Structural and functional investigation of the SRSF1-mediated nuclear export of mRNAs
Award Number
BB/S005579/1Status / Stage
CompletedDates
1 August 2019 -31 March 2023
Duration (calculated)
03 years 07 monthsFunder(s)
BBSRC (UKRI)Funding Amount
£398,560.00Funder/Grant study page
BBSRC UKRIContracted Centre
University of LeicesterPrincipal Investigator
Dr Cyril DominguezPI Contact
cd180@leicester.ac.ukPI ORCID
0000-0003-3575-8922WHO Catergories
Understanding Underlying DiseaseDisease Type
Dementia (Unspecified)CPEC Review Info
| Reference ID | 674 |
|---|---|
| Researcher | Reside Team |
| Published | 07/07/2023 |
Data
| Award Number | BB/S005579/1 |
|---|---|
| Status / Stage | Completed |
| Start Date | 20190801 |
| End Date | 20230331 |
| Duration (calculated) | 03 years 07 months |
| Funder/Grant study page | BBSRC UKRI |
| Contracted Centre | University of Leicester |
| Funding Amount | £398,560.00 |
Abstract
The nuclear export of RNAs is indispensable to eukaryotic life. The export adaptor SRSF1 plays multiple roles in the expression of genes coupling alternative splicing to mRNA nuclear export through interactions with the nucleoporin-binding factor NXF1 which heterodimerizes with p15. We showed that SRSF1 triggers pathological nuclear export of C9ORF72-repeat transcripts in the most common forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We previously proposed that the function of export adaptors is to remodel NXF1 into a high RNA affinity mode that triggers handover of the RNA from the adaptor to NXF1 licensing thereby the nuclear export process. Here, we aim to structurally and functionally characterize the interactions of SRSF1 with NXF1 and C9ORF72-repeat RNAs and investigate the conformational rearrangements leading to RNA handover. We will determine the molecular basis for the interactions of SRSF1 with NXF1 and C9ORF72-repeat RNAs using solution-state nuclear magnetic resonance spectroscopy (NMR). Co-immunoprecipitation and pull down assays will complement the structural investigation of the SRSF1:NXF1 binding site. RNA immunoprecipitation in human C9ORF72-ALS/FTD cell models, electrophoretic mobility shift assays and isothermal titration calorimetry using recombinant proteins and RNA oligonucleotides will support the NMR structures of SRSF1 bound to G-quadruplex and double-stranded C9ORF72-repeat RNAs. Finally we will use NMR, protein:protein/ protein:RNA crosslinking assays coupled with mass spectrometry, X-ray crystallography, SAXS and cryo-electron microscopy to investigate the structural remodeling of SRSF1:NXF1:p15:C9ORF72-repeat RNA nuclear export complexes. Functional implications on the nuclear export of C9ORF72-repeat transcripts will be investigated in C9ORF72-ALS/FTD patient-derived neurons and Drosophila using qRT-PCR quantification of nuclear/cytoplasmic mRNA levels, RNA-FISH and locomotor function analysis.
Aims
Here, we aim to structurally and functionally characterize the interactions of SRSF1 with NXF1 and C9ORF72-repeat RNAs and investigate the conformational rearrangements leading to RNA handover.