title: Nuclear RNA sequencing of the mouse erythroid cell transcriptome.
abstract: In addition to protein coding genes a substantial proportion of mammalian genomes are transcribed. However, most transcriptome studies investigate steady-state mRNA levels, ignoring a considerable fraction of the transcribed genome. In addition, steady-state mRNA levels are influenced by both transcriptional and posttranscriptional mechanisms, and thus do not provide a clear picture of transcriptional output. Here, using deep sequencing of nuclear RNAs (nucRNA-Seq) in parallel with chromatin immunoprecipitation sequencing (ChIP-Seq) of active RNA polymerase II, we compared the nuclear transcriptome of mouse anemic spleen erythroid cells with polymerase occupancy on a genome-wide scale. We demonstrate that unspliced transcripts quantified by nucRNA-seq correlate with primary transcript frequencies measured by RNA FISH, but differ from steady-state mRNA levels measured by poly(A)-enriched RNA-seq. Highly expressed protein coding genes showed good correlation between RNAPII occupancy and transcriptional output; however, genome-wide we observed a poor correlation between transcriptional output and RNAPII association. This poor correlation is due to intergenic regions associated with RNAPII which correspond with transcription factor bound regulatory regions and a group of stable, nuclear-retained long non-coding transcripts. In conclusion, sequencing the nuclear transcriptome provides an opportunity to investigate the transcriptional landscape in a given cell type through quantification of unspliced primary transcripts and the identification of nuclear-retained long non-coding RNAs.
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada. ja.mitchell@utoronto.ca
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Mitchell
authors.forename: Ieuan
authors.initials: I
authors.lastname: Clay
authors.forename: David
authors.initials: D
authors.lastname: Umlauf
authors.forename: Chih-Yu
authors.initials: CY
authors.lastname: Chen
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Moir
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Eskiw
authors.forename: Stefan
authors.initials: S
authors.lastname: Schoenfelder
authors.forename: Lyubomira
authors.initials: L
authors.lastname: Chakalova
authors.forename: Takashi
authors.initials: T
authors.lastname: Nagano
authors.forename: Peter
authors.initials: P
authors.lastname: Fraser
mesh_terms: D000818:Animals; D047369:Chromatin Immunoprecipitation; D041905:Erythroid Cells / Q000378:metabolism*; D005786:Gene Expression Regulation; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D051379:Mice; D011485:Protein Binding; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D062085:RNA, Long Noncoding / Q000235:genetics / Q000378:metabolism; D015138:RNA, Nuclear / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D015203:Reproducibility of Results; D017423:Sequence Analysis, RNA; D014158:Transcription, Genetic; D059467:Transcriptome
chemical_list: D062085:RNA, Long Noncoding; D015138:RNA, Nuclear; D012319:RNA Polymerase II