European Molecular Biology Laboratory (EMBL)'s video: Keynote Lecture: Lighting Up the Complex Life

Presenter: David Tollervey, The University of Edinburgh, UK From the EMBO | EMBL Symposium: The Complex Life of RNA 7 - 9 October 2020, Virtual ABSTRACT: To illuminate RNA biology in a cellular context, we developed techniques for mapping protein-binding sites on all RNAs (CRAC), the RNA-bound proteome (TRAPP) and precise sites of RNA-interactions on proteins (iTRAPP), all combined with mathematical modelling. Applying CRAC to RNA polymerases allowed high-resolution, strand-specific mapping of transcriptionally engaged RNA polymerase I, II and III. Elongation by RNA polymerases is based on a Brownian Ratchet, rather than an energy-driven mechanism, making it prone to pausing and backtracking. We combined in vivo RNAPI profiling with in vitro biochemical analyses and a quantitative, mechanistic mathematical model of transcription elongation. These revealed that folding of the nascent pre-rRNA close to the transcribing polymerase has a dominant effect on the elongation rate, with a modest contribution from the stability of the RNA-DNA duplex in the active site. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site usage, indicating regulatory significance for transcript folding. In all systems analysed, rapid alterations in RNA-protein interactions help remodel gene expression following stress, particularly translation. To identify echanisms, we applied TRAPP during short (second to minute) time courses following stress. We discovered that stress-induced translation shut-down in response to heat-shock or glucose starvation is driven by very rapid displacement of a specific set of initiation factors: eIF4A, eIF4B and Ded1. This is distinct from translation shutdown by the highly conserved, and much-studied, eIF2-phosphorylation pathway. For glucose withdrawal, the effects are startlingly fast – with complete loss of factor binding within 30 sec. Heat shock triggers mRNA degradation, selectively for ribosome synthesis factors and ribosomal proteins. We recently extended this work to SARS-CoV-2; applying TRAPP and CRAC to identify mechanisms involved in remodeling host RNA metabolism during infection. Viral infection strongly reduces RNA-binding for specific functional groups proteins.