DDX1 methylation mediated MATR3 splicing regulates intervertebral disc degeneration by initiating chromatin reprogramming

Article https://doi.org/10.1038/s41467-025-61486-7 DDX1 methylation mediated MATR3 splicing regulates intervertebral disc degeneration by initiating chromatin reprogramming Received: 2 September 2024 Check for updates 1234567890():,; 1234567890():,; Accepted: 23 June 2025 Dingchao Zhu1,2, Huaizhen Liang1,2, Bide Tong1,2, Zhi Du1,2, Gaocai Li 1, Weifeng Zhang1, Di Wu1, Xingyu Zhou1, Jie Lei1, Xiaoguang Zhang1, Liang Ma1, Bingjin Wang1, Xiaobo Feng 1, Kun Wang1, Lei Tan 1, Yu Song 1 & Cao Yang 1 Low back pain (LBP), primarily driven by intervertebral disc degeneration (IVDD), has become a core challenge in public health. DDX1, an RNA-binding protein, plays key roles in RNA metabolism but its function in IVDD remains unclear. We identify DDX1 as a substrate of methyltransferase EZH2, which methylates DDX1 at lysine 234 (K234), promoting IVDD in vitro and in vivo. EZH2 inhibition restores matrix homeostasis in nucleus pulposus (NP) cells and slows IVDD progression. Methylation at DDX1 K234 disrupts its interaction with splicing factors and RNA targets, promoting exon 14 skipping in MATR3. This truncated MATR3 disrupts nuclear architecture, increases chromatin accessibility, and activates signaling pathways such as Wnt, leading to NP cell senescence and apoptosis. Notably, delivery of MATR3-L-overexpressing mRNA via cationic lipid nanoparticles reduces NP cell degeneration and significantly alleviates IVDD, offering important insights into IVDD pathogenesis and potential therapeutic strategies. Low back pain (LBP) is a significant health burden in the elderly and the second leading cause of medical consultations in industrialized countries1. As the most common and burdensome musculoskeletal condition, an estimated 40% of LBP cases are linked to intervertebral disc degeneration (IVDD), a widespread and progressive ageassociated pathology2. Extensive research has explored the pathogenesis of IVDD, revealing a strong association between aging, apoptosis, and disc degeneration3. In addition, IVDD manifests as a proaging and apoptotic intervertebral disc (IVD) microenvironment, characterized by hypoxia, inflammation, and disturbances in metabolic and nutritional factors4. Notably, epigenomic alterations influencing chromatin architecture are key regulators of cell fate and aging. In nucleus pulposus (NP) cells, such changes may exert long-term effects across cell generations and uncover previously unrecognized aspects of the aging process5,6. However, the renewal and differentiation of NP cells in IVD tissues during degeneration are still unclear, and the related epigenomic regulatory mechanisms are still worth exploring. Alternative splicing (AS) refers to the process of generating mRNA splice isoforms through different splicing modes from one mRNA precursor, adding complexity to gene regulation by influencing protein isoform diversity, mRNA stability, and translation efficiency7. When this event occurs abnormally, different proteomes will be produced through different splicing methods, which will further affect normal cell physiological functions and may lead to pathological changes8. Previous studies have shown that senescence relies on a defined, alternatively spliced transcriptome, but this has not been comprehensively defined on a transcriptome scale, and the molecular mechanisms underlying this program are poorly understood9. Increasing evidence suggests that splicing is co-transcriptional and is directly affected by the epigenetic landscape and dynamics of the underlying transcription machinery at these sites; on the other hand, 1 Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. 2These authors e-mail: ; contributed equally: Dingchao Zhu, Huaizhen Liang, Bide Tong, Zhi Du. Nature Communications | (2025)16:6153 1 Article different mRNA transcripts generated by AS may also play a regulatory role in chromatin reprogramming10. However, the molecular couplers linking chromatin reprogramming and AS during IVDD remain unknown. RNA-binding proteins (RBP) stand out as pivotal posttranscriptional regulators due to their capacity to broadly influence the activity of multiple target RNA and their involvement in cellular senescence, where they orchestrate the expression of apoptotic genes, growth factors, and cell cycle proteins11,12. Even in minute quantities, RBP can elicit significant changes in gene expression, often attributed to aberrant patterns of post-translational modifications (PTM), among which lysine methylation has emerged as a key player13,14, impacting various biological functions such as protein-protein interactions, stability, subcellular localization, and transcription15. So far, large-scale screens have gained insight into the intricate network of interactions between RBP and their targets, laying the groundwork for a systematic exploration of post-transcriptional regulatory mechanisms, studies on RBP-mediated regulation of AS exemplify this endeavor16. Therefore, an analysis of NP cells phenotypes from the perspective of PTMmediated RBP structure and status promises to unveil previously uncharacterized post-transcriptional regulatory pathways underlying IVDD and may identify candidate molecules bridging the epigenome and AS in this pathological process. In this study, we demonstrated that EZH2-mediated DDX1 lysine methylation promotes MATR3 exon 14 skipping and facilitates NP cells senescence and apoptosis during the progression of IVDD. Functional gain and loss experiments suggest that EZH2 deficiency rescues the diminished synthesis metabolism and enhanced degradation metabolism in NP cells, thereby mitigating the IVDD progression. Mechanistically, the increased expression of EZH2 enhances the binding of DDX1, leading to the mono-methylation of lysine residue 234 on DDX1 (DDX1 K234me1). Methylated DDX1 weakens its binding to the exonintron region near MATR3 splicing sites and reduces recruitment of splicing factors. Consequently, MATR3 exon 14 skipping results in the formation of MATR3 short isoforms (MATR3-S), which genetically induce whole-genome chromatin reprogramming and aberrant activation of senescence and apoptosis signaling pathways such as Wnt and MAPK in NP cells. Importantly, a strategy based on engineered cationic lipid nanoparticles (LNPs) for the delivery of MATR3-L overexpressed mRNA drug system can effectively alleviate reduced anabolism, enhanced catabolism, as well as senescence in NP cells, and significantly alleviate IVDD progression. Results K234 methylation of DDX1 is increased in NP cells during IVDD To identify key non-histone proteins regulated by lysine methylation17, cells from non-degenerative and degenerative NP tissues, obtained from individuals with lumbar fractures, idiopathic scoliosis, or IVDD, were lysed and incubated with a pan-lysine methylation-specific antibody. LC-MS/MS was used to analyze compounds. (Fig. 1A). NP tissue degeneration was (...truncated)