Total RNA sequencing reveals nascent transcription and widespread co-transcriptional splicing in the human brain

Ameur, Adam; Zaghlool, Ammar; Halvardson, Jonatan; Wetterbom, Anna; Gyllensten, Ulf; Cavelier, Lucia; Feuk, Lars

doi:10.1038/nsmb.2143

Cited by 294 publications

(325 citation statements)

References 49 publications

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“…Three recent studies based on RNA-seq provided evidence that intronic reads might correlate with transcriptional activity. In two of these, the read coverage along introns was related to nascent transcription in combination with co-transcriptional splicing events 24 and later was used to fit a detailed transcriptional model within a single sample 25 . More recently, levels of exonic reads were found to lag 15 min behind levels of intronic reads for a set of oscillating transcripts during Caenorhabditis elegans development, suggesting that intronic levels are a proxy for nascent transcription 26 .…”

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confidence: 99%

Analysis of intronic and exonic reads in RNA-seq data characterizes transcriptional and post-transcriptional regulation

et al. 2015

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A n A ly s i s RNA-seq experiments generate reads derived not only from mature RNA transcripts but also from pre-mRNA. Here we present a computational approach called exon-intron split analysis (EISA) that measures changes in mature RNA and pre-mRNA reads across different experimental conditions to quantify transcriptional and post-transcriptional regulation of gene expression. We apply EISA to 17 diverse data sets to show that most intronic reads arise from nuclear RNA and changes in intronic read counts accurately predict changes in transcriptional activity. Furthermore, changes in posttranscriptional regulation can be predicted from differences between exonic and intronic changes. EISA reveals both transcriptional and post-transcriptional contributions to expression changes, increasing the amount of information that can be gained from RNA-seq data sets.Cellular RNAs are regulated at multiple stages, including transcription, RNA maturation and degradation. Several analytic methods have been developed to measure these processes on a transcriptomewide scale. For example, global run-on sequencing (GRO-seq) 1 uses incorporation of a nucleotide analog to enrich for nascent RNA. In Nascent-seq 2,3 , newly transcribed RNAs are isolated by purification of their complex with proteins and the DNA template. Cellular fractionation techniques 4 have also been adapted to measure nascent transcripts, which are enriched in the nucleus. mRNA half-lives have been determined, for example, by blockage of transcription followed by transcriptional profiling 5 . RNA sequencing, the most widely used method for transcriptome analysis, has been applied in numerous studies to determine steady-state mRNA levels 6,7 and alternative splicing events 8 and to identify previously unknown transcripts and noncoding RNAs 9-11 . In general, these protocols aim to enrich for mature mRNA by selection of polyadenylated RNA or by depletion of ribosomal RNA.Many computational methods (reviewed in refs. 12,13) have been developed for the analysis of RNA-seq data, to enable spliced alignment 14,15 , transcript assembly 16,17 , transcript quantification 14,18 and differential expression analysis [19][20][21] . Although RNA-seq mostly generates reads that map to exons, it also captures less abundant intronic sequences 6 . However, their interpretation has remained controversial. Some have suggested that they originate from DNA contamination and can thus be used as a quality metric for RNA-seq data 22 (see also RNA-seq guidelines of the Roadmap Epigenomics Consortium, http://www.roadmapepigenomics.org/), whereas others have hypothesized that they stem from unknown exons or intronic enhancers 6,7 . In a study based on exon arrays, probes mapping to introns were used to investigate pre-mRNA dynamics 23 . Three recent studies based on RNA-seq provided evidence that intronic reads might correlate with transcriptional activity. In two of these, the read coverage along introns was related to nascent transcription in combination with co-transcriptional splicing e...

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confidence: 99%

Analysis of intronic and exonic reads in RNA-seq data characterizes transcriptional and post-transcriptional regulation

et al. 2015

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“…First we examined the raw RNA read data for sequences that could be assembled to the intergenic spacer sequences, reasoning that polycistronic transcripts should produce intergenic regions and coding sequences in initially equal amounts. We anticipated these noncoding sequences, like introns, might be easily detectable, as intron sequences accumulate to roughly 1% of sequence reads in fission yeast (28) and are even more abundant in mammals (29). Because currently there are no known introns in Lingulodinium, we instead used read counts corresponding to the ITS, a spacer region excised during formation of mature rRNA, and found that the read ratio of ITS to mature rRNA is much higher than that of TAG spacer to mature ORF (Fig.…”

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confidence: 99%

Dinoflagellate tandem array gene transcripts are highly conserved and not polycistronic

Beauchemin

Roy

Daoust

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Dinoflagellates are an important component of the marine biota, but a large genome with high–copy number (up to 5,000) tandem gene arrays has made genomic sequencing problematic. More importantly, little is known about the expression and conservation of these unusual gene arrays. We assembled de novo a gene catalog of 74,655 contigs for the dinoflagellate Lingulodinium polyedrum from RNA-Seq (Illumina) reads. The catalog contains 93% of a Lingulodinium EST dataset deposited in GenBank and 94% of the enzymes in 16 primary metabolic KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, indicating it is a good representation of the transcriptome. Analysis of the catalog shows a marked underrepresentation of DNA-binding proteins and DNA-binding domains compared with other algae. Despite this, we found no evidence to support the proposal of polycistronic transcription, including a marked underrepresentation of sequences corresponding to the intergenic spacers of two tandem array genes. We also have used RNA-Seq to assess the degree of sequence conservation in tandem array genes and found their transcripts to be highly conserved. Interestingly, some of the sequences in the catalog have only bacterial homologs and are potential candidates for horizontal gene transfer. These presumably were transferred as single-copy genes, and because they are now all GC-rich, any derived from AT-rich contexts must have experienced extensive mutation. Our study not only has provided the most complete dinoflagellate gene catalog known to date, it has also exploited RNA-Seq to address fundamental issues in basic transcription mechanisms and sequence conservation in these algae.

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“…Another parameter for detecting de novo synthesized transcripts is the presence of intronic sequences due to incomplete cotranscriptional splicing (11). As shown in Fig.…”

Section: Gene Activation As Detected By the Appearance Of Incompletelymentioning

confidence: 99%

Fine mapping of genome activation in bovine embryos by RNA sequencing

Graf

Krebs

Zakhartchenko³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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During maternal-to-embryonic transition control of embryonic development gradually switches from maternal RNAs and proteins stored in the oocyte to gene products generated after embryonic genome activation (EGA). Detailed insight into the onset of embryonic transcription is obscured by the presence of maternal transcripts. Using the bovine model system, we established by RNA sequencing a comprehensive catalogue of transcripts in germinal vesicle and metaphase II oocytes, and in embryos at the four-cell, eight-cell, 16-cell, and blastocyst stages. These were produced by in vitro fertilization of Bos taurus taurus oocytes with sperm from a Bos taurus indicus bull to facilitate parent-specific transcriptome analysis. Transcripts from 12.4 to 13.7 × 10 3 different genes were detected in the various developmental stages. EGA was analyzed by (i) detection of embryonic transcripts, which are not present in oocytes; (ii) detection of transcripts from the paternal allele; and (iii) detection of primary transcripts with intronic sequences. These strategies revealed (i) 220, (ii) 937, and (iii) 6,848 genes to be activated from the four-cell to the blastocyst stage. The largest proportion of gene activation [i.e., (i) 59%, (ii) 42%, and (iii) 58%] was found in eight-cell embryos, indicating major EGA at this stage. Gene ontology analysis of genes activated at the four-cell stage identified categories related to RNA processing, translation, and transport, consistent with preparation for major EGA. Our study provides the largest transcriptome data set of bovine oocyte maturation and early embryonic development and detailed insight into the timing of embryonic activation of specific genes.

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Total RNA sequencing reveals nascent transcription and widespread co-transcriptional splicing in the human brain

Cited by 294 publications

References 49 publications

Analysis of intronic and exonic reads in RNA-seq data characterizes transcriptional and post-transcriptional regulation

Analysis of intronic and exonic reads in RNA-seq data characterizes transcriptional and post-transcriptional regulation

Dinoflagellate tandem array gene transcripts are highly conserved and not polycistronic

Fine mapping of genome activation in bovine embryos by RNA sequencing

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