Elucidating the structure-function relationship of the cell-type specific splicing regulator, RBPMS

The organization of RBPs into larger complexes is known to play a crucial role in regulating various aspects of RNA metabolism, including splicing. The RNA binding protein, RNA binding protein with multiple splicing (RBPMS), regulates the alternative splicing programme in differentiated vascular smooth muscle cells (VSMCs). RBPMS has an N-terminal RNA recognition motif (RRM) that mediates both RNA binding and dimerization, allowing binding to pairs of trinucleotide CAC motifs. The C-terminal tail is predicted to be unstructured, and such regions of other RNA-binding proteins are often important for higher-order structures including condensates. There are two main isoforms expressed in differentiated VSMCs, RBPMS-A and RBPMS-B, and they have unique extreme C-terminal tails. RBPMS-A is the more active isoform, both activating and repressing splicing events, whereas RBPMS-B has only been observed to activate splicing. Previously, recombinant full-length RBPMS-A was shown to be sufficient in the repression of Tpm1 exon 3, a model differentiated VSMC splicing event. Additionally, deletion of the C-terminal twenty amino acids of RBPMS-A caused a loss of higher order assembly and a reduction of splicing repressor activity. In this thesis, I describe detailed mutational analyses of the RBPMS-A C-terminal tail which revealed two clusters of aromatic residues were necessary for the activity of transfected RBPMS-A, whereas basic residues within the same region appear less important for activity. To allow analysis of the biophysical properties of RBPMS-A associated with different modes of splicing regulation, model splicing substrates were developed for in vitro RBPMS-mediated repression and activation assays. Wild-type RBPMS-A and basic mutant were fully active in both in vitro splicing assays, whereas the aromatic mutants were impaired for activity. Mass photometry and glutaraldehyde crosslinking have shown that these residues are also important for the ability of RBPMS-A to undergo higher order assembly. Remarkably, mutation of the basic residues also diminished higher-order assembly, despite these mutants still retaining full splicing activity. The C-terminus of RBPMS-B, however, was found to be antagonistic to splicing activity, with mutation of this region leading to full splicing activity to the level of RBPMS-A. Peptide studies of this region revealed that it was able to form very stable fibril-like structures, but the full-length protein forms large spherical structures. The different oligomerisation behaviours and activities of these isoforms and their mutants found in this study gives a greater insight into the mechanism of splicing regulation by RBPMS and the control of the splicing programme in smooth muscle cells.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Partridge, Ruth

Advisor dc:contributor.advisor

• Smith, Christopher

• Alternative Splicing
• RBPMS
• Vascular Smooth Muscle

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/9ab29cda-e30f-4e46-b4c1-e98d3cd7c6f3/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.116961

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/381953