An update on recent methods applied for deciphering the diversity of the noncoding RNA genome structure and function.

The explosion of high throughput sequencing technologies marked a turn in our way of understanding the complexity and diversity of the transcriptome, including noncoding transcription dependent on RNA polymerase II. Many new ncRNA populations were described in recent years, including for example TSS RNAs, lincRNAs, eRNAs, PROMPTS and several others. Besides the advances in the average depth coverage of RNA-seq experiments, various additional protocols are now available that can be used to address qualitative and quantitative aspects of the noncoding transcriptome complexity and function. In this review, we will focus on methods allowing isolation and characterization of complex RNA populations using sequencing based approaches, including conventional strategies already used for coding genome and more specific developments allowing, for example, the study of nascent strand transcription, protein-bound or structured RNAs.

[1]  J. Mattick,et al.  Refining transcriptional programs in kidney development by integration of deep RNA-sequencing and array-based spatial profiling , 2011, BMC Genomics.

[2]  Lior Pachter,et al.  Sequence Analysis , 2020, Definitions.

[3]  M. Gerstein,et al.  RNA-Seq: a revolutionary tool for transcriptomics , 2009, Nature Reviews Genetics.

[4]  V. Pirrotta,et al.  A Polycomb response element in the Ubx gene that determines an epigenetically inherited state of repression. , 1994, The EMBO journal.

[5]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[6]  D. Haussler,et al.  FragSeq: transcriptome-wide RNA structure probing using high-throughput sequencing , 2010, Nature Methods.

[7]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[8]  Manolis Kellis,et al.  PhyloCSF: a comparative genomics method to distinguish protein coding and non-coding regions , 2011, Bioinform..

[9]  Lennart Martens,et al.  LNCipedia: a database for annotated human lncRNA transcript sequences and structures , 2012, Nucleic Acids Res..

[10]  A. Morillon,et al.  Pervasive transcription - Lessons from yeast. , 2011, Biochimie.

[11]  H. Lodish,et al.  Regulation of mammalian cell differentiation by long non‐coding RNAs , 2012, EMBO reports.

[12]  H. Th. Marc Timmers,et al.  Analysis of the open region of RNA polymerase II transcription complexes in the early phase of elongation , 2001, Nucleic Acids Res..

[13]  Renato Paro,et al.  Mixture models and wavelet transforms reveal high confidence RNA-protein interaction sites in MOV10 PAR-CLIP data , 2012, Nucleic acids research.

[14]  S. Spicuglia,et al.  H3K4 tri‐methylation provides an epigenetic signature of active enhancers , 2011, The EMBO journal.

[15]  Michael F. Lin,et al.  Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals , 2009, Nature.

[16]  S. Sunkin,et al.  Specific expression of long noncoding RNAs in the mouse brain , 2008, Proceedings of the National Academy of Sciences.

[17]  Robert Tibshirani,et al.  Genome-wide measurement of RNA folding energies. , 2012, Molecular cell.

[18]  Tyson A. Clark,et al.  HITS-CLIP yields genome-wide insights into brain alternative RNA processing , 2008, Nature.

[19]  B. Williams,et al.  Mapping and quantifying mammalian transcriptomes by RNA-Seq , 2008, Nature Methods.

[20]  N. Friedman,et al.  Metabolic labeling of RNA uncovers principles of RNA production and degradation dynamics in mammalian cells , 2011, Nature Biotechnology.

[21]  L. Steinmetz,et al.  Extensive degradation of RNA precursors by the exosome in wild-type cells. , 2012, Molecular cell.

[22]  Jay Shendure,et al.  Computational discovery of sense-antisense transcription in the human and mouse genomes , 2002, Genome Biology.

[23]  S. Batalov,et al.  Antisense Transcription in the Mammalian Transcriptome , 2005, Science.

[24]  Grace X. Y. Zheng,et al.  Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs , 2010, Nature Structural &Molecular Biology.

[25]  Tim R. Mercer,et al.  Differentiating Protein-Coding and Noncoding RNA: Challenges and Ambiguities , 2008, PLoS Comput. Biol..

[26]  M. Gut,et al.  Supplemental information for : “ CpG islands and GC content dictate nucleosome depletion in a transcription independent manner at mammalian promoters ” , 2012 .

[27]  Leighton J. Core,et al.  Precise Maps of RNA Polymerase Reveal How Promoters Direct Initiation and Pausing , 2013, Science.

[28]  S. Kaufmann,et al.  Ultrashort and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution , 2012, Genome research.

[29]  D. Tollervey,et al.  Transcriptome-wide Analysis of Exosome Targets , 2012, Molecular cell.

[30]  David R. Kelley,et al.  Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks , 2012, Nature Protocols.

[31]  Martin S. Taylor,et al.  Genome-wide analysis of mammalian promoter architecture and evolution , 2006, Nature Genetics.

[32]  Leighton J. Core,et al.  The polycomb group mutant esc leads to augmented levels of paused Pol II in the Drosophila embryo. , 2011, Molecular cell.

[33]  J. Kocher,et al.  CPAT: Coding-Potential Assessment Tool using an alignment-free logistic regression model , 2013, Nucleic acids research.

[34]  David G. Knowles,et al.  The GENCODE v7 catalog of human long noncoding RNAs: Analysis of their gene structure, evolution, and expression , 2012, Genome research.

[35]  M. Tewari,et al.  MicroRNA profiling: approaches and considerations , 2012, Nature Reviews Genetics.

[36]  Nicholas T. Ingolia,et al.  Ribosome Profiling of Mouse Embryonic Stem Cells Reveals the Complexity and Dynamics of Mammalian Proteomes , 2011, Cell.

[37]  William Stafford Noble,et al.  Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project , 2007, Nature.

[38]  Yong Zhang,et al.  CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine , 2007, Nucleic Acids Res..

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

[40]  David R. Kelley,et al.  Transposable elements reveal a stem cell-specific class of long noncoding RNAs , 2012, Genome Biology.

[41]  B. Bernstein,et al.  Charting histone modifications and the functional organization of mammalian genomes , 2011, Nature Reviews Genetics.

[42]  A. Mortazavi,et al.  Computation for ChIP-seq and RNA-seq studies , 2009, Nature Methods.

[43]  J. Rinn,et al.  Ab initio reconstruction of transcriptomes of pluripotent and lineage committed cells reveals gene structures of thousands of lincRNAs , 2010, Nature biotechnology.

[44]  Albin Sandelin,et al.  Biogenic mechanisms and utilization of small RNAs derived from human protein-coding genes , 2011, Nature Structural &Molecular Biology.

[45]  Joseph R. Ecker,et al.  Genome-Wide High-Resolution Mapping of Exosome Substrates Reveals Hidden Features in the Arabidopsis Transcriptome , 2007, Cell.

[46]  A. Sandelin,et al.  PROMoter uPstream Transcripts share characteristics with mRNAs and are produced upstream of all three major types of mammalian promoters , 2011, Nucleic acids research.

[47]  Mikkel H. Schierup,et al.  RNA Exosome Depletion Reveals Transcription Upstream of Active Human Promoters , 2008, Science.

[48]  C. Glass,et al.  Reprogramming Transcription via Distinct Classes of Enhancers Functionally Defined by eRNA , 2011, Nature.

[49]  T. Hughes,et al.  Most “Dark Matter” Transcripts Are Associated With Known Genes , 2010, PLoS biology.

[50]  R. Elkon,et al.  eRNAs are required for p53-dependent enhancer activity and gene transcription. , 2013, Molecular cell.

[51]  Paulo P. Amaral,et al.  The Reality of Pervasive Transcription , 2011, PLoS biology.

[52]  Manel Esteller,et al.  Cis-acting noncoding RNAs: friends and foes , 2012, Nature Structural &Molecular Biology.

[53]  David R. Kelley,et al.  Long noncoding RNAs regulate adipogenesis , 2013, Proceedings of the National Academy of Sciences.

[54]  J. Rinn,et al.  lincRNAs act in the circuitry controlling pluripotency and differentiation , 2011, Nature.

[55]  Takefumi Kondo,et al.  Lilliputians get into the limelight: Novel class of small peptide genes in morphogenesis , 2008, Development, growth & differentiation.

[56]  C. Wahlestedt,et al.  Regulation of chromatin structure by long noncoding RNAs: focus on natural antisense transcripts. , 2012, Trends in genetics : TIG.

[57]  T. Babak,et al.  A quantitative atlas of polyadenylation in five mammals , 2012, Genome research.

[58]  N. Friedman,et al.  Comprehensive comparative analysis of strand-specific RNA sequencing methods , 2010, Nature Methods.

[59]  Yong Zhang,et al.  NATsDB: Natural Antisense Transcripts DataBase , 2006, Nucleic Acids Res..

[60]  M. Landthaler,et al.  Transcriptome-wide analysis of protein-RNA interactions using high-throughput sequencing. , 2012, Seminars in cell & developmental biology.

[61]  Derek Y. Chiang,et al.  MapSplice: Accurate mapping of RNA-seq reads for splice junction discovery , 2010, Nucleic acids research.

[62]  M. Gut,et al.  Transcription initiation platforms and GTF recruitment at tissue-specific enhancers and promoters , 2011, Nature Structural &Molecular Biology.

[63]  Jernej Ule,et al.  CLIP Identifies Nova-Regulated RNA Networks in the Brain , 2003, Science.

[64]  Howard Y. Chang,et al.  Genomic maps of long noncoding RNA occupancy reveal principles of RNA-chromatin interactions. , 2011, Molecular cell.

[65]  Cole Trapnell,et al.  Targeted RNA sequencing reveals the deep complexity of the human transcriptome , 2011, Nature Biotechnology.

[66]  Bronwen L. Aken,et al.  Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome , 2012, Genome research.

[67]  Mahadeb Pal,et al.  The role of the transcription bubble and TFIIB in promoter clearance by RNA polymerase II. , 2005, Molecular cell.

[68]  J. Weissman,et al.  Nascent transcript sequencing visualizes transcription at nucleotide resolution , 2011, Nature.

[69]  Siu-Ming Yiu,et al.  SOAPsplice: Genome-Wide ab initio Detection of Splice Junctions from RNA-Seq Data , 2011, Front. Gene..

[70]  Howard Y. Chang,et al.  Long noncoding RNA HOTAIR reprograms chromatin state to promote cancer metastasis , 2010, Nature.

[71]  J. Mattick,et al.  Genome-wide analysis of long noncoding RNA stability , 2012, Genome research.

[72]  Piero Carninci,et al.  Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat , 2012, Nature.

[73]  Scott B. Dewell,et al.  Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP , 2010, Cell.

[74]  Heng Li,et al.  A survey of sequence alignment algorithms for next-generation sequencing , 2010, Briefings Bioinform..

[75]  Brad A Chapman,et al.  The genomic binding sites of a noncoding RNA , 2011, Proceedings of the National Academy of Sciences.

[76]  Leighton J. Core,et al.  A Rapid, Extensive, and Transient Transcriptional Response to Estrogen Signaling in Breast Cancer Cells , 2011, Cell.

[77]  Annick Harel-Bellan,et al.  Argonaute proteins couple chromatin silencing to alternative splicing , 2012, Nature Structural &Molecular Biology.

[78]  J. Rinn,et al.  Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression , 2009, Proceedings of the National Academy of Sciences.

[79]  N. Akimitsu,et al.  The RNA Degradation Pathway Regulates the Function of GAS5 a Non-Coding RNA in Mammalian Cells , 2013, PloS one.

[80]  C. Ponting,et al.  Evolution and Functions of Long Noncoding RNAs , 2009, Cell.

[81]  Sebastian D. Mackowiak,et al.  Circular RNAs are a large class of animal RNAs with regulatory potency , 2013, Nature.

[82]  D. Bartel,et al.  Long noncoding RNAs in C. elegans , 2012, Genome research.

[83]  Salvatore Spicuglia,et al.  Chromatin signatures of active enhancers , 2012, Nucleus.

[84]  K. Shinozaki,et al.  Genome-wide suppression of aberrant mRNA-like noncoding RNAs by NMD in Arabidopsis , 2009, Proceedings of the National Academy of Sciences.

[85]  L. Maquat,et al.  The Pioneer Round of Translation: Features and Functions , 2010, Cell.

[86]  J. Ragoussis,et al.  A Large Fraction of Extragenic RNA Pol II Transcription Sites Overlap Enhancers , 2010, PLoS biology.

[87]  M. Esteller.  Non-coding RNAs in human disease , 2011, Nature Reviews Genetics.

[88]  D. Bartel,et al.  Formation, Regulation and Evolution of Caenorhabditis elegans 3′UTRs , 2010, Nature.

[89]  Leighton J. Core,et al.  Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters , 2008, Science.

[90]  G. Natoli,et al.  Noncoding transcription at enhancers: general principles and functional models. , 2012, Annual review of genetics.

[91]  D. Black,et al.  Transcript Dynamics of Proinflammatory Genes Revealed by Sequence Analysis of Subcellular RNA Fractions , 2012, Cell.

[92]  J. Rinn,et al.  A Large Intergenic Noncoding RNA Induced by p53 Mediates Global Gene Repression in the p53 Response , 2010, Cell.

[93]  Piero Carninci,et al.  CAGE (cap analysis of gene expression): a protocol for the detection of promoter and transcriptional networks. , 2012, Methods in molecular biology.

[94]  G. Kreiman,et al.  Widespread transcription at neuronal activity-regulated enhancers , 2010, Nature.

[95]  Nadav S. Bar,et al.  Landscape of transcription in human cells , 2012, Nature.

[96]  Leighton J. Core,et al.  Divergent transcription: A new feature of active promoters , 2009, Cell cycle.

[97]  Howard Y. Chang,et al.  Control of somatic tissue differentiation by the long non-coding RNA TINCR , 2012, Nature.

[98]  Kentaro Yoshida,et al.  SuperSAGE: powerful serial analysis of gene expression. , 2012, Methods in molecular biology.

[99]  John S. Mattick,et al.  lncRNAdb: a reference database for long noncoding RNAs , 2010, Nucleic Acids Res..

[100]  Brian P. Brunk,et al.  Comparative analysis of RNA-Seq alignment algorithms and the RNA-Seq unified mapper (RUM) , 2011, Bioinform..

[101]  E. Liu,et al.  Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses. , 2009, Genome research.

[102]  Olivier Elemento,et al.  Faster sequencers, larger datasets, new challenges , 2012, Genome Biology.

[103]  T. Hughes,et al.  Establishing legitimacy and function in the new transcriptome. , 2009, Briefings in functional genomics & proteomics.

[104]  D. Bartel,et al.  Extensive alternative polyadenylation during zebrafish development , 2012, Genome research.

[105]  Hui Xiao,et al.  NONCODE v3.0: integrative annotation of long noncoding RNAs , 2011, Nucleic Acids Res..

[106]  Toshiro K. Ohsumi,et al.  Genome-wide identification of polycomb-associated RNAs by RIP-seq. , 2010, Molecular cell.

[107]  J. Andrau,et al.  Genome-wide RNA polymerase II: not genes only! , 2008, Trends in biochemical sciences.

[108]  Gene W. Yeo,et al.  Divergent Transcription from Active Promoters , 2008, Science.

[109]  David G. Knowles,et al.  Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs , 2012, Genome research.

[110]  Doron Betel,et al.  Genome-wide identification of miRNA targets by PAR-CLIP. , 2012, Methods.

[111]  Cole Trapnell,et al.  Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. , 2011, Genes & development.

[112]  K. Hansen,et al.  Biases in Illumina transcriptome sequencing caused by random hexamer priming , 2010, Nucleic acids research.

[113]  Serban Nacu,et al.  Fast and SNP-tolerant detection of complex variants and splicing in short reads , 2010, Bioinform..

[114]  C. Kai,et al.  CAGE: cap analysis of gene expression , 2006, Nature Methods.

[115]  Olivier Voinnet,et al.  The long and the short of noncoding RNAs. , 2009, Current opinion in cell biology.

[116]  Y. Xing,et al.  Detection of splice junctions from paired-end RNA-seq data by SpliceMap , 2010, Nucleic acids research.

[117]  W. Wong,et al.  Improving PacBio Long Read Accuracy by Short Read Alignment , 2012, PloS one.

[118]  J. Rinn,et al.  Modular regulatory principles of large non-coding RNAs , 2012, Nature.

[119]  William Ritchie,et al.  Nuclear-localized tiny RNAs are associated with transcription initiation and splice sites in metazoans , 2010, Nature Structural &Molecular Biology.

[120]  Bronwen L. Aken,et al.  GENCODE: The reference human genome annotation for The ENCODE Project , 2012, Genome research.

[121]  J. Kjems,et al.  Natural RNA circles function as efficient microRNA sponges , 2013, Nature.

[122]  M. Esteller,et al.  Intronic RNAs mediate EZH2 regulation of epigenetic targets , 2012, Nature Structural &Molecular Biology.

[123]  Y. Ruan,et al.  Genome wide full-length transcript analysis using 5' and 3' paired-end-tag next generation sequencing (RNA-PET). , 2012, Methods in molecular biology.

[124]  L. Steinmetz,et al.  Functional consequences of bidirectional promoters. , 2011, Trends in genetics : TIG.

[125]  Aya Kojima,et al.  fRNAdb: a platform for mining/annotating functional RNA candidates from non-coding RNA sequences , 2006, Nucleic Acids Res..

[126]  M. Selbach,et al.  Global quantification of mammalian gene expression control , 2011, Nature.

[127]  S. Marquardt,et al.  Distinct RNA degradation pathways and 3' extensions of yeast non-coding RNA species , 2011, Transcription.

[128]  Howard Y. Chang,et al.  Extensive and coordinated transcription of noncoding RNAs within cell cycle promoters , 2011, Nature Genetics.

[129]  Leighton J. Core,et al.  Defining the status of RNA polymerase at promoters. , 2012, Cell reports.

[130]  Charles Gawad,et al.  Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types , 2012, PloS one.

[131]  Howard Y. Chang,et al.  Genome-wide measurement of RNA secondary structure in yeast , 2010, Nature.

[132]  J. Rinn,et al.  Ab initio reconstruction of transcriptomes of pluripotent and lineage committed cells reveals gene structures of thousands of lincRNAs , 2010, Nature Biotechnology.

[133]  B. Séraphin,et al.  Cryptic Pol II Transcripts Are Degraded by a Nuclear Quality Control Pathway Involving a New Poly(A) Polymerase , 2005, Cell.

[134]  James B. Brown,et al.  Long noncoding RNAs are rarely translated in two human cell lines , 2012, Genome research.