RNAfate · Identifying RNA fate checkpoints by resolving the high-resolution spatiotemporal binding dynamics of CBC containing complexes

High-throughput transcriptomic analyses in human cell lines have found that >80% of the genome is transcriptionallyactive. A major part of this massive genomic output is derived from RNA polymerase II (RNAPII) activity; such as, mRNA,sn(o)RNA and long non-coding RNA. However, although these transcripts all contain 5’-m7G caps, which are commonhallmarks of RNAPII-derived transcripts, their fates differ substantially as some are rapidly degraded while others remainstable and exercise diverse functions in the cell. What is the underlying mechanism? Transcript fate decisions are ultimatelydictated by the proteins with which the nascent RNA associate. Central to this process is the cap-binding complex (CBC).Through its early association with the 5’-m7G cap, the CBC directs a plethora of nuclear RNA metabolic events by servingas a landing pad to recruit productive and/or destructive factors. Therefore, composition of the early RNA-protein particleplays an essential role in dictating RNA fate, and the CBC and its cofactors pose an interesting dichotomous system to studyas a model for sorting mechanisms dictating RNA fate.In my project, I will delineate the spatiotemporal recruitment kinetics of selected RNA metabolic factors to identify when RNAfate decisions are made during transcription and how RNA/DNA elements contribute. To resolve the sequential loading ofthe CBC and its cofactors onto elongating transcripts, I will develop time course UV cross-linking and immunoprecipitation(CLIP) experiments, combining metabolic labelling of RNA, using the photoactivatable ribonucleoside analogue 4-sU, with anew and unprecedentedly high powered UV cross-linking technology employed at multiple short time increments. This willfor the first time enable the study of in vivo RNA binding kinetics of RNA-binding proteins with a temporal resolutionnecessary to characterise co-transcriptional RNA fate decisions.