Abstract
BackgroundThe majority of published gene-expression studies have used RNA isolated from whole cells, overlooking the potential impact of including nuclear transcriptome in the analyses. In this study, mRNA fractions from the cytoplasm and from whole cells (total RNA) were prepared from three human cell lines and sequenced using massive parallel sequencing.ResultsFor all three cell lines, of about 15000 detected genes approximately 400 to 1400 genes were detected in different amounts in the cytoplasmic and total RNA fractions. Transcripts detected at higher levels in the total RNA fraction had longer coding sequences and higher number of miRNA target sites. Transcripts detected at higher levels in the cytoplasmic fraction were shorter or contained shorter untranslated regions. Nuclear retention of transcripts and mRNA degradation via miRNA pathway might contribute to this differential detection of genes. The consequence of the differential detection was further investigated by comparison to proteomics data. Interestingly, the expression profiles of cytoplasmic and total RNA correlated equally well with protein abundance levels indicating regulation at a higher level.ConclusionsWe conclude that expression levels derived from the total RNA fraction be regarded as an appropriate estimate of the amount of mRNAs present in a given cell population, independent of the coding sequence length or UTRs.
Highlights
The majority of published gene-expression studies have used RNA isolated from whole cells, overlooking the potential impact of including nuclear transcriptome in the analyses
In addition to nuclear retention, the gene level is regulated by other mechanisms and one of them is the degradation of messenger RNA (mRNA) by the exosome complex [8,9]
We investigated the effect of the length and structure of untranslated regions and the length of the coding sequences on the transcript levels in total and cytoplasmic RNA. miRNA-mediated degradation of transcripts and its role in transcript regulation was investigated, as well as the effect on the correlation with protein levels
Summary
The majority of published gene-expression studies have used RNA isolated from whole cells, overlooking the potential impact of including nuclear transcriptome in the analyses. The majority of published transcriptome data have used RNA extracted from the whole cell (total RNA), assuming a negligible contribution of nuclear RNA to the total RNA population. Messenger ribonucleoproteins are co-transcriptionally recruited to mRNA, and direct the export of mRNAs via their interaction with mRNA export factors and nuclear pore complexes [4] This process is regulated at many levels, yielding a dynamic steady-state mRNA population that is maintained by synthesis and turnover, at varying rates for each individual transcript [5]. In addition to nuclear retention, the gene level is regulated by other mechanisms and one of them is the degradation of mRNA by the exosome complex [8,9]
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