Fig 1.
Genetic findings in a family with UBA5 mutations.
(A) The pedigree of family 1 with autosomal recessive spinocerebellar ataxia. (B) Brain magnetic resonance imaging of patient II:2. Panel (left): axial T1-weighted image showing atrophy of the cerebellar vermis. Panel (right): midline sagittal T1-weighted image showing cerebellar atrophy, particularly in the superior vermis, with enlargement of the fourth ventricle. (C) The UBA5 variants [c. 568C > T; c. 760A > G], [p. Arg246X (R246X); p. Lys310Glu (K310E)] segregated in this family. Red arrows indicate the mutation sites. (D) Two mutations (red) affected amino acids that are highly conserved across species.
Table 1.
The phenotypic characteristics of patients with intermediate.
Fig 2.
The subcellular localization, manner of degradation, and stability of mutant UBA5 and its interaction with UFM1.
(A) Immunostaining of overexpressed UBA5 and its mutants, as well as endogenous UBA5. (B) Immunostaining of endogenous UBA5 in HEK293A and HeLa cells. (C) Flag-UBA5 or related mutants were overexpressed via plasmid transfection in HEK293A cells. After 24 h, the cells were treated with MG132 (100μg/ml) or Bafilomycin A1 (BafAl, 100μg/ml). (D) UBA5 stability analysis. (E) Interactions of UBA5 mutants with Ufm1.
Fig 3.
RNAi-mediated knockdown of UBA5 and other molecules of UFM1 pathway induce neurodegeneration.
(A) Knockdown of UBA5 and other crucial molecules of UFM1 pathway, UFM1 and UFC1, in Drosophila exhibit abnormal vertical-turned wings that fail to fold, whereas knockdown of UFL1 is fatal. The wing phenotypes are: normal (a), unilateral wing abnormality (b), bilateral wing abnormality (c). (B) Percentage of abnormal wing phenotypes. (C) Comparison of climbing abilities shows climbing disabilities of the RNAi flies. (D) Comparison of flight abilities presents flight declines of the RNAi flies. (E) The life spans of the RNAi flies are significantly shortened. (F) The confocal images of larval muscle exhibit reduced type Ib bouton number and increased bouton size of the RNAi flies. (G) The statistical graph of bouton numbers. (H) The statistical graph of bouton sizes. All the indicated genotypes in panel B-E are: DA-GAL4>attP2, DA-GAL4>UBA5 RNAi, DA-GAL4>UFM1 RNAi, DA-GAL4>UFC1 RNAi, DA-GAL4>UFL1 RNAi. All the indicated genotypes in panel F-H are: OK6-GAL4>attP2, OK6-GAL4>UBA5 RNAi, OK6-GAL4>UFM1 RNAi, OK6-GAL4>UFC1 RNAi. *** = p < 0.0001 and ** = p < 0.01, Student’s t-test and Mann–Whitney test.
Fig 4.
Overexpression of wild-type UBA5 significantly restores neural lesions of UBA5 RNAi flies while mutant UBA5 could not confer effective rescue.
(A) Percentage of abnormal wing phenotypes. (B) Comparison of climbing abilities. (C) The confocal images of larval muscle. (D) The statistical graph of bouton numbers. (E) The statistical graph of bouton sizes. All the indicated genotypes in panel A-B are: DA-GAL4>attP2, DA-GAL4>UBA5 RNAi, DA-GAL4>UBA5 RNAi+dUBA5-1, DA-GAL4>UBA5 RNAi+dUBA5-2, DA-GAL4>UBA5 RNAi+hUBA5-1, DA-GAL4>UBA5 RNAi+hUBA5-2, DA-GAL4>UBA5 RNAi+hUBA5 R246X-1, DA-GAL4>UBA5 RNAi+hUBA5 R246X-2, DA-GAL4>UBA5 RNAi+hUBA5 K310E-1, DA-GAL4>UBA5 RNAi+hUBA5 K310E-2. All the indicated genotypes in panel C-E are: OK6-GAL4>attP2, OK6-GAL4>UBA5 RNAi, OK6-GAL4>UBA5 RNAi+dUBA5-1, OK6-GAL4>UBA5 RNAi+hUBA5-1, OK6-GAL4>UBA5 RNAi+hUBA5 R246X-1, OK6-GAL4>UBA5 RNAi+hUBA5 K310E-1. *** = p < 0.0001, ** = p < 0.01 and ns = no significance, Student’s t-test and Mann–Whitney test.