,17 Michael J. Brownstein,10 Andreas Bosio,8 Arndt Borkhardt,6,28 James J. Russo,11 Chris Sander,3 Mihaela Zavolan,2,19,* and Thomas Tuschl1,* 1 Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, Box 186, The Rockefeller University, New York, NY 10021, USA 2 Biozentrum, University of Basel, CH-4056 Basel, Switzerland 3 Computational Biology Center, Memorial Sloan Kettering Cancer Center, New York, NY 10021, USA 4 Dipartimento di Biotecnologie Cellulari ed Ematologia, Universita ` di Roma ������La Sapienza,������ 00185 Roma, Italy 5 Division of Molecular Genetics B060, Deutsches Krebsforschungszentrum (DKFZ), 69120 Heidelberg, Germany 6 Oncology and Hematology Department, Dr. v. Hauner Children���s Hospital, University of Munich, 80337 Munich, Germany 7 Renal Division, University Hospital Freiburg, 79106 Freiburg, Germany 8 Miltenyi Biotec GmbH, 50829 Cologne, Germany 9 TIGR (The Institute for Genomic Research), Rockville, MD 20850, USA 10 J. Craig Venter Institute, Functional Genomics, Rockville, MD 20850, USA 11 Columbia Genome Center, Russ Berrie Pavilion, New York, NY 10032, USA 12 Dipartimento di Biologia e Patologia Cellulare e Molecolare, Universita��� di Napoli Federico II, 80131 Napoli, Italy 13 Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University Medical Center, 40225 Dusseldorf,�� Germany 14 Laboratory of Lymphocyte Biology, The Rockefeller University, New York, NY 10021, USA 15 Laboratory of Neuromorphology, Hungarian Academy of Sciences-Semmelweis University, Budapest, Hungary 16 Ullman Bldg Room 509, Albert Einstein College of Medicine, Bronx, NY 10461, USA 17 Department of Pathology and Laboratory Medicine, the Joan and Sanford I. Weill Medical College of Cornell University, New York, NY 10021, USA 18 Division of Clinical Pharmacology, University Hospital, University of Bonn, 53105 Bonn, Germany 19 Swiss Institute of Bioinformatics, 1015 Lausanne, Switzerland 20 IRGS, Biogem s.c.ar.l., 83031, Ariano Irpino (AV), Italy 21 Department of Chemical Engineering, Columbia University, New York, NY 10027, USA 22 DNA Sequencing Facility, NINDS, NIH, Bethesda, MD 20892, USA 23 Division of Clinical Pharmacology, Department of Internal Medicine, University of Munich, 80366 Munich, Germany 24 Present address: Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA 01605, USA. 25 Present address: IBM-CNRS, 12, rue du General �� �� Zimmer, 678084 Strasbourg Cedex, France. 26 Present address: Cold Spring Harbor Laboratory, Watson School of Biological Sciences and Howard Hughes Medical Institute, Cold Spring Harbor, NY 11724, USA. 27 Present address: Laboratory of Developmental Neurogenetics, Rockefeller University, New York, NY 10021, USA. 28 Present address: Department of Pediatric Oncology, Hematology and Immunology, Heinrich Heine University Medical Center, 40225 Dusseldorf, �� Germany. 29 Present address: Computational Biology Group, Department of Medical Genetics, University of Lausanne, 1005 Lausanne, Switzerland. *Correspondence: mihaela.zavolan@unibas.ch (M.Z.), ttuschl@rockefeller.edu (T.T.) DOI 10.1016/j.cell.2007.04.040 SUMMARY MicroRNAs (miRNAs) are small noncoding regulatory RNAs that reduce stability and/or translation of fully or partially sequence- complementary target mRNAs. In order to iden- tify miRNAs and to assess their expression patterns, we sequenced over 250 small RNA libraries from 26 different organ systems and cell types of human and rodents that were en- riched in neuronal as well as normal and malig- nant hematopoietic cells and tissues. We pres- ent expression profiles derived from clone count data and provide computational tools Cell 129, 1401���1414, June 29, 2007 ��2007 Elsevier Inc. 1401

for their analysis. Unexpectedly, a relatively small set of miRNAs, many of which are ubi- quitously expressed, account for most of the differences in miRNA profiles between cell lineages and tissues. This broad survey also provides detailed and accurate information about mature sequences, precursors, genome locations, maturation processes, inferred tran- scriptional units, and conservation patterns. We also propose a subclassification scheme for miRNAs for assisting future experimental and computational functional analyses. INTRODUCTION MicroRNAs (miRNAs) are small ( 22 nucleotides) noncod- ing regulatory RNA molecules encoded by plants, animals, and some viruses (reviewed in Bartel, 2004 Berezikov and Plasterk, 2005 Cullen, 2006 Mallory and Vaucheret, 2006). They were first discovered in Caeno- rhabditis elegans and were shown to regulate expression of partially complementary mRNAs (Lee et al., 1993 Wightman et al., 1993 Moss et al., 1997). Most miRNAs are evolutionarily conserved in related species and some even show conservation between invertebrates and verte- brates (Pasquinelli et al., 2000 Lagos-Quintana et al., 2001 Lau et al., 2001 Lee and Ambros, 2001). Many miRNAs have well-defined developmental and cell-type- specific expression patterns (reviewed in Wienholds and Plasterk, 2005). However, for most mammalian miRNAs the relative abundance and specificity of expression remain to be investigated. miRNAs regulate a variety of developmental and physi- ological processes (reviewed in Cao et al., 2006 Plasterk, 2006 Shivdasani, 2006). The analysis of miRNA function in animals is either performed genetically or by delivery of synthetic miRNA precursors or antisense oligonucleo- tides (antagomirs reviewed recently in Krutzfeldt �� et al., 2006). Such analysis revealed that 100 to 200 target mRNAs are repressed and destabilized by a single miRNA (Krutzfeldt �� et al., 2005 Lim et al., 2005 Linsley et al., 2007). Other mRNAs appear to be under selective pres- sure to avoid complementarity to coexpressed highly abundant miRNAs (Farh et al., 2005 Stark et al., 2005 Sood et al., 2006). Many computational studies have been conducted to define miRNA regulatory networks (reviewed in Rajewsky, 2006), yet most molecular targets of miRNAs remain experimentally undefined. Posttranscriptional editing of some double-stranded precursor miRNAs by adenosine deamination (Luciano et al., 2004 Pfeffer et al., 2005 Blow et al., 2006 Kawa- hara et al., 2007) can further control targeting specificity as well as modulate the stability and processing of miRNA precursor transcripts (Gottwein et al., 2006 Yang et al., 2006). Polymorphic sequence variation identified in some other pre-miRNA sequences, in contrast, had no effect on miRNA processing (Iwai and Naraba, 2005 Diederichs and Haber, 2006). Regulated processing of miRNA precursor transcripts has also been reported in the context of cell-type and stage-specific expression (Obernosterer et al., 2006 Thomson et al., 2006). The increasing number of studies addressing the role of miRNAs in development and in various diseases including cancer emphasizes the need for a comprehensive catalog of accurate sequence, expression, and conservation information for the large number of recently proposed miRNAs. Here we present a database and analysis of over 250 small RNA cDNA libraries obtained by cloning and sequencing. We have developed interactive analysis tools and illustrate their utility in discovering miRNA ex- pression changes associated with hematopoietic and ner- vous system differentiation and malignant transformation. RESULTS miRNA Profiling by Sequence Analysis of Small RNA Clone Libraries We cloned and sequenced more than 330,000 indepen- dent small RNA sequences from 256 small RNA libraries prepared from 26 distinct organ systems and cell types of human and/or rodents (Table S1) and also reanalyzed some previously described small RNA libraries (Lagos- Quintana et al., 2002, 2003 Poy et al., 2004). Each library was covered by about 1,300 clones and contained on average 65% miRNA sequences representing 70 to 75 distinct mature miRNAs (Tables S2���S4). Our small RNA annotation procedure and miRNA profile analysis (Figure S1) kept track of small RNA clones that mapped equally well to more than one miRNA precursor (Tables S5���S8). About one-third of all miRNA clones mapped to multicopy miRNA genes with indistinguishable mature sequences, while another 1% mapped to two or more paralogs with related, but not identical, mature miRNA sequences. Clone counts of miRNAs were collected in two distinct table formats either distributing the clones between miRNA genes or precursors (Tables S9���S11) or collecting them as unique mature sequences (Tables S12���S14). In the annotation process, we identified 33 new miRNAs (Table 1), and we confirmed the expression of many evolutionarily conserved miRNAs previously only cloned in other species. We provide evidence forexpressionof 340,303, and205 distinct mature miRNAs from human, mouse, and rat these are encoded by 395, 363, and 231 different miRNA genes, respectively. When including orthology rela- tionships for cloned miRNAs (Table S15), we obtained a final list of 416, 386, and 325 miRNA genes present in human, mouse, and rat contained in 214, 190, and 168 transcription units, respectively. The 8.2 release of miRBase includes 84, 7, and 13 additional miRNA gene candidates for human, mouse, and rat, respectively, for which we havenot found supporting evidence inthis study. About 80% of these miRNA candidates were initially iden- tified at very low or single clone counts in single libraries or 1402 Cell 129, 1401���1414, June 29, 2007 ��2007 Elsevier Inc.