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

Objective

High-throughput transcriptomic analyses in human cell lines have found that >80% of the genome is transcriptionally
active. 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 common
hallmarks of RNAPII-derived transcripts, their fates differ substantially as some are rapidly degraded while others remain
stable and exercise diverse functions in the cell. What is the underlying mechanism? Transcript fate decisions are ultimately
dictated 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 serving
as a landing pad to recruit productive and/or destructive factors. Therefore, composition of the early RNA-protein particle
plays an essential role in dictating RNA fate, and the CBC and its cofactors pose an interesting dichotomous system to study
as 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 RNA
fate decisions are made during transcription and how RNA/DNA elements contribute. To resolve the sequential loading of
the 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 a
new and unprecedentedly high powered UV cross-linking technology employed at multiple short time increments. This will
for the first time enable the study of in vivo RNA binding kinetics of RNA-binding proteins with a temporal resolution
necessary to characterise co-transcriptional RNA fate decisions.

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Topic(s)

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Funding Scheme

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MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)

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Call for proposal

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(opens in new window) H2020-MSCA-IF-2017

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Coordinator

AARHUS UNIVERSITET
Net EU contribution

Net EU financial contribution. The sum of money that the participant receives, deducted by the EU contribution to its linked third party. It considers the distribution of the EU financial contribution between direct beneficiaries of the project and other types of participants, like third-party participants.

€ 200 194,80
Address
NORDRE RINGGADE 1
8000 Aarhus C
Denmark

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Region
Danmark Midtjylland Østjylland
Activity type
Higher or Secondary Education Establishments
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Total cost

The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.

€ 200 194,80
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