Talk on Saturday 09:15-09:30am submitted by Brian Magnuson
The life and times of transcribed RNA as told through global nascent RNA sequencing
Brian Magnuson (School of Public Health Environmental Health Sciences, University of Michigan, Ann Arbor, MI), Artur Veloso (Novartis Institutes of Biomedical Sciences, Cambridge, MA), Michelle Paulsen (Department of Radiation Oncology, University of Michigan, Ann Arbor, MI), Frederick Derheimer (Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN ), Zahid Bonday (Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN), Mats Ljungman (Department of Radiation Oncology, University of Michigan, Ann Arbor, MI)
RNA is born, matures, and dies through a number of processes – initiation, elongation, termination, processing, engagement, degradation – all of which can be regulated and ultimately define RNA fate. Global analysis using RNA-seq identifies changes in transcript abundance, splicing patterns, and other characteristics, but only from the steady-state pool of RNA, which is a mixture of young, old, and predominantly stable RNA. To address this, we developed two complementary methods: Bru-seq and BruChase-seq. Bru-seq is the brief metabolic labeling of RNA in live cells with bromouridine followed by immunoprecipitation and sequencing. BruChase-seq incorporates a chase period after RNA labeling in a paired sample. Given the initial synthesis state of RNA, we can follow the fate of RNA as it ages and, importantly, infer whether genes are regulated pre- or post-transcriptionally. The protein arginine methyltransferase 5 (PRMT5), an enzyme that modifies histones, transcription factors, splicing factors, and signaling proteins, has the potential to regulate RNA at multiple levels. For this reason, Bru- and BruChase-seq are well-suited to study the multi-faceted PRMT5. We have found, through knockdown and small molecule inhibition in human melanoma cells, that PRMT5 indeed regulates genes at the levels of transcription, splicing, and stability.
While Bru-seq is capable of capturing primary transcription units of genes that are unstable or highly processed (e.g. miRNA), some RNA species, such as enhancer RNA, are often missed. We found that irradiating cells prior to labeling with UVC light enhanced signal near transcription start sites, putative enhancers, and PROMPTs. UV light inhibits elongation but not initiation and therefore polymerases continue to synthesize RNA near initiation sites. We found that in TNF-treated normal human fibroblasts, BruUV-seq signal near gene TSSs is responsive to stimuli and correlates to changes in nascent (Bru-seq) signal. Furthermore, changes in putative active enhancer elements change with nearby transcribing genes and thus a potential application of BruUV-seq is to identify enhancer dynamics within an experiment. Together, Bru-seq and its derivatives provide a complementary and comprehensive picture of RNA regulation at multiple levels throughout its lifespan.
Keywords: gene regulation, rna sequencing, methyltransferase