A-44G Transition In SMN2 Intron 6 Protects Patients With Spinal Muscular Atrophy

Background and objective:Spinal muscular atrophy(SMA)is a devastating inheritable disorder,characterized by loss of spinal cord a-motor neurons,which results in widespread atrophy of skeletal muscles,especially those of the limbs and trank.SMA is caused by reduced expression of survivor of motor neuron(SMN).In SMA patients,SMN1 gene encoding SMN protein fails to produce normal SMN protein because of loss of function mutation,and homologous SMN2 gene also expresses only a full length of SMN protein because of exon 7 C6T changes.SMA is subdivided into four types,based on the clinical severity and age of onset,with type I being the most severe one,and type IV being the adult-onset form.The copy number of SMN2 is a key modifier of the disease.In general,patients with the type I form have 1 or 2 copies of SMN2;most patients with the intermediate type II form have 3 copies of SMN2;and most patients with the milder type III form have 3-4 copies of the gene.However,there are many cases in which SMN2 copy number is not inversely correlated with clinical severity.Thomas Prior et al.previously showed that the c.859G>C mutation in exon 7 promotes inclusion of the exon and accounts for the much milder phenotype in several patients who carry 2 copies of SMN2.In this study,we followed a set of 53 patients with 0 copies of SMN1 and 3 copies of SMN2,who were older than 20 years at the time of molecular diagnostic testing.Genomic sequencing revealed one conversion(SMN1 to SMN2)event in 5/53 patients.We compared these findings to a more severe group of 90 SMA patients younger than 6 months at the time of testing,which were negative for the conversion and also for the 859 change.Therefore,we hypothesized that SMN1C6T exon 7 splicing may be improved,resulting in an increased expression of SMN protein,thereby alleviating the patient’s condition.Methods:Genotype analysis of SMA patients by gene sequencing;Construction of SMN1/2 minigene vectors for alternative splicing assay;Site-directed mutagenesis was used to analyze the key changes that affect the inclusion of exon 7 in SMN1/2;to analyze the effect of RNA 2ary structure on SMN1/2 splicing changes by correlation between exon 7 inclusion and minimum free energy;Analysis of cis-acting elements affecting splicing by deletion mutagenesis;Identification of the RNA binding protein by RNA-affinity chromatography and mass spectrometry;To verify the regulatory effects of RNA binding proteins on splicing by MS2-tethered assay;The analysis of the domain of RNA binding protein explains the mechanism of splicing;Overexpression and knockdown of RNA binding proteins validate their regulatory roles in alternative splicing of SMN1/2 genes.Results:SMN1C6T pre-mRNA splices exon 7 more efficiently than SMN2 pre-mRNA;The A-44G change is responsible for the moderate improvement of exon 7 splicing;the-44 region is intronic splicing silencer;HuR represses splicing when bound to the silencer,the G-44A transition moderately increases of HuR;RRM1 and RRM2 mediate repression activity by HuR;Over-expression of HuR inhibits exon 7 inclusion and knock-down of HuR improves splicing.Conclusion:We have found a new mechanism for improving the level of SMN protein and alleviating the condition of SMA patients.The A-44G change markedly decreases the binding affinity of HuR,resulting in a moderate increase in exon 7 inclusion.