Functional characterization of Snurportin-1 C-terminal domain in the context of a novel muscular dystrophy

SNUPN gene encodes for a nuclear import adaptor protein Snurportin 1 (SPN1) which participates in the nuclear transport of small nuclear ribonucleoproteins (snRNPs), by specifically binding to their m3G-cap. Nucleocytoplasmic shuttling is a highly versatile process that plays a major role in the maturation of pre-mRNA before translation, by facilitating the formation of the spliceosome complex through the transport of snRNPs. The spliceosome is a large RNA-protein complex that facilitates the removal of the introns from nuclear pre-mRNA. Mutations in proteins involved in the nucleocytoplasmic machinery can impair spliceosome formation, leading to abnormal splicing, and are reported to be causative for various genetic disorders, including muscular dystrophies. Muscular Dystrophies includes a variety of genetically and phenotypically heterogeneous disorders characterized by progressive muscle weakness, often manifesting in infancy or early childhood. To this date, muscular dystrophies have been associated with more than 85 genes, however, 40% of the cases do not have a definitive molecular diagnosis. Our group recruited eighteen patients from fifteen unrelated families diagnosed with muscular dystrophy. All patients presented with muscular weakness, sporadically accompanied by either neurological defects and/or cataracts. By performing whole exome sequencing we uncovered novel germline homozygous or compound heterozygous variants in the SNUPN gene across all patients. Notably, no pathogenic variants of this gene have previously been linked to muscular defects. We hypothesized that SNUPN mutations result in structural defects in SPN1 that impair the nucleocytoplasmic transport of the UsnRNPs. Interestingly, all the SPN1 pathogenic variants are localized in the poorly defined C-terminal region of the protein. This region is known to be taking part in intramolecular interactions. The primary aim of this study was to develop tools and conduct in vitro assays to further characterize the function of the C-terminal region of SPN1 and ultimately investigate the protein's ability to carry out intermolecular interactions via its C-terminal region. To address this question, we first generated SNUPN wild-type and mutant DNA constructs in the pCS2+ mammalian expression vector by molecular cloning and site-directed mutagenesis. Next, biochemical experiments were carried out by overexpressing WT and mutants of SPN1 in HEK293T/HeLa cells to compare their levels, localization, and interactions. Through these experiments, we aimed to unravel the importance of the C-terminal region of the protein and the impact of the mutations on SPN1's function in self-oligomerization. We hypothesize that pathogenic SNUPN variants may disrupt self-oligomerization of the C-terminal region of SPN1 thereby altering the cellular function of the protein. This may ultimately impair the nucleocytoplasmic shuttling of the UsnRNPs' leading to spliceosome defects and the observed phenotype in patients. Overall, this study aims to highlight the essential role of the uncharacterized C-terminal region of the SPN1 protein.