Efficient targeted transcript discovery via array-based normalization of RACE libraries

Djebali, Sarah; Kapranov, Philipp; Foissac, Sylvain; Lagarde, Julien; Reymond, Alexandre; Ucla, Catherine; Wyss, Carine; Drenkow, Jörg; Dumais, Erica; Murray, Ryan R.; Lin, Chenwei; Szeto, David; Denœud, France; Calvo, Miquel; Frankish, Adam; Harrow, Jennifer; Makrythanasis, Periklis; Vidal, Marc; Salehi‐Ashtiani, Kourosh; Antonarakis, Stylianos E.; Gingeras, T; Guigó, Roderic

doi:10.1038/nmeth.1216

Cited by 41 publications

(51 citation statements)

References 24 publications

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“…We validated 72% of the assessed model confirming that they are bona fide transcribed units missing from the current annotations. Together these observations support the notion that the human genome is pervasively transcribed as suggested by multiple authors (Kapranov et al 2002;Denoeud et al 2007; The ENCODE Project Consortium 2007; Djebali et al 2008Djebali et al , 2012bClark et al 2011). Further studies are warranted to identify the molecular roles of the new genes identified by RNA-seq (and validated by RT-PCR-seq) and the novel exons of known genes pinpointed by RT-PCR-seq.…”

Section: Wwwgenomeorgsupporting

confidence: 73%

Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome

Howald

Tanzer²,

Chrast

et al. 2012

Genome Res.

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Within the ENCODE Consortium, GENCODE aimed to accurately annotate all protein-coding genes, pseudogenes, and noncoding transcribed loci in the human genome through manual curation and computational methods. Annotated transcript structures were assessed, and less well-supported loci were systematically, experimentally validated. Predicted exon-exon junctions were evaluated by RT-PCR amplification followed by highly multiplexed sequencing readout, a method we called RT-PCR-seq. Seventy-nine percent of all assessed junctions are confirmed by this evaluation procedure, demonstrating the high quality of the GENCODE gene set. RT-PCR-seq was also efficient to screen gene models predicted using the Human Body Map (HBM) RNA-seq data. We validated 73% of these predictions, thus confirming 1168 novel genes, mostly noncoding, which will further complement the GENCODE annotation. Our novel experimental validation pipeline is extremely sensitive, far more than unbiased transcriptome profiling through RNA sequencing, which is becoming the norm. For example, exon-exon junctions unique to GENCODE annotated transcripts are five times more likely to be corroborated with our targeted approach than with extensive large human transcriptome profiling. Data sets such as the HBM and ENCODE RNA-seq data fail sampling of low-expressed transcripts. Our RT-PCR-seq targeted approach also has the advantage of identifying novel exons of known genes, as we discovered unannotated exons in~11% of assessed introns. We thus estimate that at least 18% of known loci have yet-unannotated exons. Our work demonstrates that the cataloging of all of the genic elements encoded in the human genome will necessitate a coordinated effort between unbiased and targeted approaches, like RNA-seq and RT-PCR-seq.

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Section: Wwwgenomeorgsupporting

confidence: 73%

Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome

Howald

Tanzer²,

Chrast

et al. 2012

Genome Res.

Self Cite

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“…A systematic analysis of the location of the 59 termini of coding genes expressed in various cell lines was initiated as part of the ENCODE pilot project ( Denoeud et al 2007; The ENCODE Project Consortium 2007; Tress et al 2007;Djebali et al 2008). This project discovered that gene boundaries extend well beyond the annotated termini in 65% of cases, often extending into neighboring genes, leading to the production of chimeric RNAs (Gingeras 2009).…”

Section: [Supplemental Materials Is Available For This Article]mentioning

confidence: 99%

“…This project discovered that gene boundaries extend well beyond the annotated termini in 65% of cases, often extending into neighboring genes, leading to the production of chimeric RNAs (Gingeras 2009). A more recent revision of this analysis focusing on chromosomes 21 and 22 revealed additional cases of chimeric transcripts not only connecting neighboring genes but, rather, encompassing distal genes (Djebali et al 2008(Djebali et al , 2012. Characterization of these chimeric transcripts has highlighted that the information stored in the genome and expressed in the transcriptome is not as linear as previously believed Gingeras 2009).…”

Section: [Supplemental Materials Is Available For This Article]mentioning

confidence: 99%

Chimeras taking shape: Potential functions of proteins encoded by chimeric RNA transcripts

Frenkel‐Morgenstern¹,

Lacroix²,

Ezkurdia³

et al. 2012

Genome Res.

124

154

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Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans.

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“…This approach revealed that most genes do not possess a single TSS, but instead exhibit sets of closely spaced TSSs that will be referred to as Transcription start regions (TSRs) in the following. While the largest number of TSS profiling studies have been performed in mammalian (i.e., human and mouse) systems [Djebali et al 2008; FANTOM Consortium and the RIKEN PMI and CLST (DGT) 2014], CAGE has also been performed in nonmammalian metazoans, including fruit fly (Hoskins et al 2011), nematode (Nepal et al 2013), and zebrafish (Haberle et al 2014). Overall, these studies indicate that the majority of core promoters in metazoan genomes lack TATA elements (Lenhard et al 2012), an unanticipated finding given previously established models of transcription initiation.…”

mentioning

confidence: 99%

Promoter Architecture and Sex-Specific Gene Expression in Daphnia pulex

et al. 2016

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Large-scale transcription start site (TSS) profiling produces a high-resolution, quantitative picture of transcription initiation and core promoter locations within a genome. However, application of TSS profiling to date has largely been restricted to a small set of prominent model systems. We sought to characterize the cis-regulatory landscape of the water flea Daphnia pulex, an emerging model arthropod that reproduces both asexually (via parthenogenesis) and sexually (via meiosis). We performed Cap Analysis of Gene Expression (CAGE) with RNA isolated from D. pulex within three developmental states: sexual females, asexual females, and males. Identified TSSs were utilized to generate a "Daphnia Promoter Atlas," i.e., a catalog of active promoters across the surveyed states. Analysis of the distribution of promoters revealed evidence for widespread alternative promoter usage in D. pulex, in addition to a prominent fraction of compactly-arranged promoters in divergent orientations. We carried out de novo motif discovery using CAGEdefined TSSs and identified eight candidate core promoter motifs; this collection includes canonical promoter elements (e.g., TATA and Initiator) in addition to others lacking obvious orthologs. A comparison of promoter activities found evidence for considerable statespecific differential gene expression between states. Our work represents the first global definition of transcription initiation and promoter architecture in crustaceans. The Daphnia Promoter Atlas presented here provides a valuable resource for comparative study of cis-regulatory regions in metazoans, as well as for investigations into the circuitries that underpin meiosis and parthenogenesis.

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Efficient targeted transcript discovery via array-based normalization of RACE libraries

Cited by 41 publications

References 24 publications

Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome

Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome

Chimeras taking shape: Potential functions of proteins encoded by chimeric RNA transcripts

Promoter Architecture and Sex-Specific Gene Expression in Daphnia pulex

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