The effect of heterogeneous Transcription Start Sites (TSS) on the translatome: implications for the mammalian cellular phenotype

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.

Transcriptome‐Wide Analysis Reveals the Landscape of Aberrant Alternative Splicing Events in Liver Cancer

“…SE events are supposed to be the most common splicing patterns in the mammalian transcriptome, in part because of the investigated areas that are restricted within intragenic regions and the limits of sequencing technology . With the improvement of high‐throughput RNA‐seq technology and abundant transcript annotation, an increasing number of transcript variants generated from the alternative use of transcription start sites are found to play roles in tumors . DNA repeats are reported to be among the most variable loci in the human genome, whose instability is associated with gene expression changes and involved in human carcinomas .…”

“…(11,36) With the improvement of high-throughput RNA-seq technology and abundant transcript annotation, an increasing number of transcript variants generated from the alternative use of transcription start sites are found to play roles in tumors. (37)(38)(39) DNA repeats are reported to be among the most variable loci in the human genome, whose instability is associated with gene expression changes and involved in human carcinomas. (40,41) We searched the upstream regions of differential AF events for DNA repeat sequences.…”

Bcr‐Abl regulation of sphingomyelin synthase 1 reveals a novel oncogenic‐driven mechanism of protein up‐regulation

“…A limited number of studies have described the alteration in levels of cancer-associated full-length proteins as a consequence of a promoter shift. Similar to SGMS1, increased expression of Axin2 and mdm2 has been associated with a shift in promoter use, which resulted in increased translational efficiency (84)(85)(86)(87)(88)(89), whereas, in contrast to SMS1, lower BRCA1 (breast cancer gene 1) levels in a subset of breast cancers may be a consequence of inefficient translation as a result of a shift in promoter use toward a longer 59 UTR (90,91). However, in reported cases, the trigger for the shift in promoter use is yet to be identified.…”

Decoding sORF translation – from small proteins to gene regulation

“…Lack of this 5'-most exon in differentiated myotubes could contribute to the increased Cryab protein production. 120,121 Because alternative TSS usage is common during vertebrate embryogenesis 122,123 and in different cell types, [124][125][126][127] such a transcription-based control of uORF regulation could have broad implications in developmental regulation of gene expression.…”