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

Abstract

<b>Background:</b> X-linked spinal muscular atrophy (XL-SMA) results from mutations in the Ubiquitin-Like Modifier Activating Enzyme 1 ( <i>UBA1</i>). Previously, four novel closely clustered mutations have been shown to cause this fatal infantile disorder affecting only males. These mutations, three missense and one synonymous, all lie within Exon15 of the <i>UBA1</i> gene, which contains the active adenylation domain (AAD). <b>Methods:</b> In this study, our group characterized the three known missense variants <i>in vitro</i>. Using a novel Uba1 assay and other methods, we investigated Uba1 adenylation, thioester, and transthioesterification reactions <i>in vitro</i> to determine possible biochemical effects of the missense variants. <b>Results:</b> Our data revealed that only one of the three XL-SMA missense variants impairs the Ubiquitin-adenylating ability of Uba1. Additionally, these missense variants retained Ubiquitin thioester bond formation and transthioesterification rates equal to that found in the wild type. <b>Conclusions:</b> Our results demonstrate a surprising shift from the likelihood of these XL-SMA mutations playing a damaging role in Uba1's enzymatic activity with Ubiquitin, to other roles such as altering <i>UBA1</i> mRNA splicing via the disruption of splicing factor binding sites, similar to a mechanism in traditional SMA, or disrupting binding to other important <i>in vivo</i> binding partners. These findings help to narrow the search for the areas of possible dysfunction in the Ubiquitin-proteasome pathway that ultimately result in XL-SMA. Moreover, this investigation provides additional critical understanding of the mutations' biochemical mechanisms, vital for the development of future effective diagnostic assays and therapeutics.