No abstract

The most common causative diagnosis of hereditary small-vessel-disease of the brain, CADASIL, is due to highly stereotyped mutations in the NOTCH3 receptor. NOTCH3 has 33 exons but all CADASIL mutations occur within the Epidermal Growth Factor-like Repeats encoded by exons 2-24, lead to an odd number of cysteine residues and are associated with GOM deposits and abnormal NOTCH3 protein accumulation. The majority of CADASIL mutations appear to retain normal level of signaling activity, while very few mutations show reduced activity. Herein we identified a novel heterozygous missense mutation (c.4544T>C) in exon 25 of NOTCH3 in a patient with cerebral small-vessel-disease but lacking GOM deposits and NOTCH3 accumulation. The mutation should result in a p.L1515P substitution in the evolutionarily highly conserved juxtamembranous region of NOTCH3, which constitutes the heterodimerization domain. The p.L1515P mutant exhibits increased canonical NOTCH3 signaling, although in a ligand-independent fashion. Biochemical analysis suggests that the mutation renders NOTCH3 hyperactive through destabilization of the heterodimer. Therefore, our study suggests that the p.L1515P mutation falls in a novel mechanistic class of NOTCH3 mutations and that NOTCH3 activating mutations should be further considered for molecular analysis of patients with cerebral small-vessel-disease.

Pyruvate dehydrogenase (PDH) complex deficiency is a major cause of lactic acidosis and Leigh's encephalomyelopathies in infancy and childhood, resulting in early death in the majority of patients. Most of the molecular defects have been localized in the coding regions of the E1␣ PDH gene. Recently, we identified a novel mutation of the E1␣ PDH gene in a patient with an encephalopathy and lactic acidosis. This mutation, located downstream of exon 7, activates a cryptic splice donor and leads to the retention of intronic sequences. Here, we demonstrate that the mutation results in an increased binding of the SR protein SC35. Consistently, ectopic overexpression of this splicing factor enhanced the use of the cryptic splice site, whereas small interfering RNA-mediated reduction of the SC35 protein levels in primary fibroblasts from the patient resulted in the almost complete disappearance of the aberrantly spliced E1␣ PDH mRNA. Our findings open the exciting prospect for a novel therapy of an inherited disease by altering the level of a specific splicing factor.Removal of intervening sequences (introns) from precursor messenger RNAs (pre-mRNA) is an essential step in the expression of most metazoan protein-encoding genes, often regulated in a cell-type-specific or developmental manner. Alternative splicing enables the same precursor to give rise to several mRNAs that code for proteins having distinct functions. Thus, the precise selection of 5Ј and 3Ј splicing sites is a way to generate diversity and may lead to the regulation of gene expression according to tissue type or during development of the organism. The results of a recently published genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays (22) indicate that at least 74% of human multiexon genes are alternatively spliced.Among the splicing factors involved in splice site choice, members of the SR protein family have been extensively studied (see references 18 and 33 for reviews). SR proteins are characterized by the presence of one or two copies of an RNA recognition motif and a carboxyl-terminal domain rich in arginine and serine residues (RS domain). The RS domain is responsible for specific protein-protein interactions between RS domain-containing proteins (25,(42)(43)(44), whereas the RNA recognition motif domain recognizes several classes of specific RNA motifs, including exonic splicing enhancers (ESEs) and intronic splicing enhancers. These sequences have been demonstrated to play a key role in both alternative and constitutive splice site selection (see references 2 and 41 for reviews). This activity is counteracted by that of splicing repressors, such as members of the hnRNP family which can bind RNA in a nonspecific way but also recognize negative regulatory elements known as exonic and intronic splicing silencers (see references 29 and 43 for reviews). Such an antagonism accounts for the ability of SR proteins to influence splice site choice in a concentration-dependent manner (28, 33).