Inherited disorders of cobalamin metabolism disrupt nucleocytoplasmic transport of mRNA through impaired methylation/phosphorylation of HuR. Mus musculus

Molecular mechanisms underlying the neurological disorders of inherited diseases of cobalamin metabolism remain obscure. Transcriptome data from a cell model with impaired cobalamin metabolism implicated dysregulated RNA metabolism and endoplasmic reticulum stress. Evidence from the cell model, Cd320 knockout mice, and fibroblasts with inborn errors of cobalamin metabolism indeed supported the findings of transcriptome. These models allowed the demonstration of RNA binding proteins (RBP) mislocalized subcellularly due to failed nucleocytoplasmic shuttling. In the case of HuR, decreased RBP interaction with nuclear receptor CRM1/exportin accounted for the failure, and HuR hypomethylation and dephosphorylation were the causes of this. Our evidence further indicated that decreased SAM and CARM1 level and increased PP2A expression were partly responsible for the post translational modifications of HuR. The consequent HuR mislocalization to the nucleus in turn reduced the expression of SIRT1 and other genes involved in brain development, neuroplasticity, myelin formation, and brain aging. This mislocalization was reversible upon treatment with siPpp2ca, cobalamin, S-adenosylmethionine, or PP2A inhibitor okadaic acid. In conclusion, our data highlight the key roles of cobalamin metabolism on HuR-mediated mRNA stability, which when disrupted produce genomic changes consistent with the effects of inborn errors of cobalamin on brain development, neuroplasticity and myelin formation. Overall design: The N1E-115 cells are neuroblastoma cells chosen here as a neuronal model. Chimeric proteins were produced by the fusion of either N-terminal (TO) or C-terminal (OT) of transcoblamin (TCN2) to oleosin. Expressed TO fusion protein has high binding affinity toward Cbl whereas OT fusion protein has no detectable Cbl binding activity. Cells were cultured in a proliferation or a differentiation medium. Biological replicates were used to obtain samples from proliferated control (WT-p ; n=4), proliferated TO (TO-p ; n=4), proliferated OT (OT-p ; n=4), differentiated control (WT-d ; n=4), differentiated TO (TO-d ; n=4) and differentiated OT cells (OT-d ; n=4). WT samples represent non-transfected N1E-115 cells. TO and OT cells were transfected with the TO and OT chimeric constructs, respectively.