SMN deficiency inhibits endochondral ossification via promoting TRAF6-induced ubiquitination degradation of YBX1 in spinal muscular atrophy

Survival of motor neuron (SMN) protein encoded by SMN1 gene, is the essential and ubiquitously expressed protein in all tissues. Prior studies demonstrated that SMN deficiency impaired bone development, but the underlying mechanism of abnormal endochondral ossification remains obscure. Here, we showed SMN is involved in hypertrophic chondrocytes differentiation through regulating RNA splicing and protein degradation via analyzing single cell RNA-sequencing data of hypertrophic chondrocytes. Of note, SMN loss induced dwarfism and delayed endochondral ossification in Smn1 depletion-severe spinal muscular atrophy (SMA) mouse model and Smn1 chondrocyte conditional knockdown mouse. Histological analysis revealed that SMN deficiency expanded the zone of hypertrophic chondrocytes in the growth plates, but delayed turnover from hypertrophic to ossification zone. Widespread changes in endochondral ossification related gene expression and alternative splicing profiles were identified via RNA sequencing of growth plate cartilages from SMA mice on postnatal day 4. Importantly, Mass spectrometry-based proteomics analysis elucidated Y-box-binding protein 1 (YBX1) as a vital SMN-binding factor, was decreased in SMA mice. YBX1 knockdown reproduced the aberrant gene expression and splicing changes observed in SMA growth plate cartilages. Comparing the binding proteins of SMN and YBX1 revealed TNF receptor-associated factor 6 (TRAF6), which promoted ubiquitination degradation of YBX1. By conditionally deleting Smn1 in chondrocytes of WT mice and overexpressing Smn1 in chondrocytes of SMA mice, we proved that SMN expression in chondrocytes is critical for hypertrophic chondrocyte-mediated endochondral ossification. Collectively, these results demonstrate that SMN deficiency contributes to rapid systemic bone dysplasia syndrome by promoting TRAF6-induced ubiquitination degradation of YBX1 in growth plate cartilages of SMA mice.