Role Of SMN1 Gene Nonsense-mediated MRNA Decay In Pathogenesis Of Spinal Muscular Atrophy

Nonsense-mediated mRNA decay(NMD)is a highly conserved quality control mechanism that exists in eukaryotes.It can prevent the accumulation of potentially toxic truncated proteins by eliminating abnormal mRNA containing premature translation-termination codon(PTC).Studies have shown that NMD plays an important role in many genetic diseases.Spinal muscular atrophy(SMA)is a neurodegenerative autosomal recessive genetic disease.The disease is caused by the deletion or mutation of the survival motor neuron 1(SMN1)gene.However,the role of NMD in SMA remains unclear.To clarify the role of SMN1 nonsense mutations in SMA,a SMN1 minigene recombinant plasmid(WT)was constructed in this study.The plasmid concludes three parts: the first part contains the whole exon l and the 5’partial intron 1(S1);the second part contains the 3’ partial intron 4,the whole exon 5 and the 5’ partial intron 5(S2);and the last part contains the 3’partial intron 6,the whole exon 7,intron 7,and partial exon 8(S3).According to the Human Gene Mutation Database,the mutants Pc MV-Mini-S123-E1(Q15X in Eoxn 1,E1),Pc MV-Mini-S123-E5(L228X in Eoxn 5,E5)and Pc MV-Mini-S123-E7(Q282X in Eoxn 7,E7)were designed by PCR mutagenesis.The transcripts of WT and mutants were detected at the RNA level.The experiments with translation inhibitor cycloheximide treatment and q RT-PCR showed that mutants E5 and E7 triggered translation-dependent NMD,and E1 escaped NMD.On the other hand,recent studies have found that alternative splicing coupled to nonsense-mediated mRNA decay(AS-NMD)is a post-transcriptional regulatory mechanism to fine-tune homeostatic expression of many RNA-binding proteins.To explore the correlation between AS-NMD and SMA,RNA sequencing of peripheral blood lymphocytes in SMA patients with homozygous deletion of SMN1gene(E0)and normal controls with two copies of SMN1 gene(NC)was performed.The r MATS software was used to analyze the differences of alternative splicing events in RNA-seq data.Exon skipping(SE)and intron retention(RI)events were analyzed between EO group and NC group.The gene ontology(GO)and kyoto encyclopedia of genes and genomes pathway(KEGG)of these splicing isoforms were analyzed by online tool Novo Magic v3.0.Personalized analysis and mapping were realized,and NCBI gene database was used to annotate these genes.The results revealed that homozygous deletion of SMN1 gene can lead to changes in some mRNA alternative splicing and some gene expression.The differential genes with SE affect ribonucleoprotein assembly,intracellular transport,translation,acetylation modification,mitochondrial energy metabolism and ubiquitin-regulated degradation pathway,while the differential genes with RI not only participate in ubiquitin regulation,endocytosis,translation and mitochondrial metabolism,but also regulate acetylation modification related to cytoskeleton and metabolic enzyme activity.In conclusion,SMN1 deletion can change the alternative splicing of some genes,and then affect RNA synthesis,assembly and degradation of related proteins,resulting in protein homeostasis imbalance.This provides a new clue to the pathogenesis of SMA.