Abstract
BackgroundThe genetic program, as manifested as the cellular phenotype, is in large part dictated by the cell’s protein composition. Since characterisation of the proteome remains technically laborious it is attractive to define the genetic expression profile using the transcriptome. However, the transcriptional landscape is complex and it is unclear as to what extent it reflects the ribosome associated mRNA population (the translatome). This is particularly pertinent for genes using multiple transcriptional start sites (TSS) generating mRNAs with heterogeneous 5′ transcript leaders (5′TL). Furthermore, the relative abundance of the TSS gene variants is frequently cell-type specific. Indeed, promoter switches have been reported in pathologies such as cancer. The consequences of this 5′TL heterogeneity within the transcriptome for the translatome remain unresolved. This is not a moot point because the 5′TL plays a key role in regulating mRNA recruitment onto polysomes.ResultsIn this article, we have characterised both the transcriptome and translatome of the MCF7 (tumoural) and MCF10A (non-tumoural) cell lines. We identified ~550 genes exhibiting differential translation efficiency (TE). In itself, this is maybe not surprising. However, by focusing on genes exhibiting TSS heterogeneity we observed distinct differential promoter usage patterns in both the transcriptome and translatome. Only a minor fraction of these genes belonged to those exhibiting differential TE. Nonetheless, reporter assays demonstrated that the TSS variants impacted on the translational readout both quantitatively (the overall amount of protein expressed) and qualitatively (the nature of the proteins expressed).ConclusionsThe results point to considerable and distinct cell-specific 5′TL heterogeneity within both the transcriptome and translatome of the two cell lines analysed. This observation is in-line with the ribosome filter hypothesis which posits that the ribosomal machine can selectively filter information from within the transcriptome. As such it cautions against the simple extrapolation transcriptome → proteome. Furthermore, polysomal occupancy of specific gene 5′TL variants may also serve as novel disease biomarkers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2179-8) contains supplementary material, which is available to authorized users.
Highlights
The genetic program, as manifested as the cellular phenotype, is in large part dictated by the cell’s protein composition
Comparison of total and polysomal mRNA populations in MCF7 and MCF10A cells reveals signs of translational regulation Biological triplicates of the polysomal and total RNAs from each cell line were processed for RNA sequencing (RNAseq)
The cell lines displayed a significantly altered transcriptome with over 8000 genes being differentially expressed in both the total and polysomal RNA. This difference was striking for some genes, for example TGFB1 and MMP14, were found to be over one thousand fold lower expressed in MCF7 compared to MCF10A (Additional file 2A/B)
Summary
The genetic program, as manifested as the cellular phenotype, is in large part dictated by the cell’s protein composition. If one accepts that the end readout of the genetic program, as manifested as the cellular phenotype, is dictated in large part by the proteome, it would appear more judicious to directly characterise the cells protein composition, this remains technically difficult Such switches in the cellular phenotype are at the core of the non-tumoural to tumoural changes observed in cancer. The TL contains multiple elements that can modulate the translational readout both quantitatively (overall amount of protein expressed) and qualitatively (the sequence of the proteins expressed), and all are hard-wired into cellular signalling pathways regulating proliferation, survival and development [3] These include RNA structure, internal ribosome entry sites (IRESes), protein binding sites, upstream AUG codons (uAUG) which may be responsible for the translation of upstream open reading frames (uORFs) (Fig. 1a) [9, 10]. The impact of global changes on TSS selection (the cells TSS fingerprint) on the mammalian proteome remains unclear despite evidence from yeast that such changes correlate with a major shift in translational activity [17]
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