Seq-ing answers: uncovering the unexpected in global gene regulation

Abstract

The development of techniques for measuring gene expression globally has greatly expanded our understanding of gene regulatory mechanisms in depth and scale. We can now quantify every intermediate and transition in the canonical pathway of gene expression-from DNA to mRNA to protein-genome-wide. Employing such measurements in parallel can produce rich datasets, but extracting the most information requires careful experimental design and analysis. Here, we argue for the value of genome-wide studies that measure multiple outputs of gene expression over many timepoints during the course of a natural developmental process. We discuss our findings from a highly parallel gene expression dataset of meiotic differentiation, and those of others, to illustrate how leveraging these features can provide new and surprising insight into fundamental mechanisms of gene regulation.

Keywords: Gene expression; Gene regulation; LUTI; Mass spectrometry; Non-canonical gene expression; Ribosome profiling; Transcript isoforms; Transcription; Translation; mRNA-seq; uORFs.

Fig. 1

Integrated analysis of parallel, genome-wide measurements through meiotic development reveals pervasive non-canonical regulation of gene expression. a We performed RNA sequencing (mRNA), ribosome profiling (translation) and quantitative mass spectrometry (protein) on matched lysate from a timecourse spanning meiosis in budding yeast. Hierarchical clustering is displayed with rows representing individual genes (n = 4464) that were quantified across all measurements and timepoints. Columns represent timepoints through meiotic differentiation depicted in b. b Meiosis is a conserved cellular differentiation program comprised of a coordinated series of unidirectional transitions in cell state, ultimately producing haploid gamete cells from a diploid precursor. c Above, measurements of mRNA, translation and protein abundance are depicted for a model locus regulated by transcript isoform toggling. Below, a diagram for this model gene locus is shown. Transcription from an ORF-proximal transcription start site (TSS2) mediated by a developmentally regulated transcription factor (TF2) leads to the production of a canonical, well-translated transcript and the ORF-endcoded protein is abundant. A decrease in TF2 and an increase in another transcription factor (TF1) results in a switch to transcription from an ORF-distal transcription start site (TSS1), producing a long undecoded transcript isoform (LUTI) mRNA that is poorly translated. ORF-encoded protein is reduced, despite increased transcription from this locus. As a consequence, ORF-encoding transcript abundance and ORF-encoded protein abundance show poor correlation over time. Using integrated parallel measurements outlined in a, we identified widespread use of this unconventional regulatory mechanism for hundreds of genes