The RNA-Binding Protein HuR Regulates Splicing Programs in Osteocytes

Post-transcriptional gene regulation plays a crucial role in maintaining cellular homeostasis. Alternative splicing (AS) is one such post-transcriptional gene regulatory mechanisms. In this study we used RNA binding protein (RBP) motif enrichment analysis of RNA-seq data to identify enriched RBP positions around alternatively spliced exons in primary osteocytes cultured in high glucose conditions (HG). We identified the RNA-binding protein human antigen R (HuR) as a top candidate regulator of AS in our datasets. HuR mRNA levels were reduced under hyperglycemic conditions in both MLO-Y4 cells and mice long bones, while protein expression remained unchanged. We investigated the mechanistic role of HuR in osteocyte adaptation to high glucose using HuR knockdown (KD) in MLO-Y4 cells. HuR KD led to profound differential gene expression and differential mRNA splicing changes. Functional splicing analysis revealed enrichment in pathways related to stress response, translation, and insulin signaling. Mechanistic studies confirmed AS analysis; HuR KD cells showed decreased mitochondrial mass, impaired respiratory function, reduced ATP production, and increased sensitivity to oxidative stress under challenging conditions. HuR KD cells exhibited reduced viability specifically under HG but not osmotic stress conditions. Additionally, HuR KD displayed dysregulation in key translational signals, including activation of mTOR kinase, decreased MNK1 activity and decreased in eIF4E phosphorylation while preserving global protein output. Moreover, HuR KD cells exhibited reduced insulin sensitivity as evident by reduced AKT phosphorylation in insulin stimulated cells. These findings establish HuR as a central post-transcriptional regulator of osteocyte survival and metabolic adaptation under high glucose stress, with potential implications for hyperglycemic bone fragility.