Nuclear RNA sequencing of the mouse erythroid cell transcriptome

PLoS One. 2012;7(11):e49274. doi: 10.1371/journal.pone.0049274. Epub 2012 Nov 29.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatin Immunoprecipitation
  • Erythroid Cells / metabolism*
  • Gene Expression Regulation
  • Gene Library
  • High-Throughput Nucleotide Sequencing
  • Mice
  • Protein Binding
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism
  • RNA, Long Noncoding / genetics
  • RNA, Long Noncoding / metabolism
  • RNA, Nuclear / genetics*
  • Regulatory Sequences, Nucleic Acid
  • Reproducibility of Results
  • Sequence Analysis, RNA
  • Transcription, Genetic
  • Transcriptome*

Substances

  • RNA, Long Noncoding
  • RNA, Nuclear
  • RNA Polymerase II