High-Resolution Deep Sequencing of Nascent Transcription in Yeast with BioGRO-seq

Abstract

RNA biogenesis in eukaryotic cells is a tightly regulated multilayered process in which a diverse set of players act in an orchestrated manner via complex molecular interactions to secure the initial flow of gene expression. Transcription from DNA to RNA is the essential first step in RNA biogenesis, and consists of three main phases: initiation, elongation, and termination. In each phase, transcription factors act on RNA polymerases to modulate their passage along the DNA template in a very precise manner, governed by molecular mechanisms, some of which are not yet fully understood. Genome-scale run-on-based methodologies have been developed with the aim of mapping the position of transcriptionally engaged RNA polymerases. Among them, the BioGRO methodology has been instrumental in advancing our understanding of the transcriptional dynamics in yeast. Here we take the previously known BioGRO method further by coupling it with deep sequencing. BioGRO-seq maps elongating RNA polymerases along the genome with strand specificity and single-nucleotide resolution. BioGRO-seq profiling provides insights into the biogenesis and regulation of not just the canonical protein-coding transcriptome, but also into the often more challenging to study noncoding and unstable transcriptome.