Structural and functional investigation of the SRSF1-mediated nuclear export of mRNAs

Cells form the basic living units of the body, converting food and oxygen into energy to produce proteins that serve as building blocks and molecular machines. The DNA blueprint that contain genes for making up proteins is housed in a cell centre, the nucleus, which is separated from the surrounding compartment, the cytoplasm, where proteins are manufactured. Messenger intermediate molecules (mRNAs) copied from the blueprint are transported from the nucleus into the cytoplasm across nuclear openings where each mRNA guides the assembly of one type of protein. Other RNAs are also produced from the core DNA blueprint and require passage into the cytoplasm - they do not convey information for assembling the proteins but allow building some of the gene-working machineries and modulating the production of proteins depending on the needs of the cells. The quantity and functionality of tens of thousands of proteins account overall for the normal functioning or the death of cells.

We and others have shown that the protein with the name SRSF1 has an important role in transporting mRNAs into the cytoplasm by bringing mRNAs into contact with another protein, NXF1, which drives mRNAs through the nuclear openings. In addition, we recently reported that the attachment of SRSF1 to differently shaped C9ORF72 mRNAs, involved in lethal diseases of the brain called amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), is also responsible for inducing transportation of these peculiar mRNAs into the cytoplasm. The precise mechanisms of how SRSF1 attaches to NXF1 and mRNAs for transport into the cytoplasm remain to be discovered. Our research project now aims to (i) understand precisely how SRSF1 attaches to the C9ORF72-ALS/FTD RNAs and NXF1 and (ii) how SRSF1 induces transportation of these RNAs into the cytoplasm. We will carry out experiments in human nerve cells and fruit fly models of C9ORF72-ALS/FTD as well as with combinations of pure SRSF1, NXF1 and RNA in test tubes. This will allow understanding for the first time how this dynamic process occurs at the scale of atoms. Our programme of research has three complementary objectives:

1. Discover how SRSF1 attaches to NXF1 at the atomic level.

2. Discover how SRSF1 attaches to two types of shaped C9ORF72-ALS/FTD RNAs forming special four-stranded or double-stranded structures at the atomic level.

3. Examine how SRSF1, C9ORF72-ALS/FTD RNAs and NXF1 assemble at the microscopic level to form a single composite transport machine and how important this is for promoting the transport of RNAs into the cytoplasm of human cell models and C9ORF72-ALS/FTD nerve cells and fruit fly models.

In the long term, we expect that a detailed understanding of the mechanisms by which SRSF1 allow transport of RNA though nuclear openings would have far-reaching implications for the understanding of novel biological mechanisms and the potential future development of drug inhibitors for the treatment of some neurodegenerative diseases such as C9ORF72-ALS/FTD. Several communities of academic researchers including students and scientists will directly be involved in conducting the research. Furthermore, this work will lead to inspirational communications, presentations and publications that will benefit the academic community, the general public, local schools, the tertiary sector and potentially in the longer term the biotechnology sector and the pharmaceutical industry.

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.

Results from this multidisciplinary proposal between groups with unique world-renowned expertise in structural biology and nuclear export of mRNAs are expected to transform our understanding of the molecular mechanisms driving the nucleocytoplasmic transport of RNAs. The combination of state-of-the-art structural methodologies and functional assays in human derived neurons and Drosophila will provide unambiguous conclusions that will enable maximum scientific, societal and economic impacts.

1. Professional development of people and skills
(i) Two PDRAs will respectively gain world-class expertise in structural biology methodologies and investigation of the nucleocytoplasmic transport of RNA including in vivo studies using human-derived neurons and structural investigations of protein:RNA interactions, two fields of research under-represented in the UK. Investigators will mentor the PDRAs, demonstrate particular research skills when needed and ensure the professional and personal developments of the PDRAs.
(ii) The Year-11, BSc, MSc and PhD students who will work on research projects linked to this proposal. The PDRAs will also co-supervise students to further develop their skills and CV.

2. Scientific and academic impact
This research is based on published discoveries of significant importance expected to lead to further very high impact publications. In addition, the PDRAs and the investigators will present the data at major national and international conferences, meet renowned scientists and potentially establish new collaborations or future career development opportunities for the PDRAs. We will also share our reagents upon publication, increasing even further the impact generated from the direct outcomes of this research.

3. Societal and educational impact
(i) School outreach activities: local schools and public will benefit from general concepts concerning the expression of genes, the nuclear export of RNAs, nerve cells, neurodegeneration and scientific methodologies that can be used to investigate these processes. Students will also be informed about the development of the research through sharing interesting findings and research challenges. Our research will also be communicated in school magazines.
(ii) Public engagement activities: dissemination of results through the University of Sheffield, University of Leicester and ETH-Zurich websites and institution-linked outreach activities.
(iii) Teaching in the Universities of Sheffield and Leicester: the investigators deliver lectures on gene expression and structural biology in several undergraduate and M.Sc. courses.
(iv) In the longer term, the general knowledge from this research project may inform public and tertiary sectors on future research development and priorities for the manipulation of gene expression in the biotechnological and biomedical sectors.

4. Potential economic impact
The proposed research is not expected to lead to the development of commercial opportunities in the short term. However, the outcomes from this research is likely to lead to (i) the design of modified SRSF1 or NXF1 proteins with added bio-technological/medical values and (ii) to the future identification of small molecules or cell permeable peptide inhibitors to manipulate the mRNA nuclear export pathway in physiological and disease conditions. Both drug development and gene therapy approaches using inhibitory cell permeable peptides could be used. Any commercially sensitive findings will be handled confidentially with our University Research and Innovation Services and their approved partners. The PI regularly discusses commercial opportunities with several biotech, venture and pharmaceutical companies (AveXis, Kurma Partners, Pfizer, Biogen). He is also the primary inventor of a patent application regarding the inhibition of the nuclear export of C9ORF72-repeat transcripts as a novel therapeutic strategy in C9ORF72-ALS/FTD (PCT/GB2017/051539).