Functional characterizations of rare UBA1 variants in X-linked Spinal Muscular Atrophy

Area of Science:

• Biochemistry
• Genetics
• Molecular Biology

Background:

• X-linked spinal muscular atrophy (XL-SMA) is a fatal infantile disorder caused by mutations in the Ubiquitin-Like Modifier Activating Enzyme 1 (UBA1) gene.
• Four novel mutations, including three missense and one synonymous, located in Exon15 (active adenylation domain) of UBA1 have been identified.
• These mutations exclusively affect males, highlighting the X-linked nature of the disorder.

Purpose of the Study:

• To biochemically characterize three known XL-SMA missense variants of UBA1 in vitro.
• To investigate the impact of these variants on Uba1's enzymatic activities, including adenylation, thioester formation, and transthioesterification.
• To elucidate the molecular mechanisms underlying XL-SMA pathogenesis.

Main Methods:

• In vitro characterization of three missense variants in the UBA1 gene.
• Utilized a novel Uba1 assay to assess enzyme activity.
• Investigated Uba1 adenylation, thioester, and transthioesterification reactions.

Main Results:

• Only one of the three missense variants demonstrated an impairment in Uba1's ubiquitin-adenylating ability.
• All three missense variants maintained wild-type levels for ubiquitin thioester bond formation and transthioesterification rates.
• The findings suggest that the detrimental effects of these XL-SMA mutations may not solely lie in direct enzymatic inhibition.

Conclusions:

• XL-SMA missense mutations may exert their damaging effects through mechanisms other than direct impairment of Uba1's enzymatic activity.
• Potential alternative mechanisms include altering UBA1 mRNA splicing by disrupting splicing factor binding sites or affecting interactions with other in vivo binding partners.
• These findings are crucial for understanding XL-SMA pathogenesis, developing diagnostic assays, and designing targeted therapeutics.