Molecular Mechanism Of SMN Deficiency In Activating P53 Signaling Pathway

Background and Objective:Spinal muscular atrophy(SMA)is an euromuscular disease,characterized with degeneration of α-motor neuron,leading to atrophy of skeletal muscles and eventually respiratory failure.SMA is the leading genetic cause of infantile death,with an incidence of approximately one in 6000-10000 live births.SMA is an autosomal recessive disorder that results from a homozygous deletion or mutation in the 5q13 survival of motor neuron(SMN1)gene,which encodes the survival of motor neuron(SMN)protein.SMN,an ubiquitously expressed protein,is involved in the assembly and biogenesis of numerous biomacromolecules and plays key roles in RNA metabolism.However,the molecular mechanism by which SMN deficiency causes motor neuron death is still unclear.Recently,it has been proposed that p53 activation induces motor neuron degeneration.Indeed,dysregulation of Mdm2 and Mdm4 alternative splicing was observed in mouse models,which results in reduction of the full-length functional MDM2 and MDM4 and consequently in p53 stablization.However,such a notion has never been tested in more relevant human settings.Here,we explore alterations in p53 expression and underlying mechanisms in SMN-defecient human cells.Methods:Using lentiviral shRNA to construct inducible stable cell lines with efficiently knockdown of SMN levels;qPCR and Western blot were used to detect expression of p53 at mRNA and protein levels;RT-PCR was used to detect MDM2 and MDM4 alternative splicing;CHX was used to study p53 protein degradation;Co-IP was used to demonstrate p53 ubiquitination;Overexpression plasmid pCGT7-p53 was constructed to study the degradation of p53 and the effects of different domains on p53 degradation.To define key lysine sites and their corresponding E3 ubiquitin ligases that affect p53 stability in the SMA models.site-directed mutagenesis was used to mutate single amino acid.Results:Five stable human cell lines with efficient knockdown of SMN expression were successfully established,and high levels of endogenous p53 expression were detected in HEK293(embryonic kidney)and SH-SY5Y(neuroblastoma).We found that SMN deficiency did not alter MDM2 and MDM4 alternative splicing in human cells.SMN deficiency inhibits the degradation of both endogenous p53 and T7-taged p53(T7-p53)expressed from pCGT7-p53.SMN deficiency resulted in a significant reduction in p53-bound polyubiquitin.Constructs that express a series of truncated T7-p53 mutants were successfully generated.Upon SMN depletion,a dramatic increase in protein stability was observed in the mutant that lacks the N-terminal domain.Analysis of 20 lysine-to-arginine substitutions revealed that in addition to the known C-terminal lysine sites,and the six lysine sites at the N terminus were also attributalbe to p53 stability.Conclusion:SMN deficiency in HEK293 and SH-SY5Y cells increases p53 levels at the post-transcriptional level.However,unlike what observed in animal models,increased expression of p53 in human cells dose not invovle dysregulation of Mdm2 and Mdm4 alternative splicing,but instead,is casued by enhanced stability of the protein due to reduced p53 ubiquitination.The N-terminal domain(NATD)is indispensable for p53 ubiquitination.We propose that SMN deficiency inactivates a novel ubiquitination pathway that is independent of the C-terminal lysine sites in p53 in human cells and Ser15 phosphorylation inhibits the ubiquitination pathway.