Abstract
BackgroundRecent studies have described a widespread induction of transcriptional readthrough as a consequence of various stress conditions in mammalian cells. This novel phenomenon, initially identified from analysis of RNA-seq data, suggests intriguing new levels of gene expression regulation. However, the mechanism underlying naturally occurring transcriptional readthrough, as well as its regulatory consequences, still remain elusive. Furthermore, the readthrough response to stress has thus far not been investigated outside of mammalian species, and the occurrence of readthrough in many physiological and disease conditions remains to be explored.ResultsTo facilitate a wider investigation into transcriptional readthrough, we created the DoGFinder software package, for the streamlined identification and quantification of readthrough transcripts, also known as DoGs (Downstream of Gene-containing transcripts), from any RNA-seq dataset. Using DoGFinder, we explore the dependence of DoG discovery potential on RNA-seq library depth, and show that stress-induced readthrough induction discovery is robust to sequencing depth, and input parameter settings. We further demonstrate the use of the DoGFinder software package on a new publically available RNA-seq dataset, and discover DoG induction in human PME cells following hypoxia – a previously unknown readthrough inducing stress type.ConclusionsDoGFinder will enable users to explore, in a few simple steps, the readthrough phenomenon in any condition and organism.DoGFinder is freely available at https://github.com/shalgilab/DoGFinder.
Highlights
Recent studies have described a widespread induction of transcriptional readthrough as a consequence of various stress conditions in mammalian cells
DoGFinder demonstrates sensitivity and robustness of readthrough detection to sequencing depth and recapitulates readthrough identification from various inputs To test the performance of DoGFinder, and assess its sensitivity to varying sequencing depths, we used our published nuclear-enriched rRNA-depleted strand specific paired-end RNA-seq data from mouse NIH3T3 cells that were exposed to osmotic stress (KCl, 2 h) [1]
We previously identified that stress both induces Downstream of gene-containing transcripts (DoGs) in numerous genes, and that DoGs get massively longer
Summary
Recent studies have described a widespread induction of transcriptional readthrough as a consequence of various stress conditions in mammalian cells This novel phenomenon, initially identified from analysis of RNA-seq data, suggests intriguing new levels of gene expression regulation. Using RNA-seq, we have identified massive induction of transcriptional readthrough in response to osmotic stress [1, 2], heat shock and oxidative stress [1] in mammalian cells, occurring in thousands of genes. In these conditions, thousands of DoGs, many extending. It can further merge or intersect DoG annotation files (see below), and quantify the expression levels of DoGs
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