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

Abstract

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.

References

[1] Wasserman HM, et al. . Low bone mineral density and fractures are highly prevalent in pediatric patients with spinal muscular atrophy regardless of disease severity. Neuromuscul. Disord., 2017, 27: 331-337.

[2] Vai S, et al. . Bone and spinal muscular atrophy. Bone, 2015, 79: 116-120.

[3] Aponte Ribero V, et al. . Systematic literature review of the natural history of spinal muscular atrophy: motor function, scoliosis, and contractures. Neurology, 2023, 101: e2103-e2113.

[4] Nishio, H. et al. Spinal muscular atrophy: the past, present, and future of diagnosis and treatment. Int. J. Mol. Sci. 24, 11939 (2023).

[5] Lefebvre S, et al. . Identification and characterization of a spinal muscular atrophy-determining gene. Cell, 1995, 80: 155-165.

[6] Tisdale S, et al. . SMN is essential for the biogenesis of U7 small nuclear ribonucleoprotein and 3′-end formation of histone mRNAs. Cell Rep., 2013, 5: 1187-1195.

[7] Jangi M, et al. . SMN deficiency in severe models of spinal muscular atrophy causes widespread intron retention and DNA damage. Proc. Natl. Acad. Sci. USA, 2017, 114: E2347-E2356.

[8] Doktor TK, et al. . RNA-sequencing of a mouse-model of spinal muscular atrophy reveals tissue-wide changes in splicing of U12-dependent introns. Nucleic Acids Res., 2017, 45: 395-416.

[9] Zhang Z, et al. . SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell, 2008, 133: 585-600.

[10] Chaytow H, Huang YT, Gillingwater TH, Faller KME. The role of survival motor neuron protein (SMN) in protein homeostasis. Cell Mol. Life Sci., 2018, 75: 3877-3894.

[11] Liu Q, Fischer U, Wang F, Dreyfuss G. The spinal muscular atrophy disease gene product, SMN, and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins. Cell, 1997, 90: 1013-1021.

[12] Peter CJ, et al. . The COPI vesicle complex binds and moves with survival motor neuron within axons. Hum. Mol. Genet., 2011, 20: 1701-1711.

[13] Garcera A, Bahi N, Periyakaruppiah A, Arumugam S, Soler RM. Survival motor neuron protein reduction deregulates autophagy in spinal cord motoneurons in vitro. Cell Death Dis., 2013, 4e686

[14] Han KJ, et al. . Ubiquitin-specific protease 9x deubiquitinates and stabilizes the spinal muscular atrophy protein-survival motor neuron. J. Biol. Chem., 2012, 287: 43741-43752.

[15] Bürglen L, et al. . SMN gene deletion in variant of infantile spinal muscular atrophy. Lancet, 1995, 346: 316-317.

[16] Lorson CL, Hahnen E, Androphy EJ, Wirth B. A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc. Natl. Acad. Sci. USA, 1999, 96: 6307-6311.

[17] Wu X, et al. . A-44G transition in SMN2 intron 6 protects patients with spinal muscular atrophy. Hum. Mol. Genet, 2017, 26: 2768-2780.

[18] Monani UR, et al. . A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2. Hum. Mol. Genet, 1999, 8: 1177-1183.

[19] Hensel N, et al. . Altered bone development with impaired cartilage formation precedes neuromuscular symptoms in spinal muscular atrophy. Hum. Mol. Genet, 2020, 29: 2662-2673.

[20] Hann, S. H. et al. Depletion of SMN protein in mesenchymal progenitors impairs the development of bone and neuromuscular junction in spinal muscular atrophy. Elife12, RP92731 (2024).

[21] Long, J. T. et al. Hypertrophic chondrocytes serve as a reservoir for marrow-associated skeletal stem and progenitor cells, osteoblasts, and adipocytes during skeletal development. Elife11, e76932 (2022).

[22] Tare RS, Oreffo RO, Clarke NM, Roach HI. Pleiotrophin/Osteoblast-stimulating factor 1: dissecting its diverse functions in bone formation. J. Bone Min. Res., 2002, 17: 2009-2020.

[23] Zhang J, et al. . PALMD haploinsufficiency aggravates extracellular matrix remodeling in vascular smooth muscle cells and promotes calcification. Am. J. Physiol. Cell Physiol., 2024, 327: C1012-C1022.

[24] Hsieh-Li HM, et al. . A mouse model for spinal muscular atrophy. Nat. Genet., 2000, 24: 66-70.

[25] Brighton CT. The growth plate. Orthop. Clin. North Am., 1984, 15: 571-595.

[26] Sheng L, Rigo F, Bennett CF, Krainer AR, Hua Y. Comparison of the efficacy of MOE and PMO modifications of systemic antisense oligonucleotides in a severe SMA mouse model. Nucleic Acids Res, 2020, 48: 2853-2865.

[27] Rim, Y. A., Nam, Y. & Ju, J. H. The Role of Chondrocyte Hypertrophy and Senescence in Osteoarthritis Initiation and Progression. Int. J. Mol. Sci. 21, 2358 (2020).

[28] Laron Z. Insulin-a growth hormone. Arch. Physiol. Biochem, 2008, 114: 11-16.

[29] Lakatos P, et al. . Thyroid hormones, glucocorticoids, insulin, and bone. Handb. Exp. Pharm., 2020, 262: 93-120.

[30] Ryan JJ, Danish R, Gottlieb CA, Clarke MF. Cell cycle analysis of p53-induced cell death in murine erythroleukemia cells. Mol. Cell Biol., 1993, 13: 711-719. DOI:

[31] Long F, Ornitz DM. Development of the endochondral skeleton. Cold Spring Harb. Perspect. Biol., 2013, 5: a008334

[32] Chan, W. C. W., Tan, Z., To, M. K. T. & Chan, D. Regulation and Role of Transcription Factors in Osteogenesis. Int. J. Mol. Sci.22, 5445 (2021).

[33] Li DK, Tisdale S, Lotti F, Pellizzoni L. SMN control of RNP assembly: from post-transcriptional gene regulation to motor neuron disease. Semin. Cell Dev. Biol., 2014, 32: 22-29.

[34] Xiao Y, et al. . Splicing factor YBX1 regulates bone marrow stromal cell fate during aging. EMBO J., 2023, 42e111762

[35] Stark C, et al. . BioGRID: a general repository for interaction datasets. Nucleic Acids Res., 2006, 34: D535-D539.

[36] Kim EK, Choi EJ. SMN1 functions as a novel inhibitor for TRAF6-mediated NF-kappaB signaling. Biochim. Biophys. Acta Mol. Cell Res., 2017, 1864: 760-770.

[37] Brenke JK, et al. . Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity. J. Biol. Chem., 2018, 293: 13191-13203.

[38] Wishart TM, et al. . SMN deficiency disrupts brain development in a mouse model of severe spinal muscular atrophy. Hum. Mol. Genet., 2010, 19: 4216-4228.

[39] Liu H, Beauvais A, Baker AN, Tsilfidis C, Kothary R. Smn deficiency causes neuritogenesis and neurogenesis defects in the retinal neurons of a mouse model of spinal muscular atrophy. Dev. Neurobiol., 2011, 71: 153-169.

[40] Sheng L, et al. . Downregulation of Survivin contributes to cell-cycle arrest during postnatal cardiac development in a severe spinal muscular atrophy mouse model. Hum. Mol. Genet., 2018, 27: 486-498.

[41] Park J, et al. . Dual pathways to endochondral osteoblasts: a novel chondrocyte-derived osteoprogenitor cell identified in hypertrophic cartilage. Biol. Open, 2015, 4: 608-621.

[42] Zhou X, et al. . Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice. PLoS Genet., 2014, 10: e1004820

[43] Chen Z, Yue SX, Zhou G, Greenfield EM, Murakami S. ERK1 and ERK2 regulate chondrocyte terminal differentiation during endochondral bone formation. J. Bone Min. Res., 2015, 30: 765-774.

[44] Rudnik-Schoneborn S, et al. . Digital necroses and vascular thrombosis in severe spinal muscular atrophy. Muscle Nerve, 2010, 42: 144-147.

[45] Zhou H, et al. . A novel morpholino oligomer targeting ISS-N1 improves rescue of severe spinal muscular atrophy transgenic mice. Hum. Gene Ther., 2013, 24: 331-342.

[46] Somers E, et al. . Vascular defects and spinal cord hypoxia in spinal muscular atrophy. Ann. Neurol., 2016, 79: 217-230.

[47] Zhou, H. et al. Microvasculopathy in spinal muscular atrophy is driven by a reversible autonomous endothelial cell defect. J. Clin. Invest.132, e153430 (2022).

[48] White, A. L. & Bix, G. J. VEGFA isoforms as pro-angiogenic therapeutics for cerebrovascular diseases. Biomolecules13, 702 (2023).

[49] Shiying W, et al. . The different effects of VEGFA121 and VEGFA165 on regulating angiogenesis depend on phosphorylation sites of VEGFR2. Inflamm. Bowel Dis., 2017, 23: 603-616.

[50] Inada M, et al. . Maturational disturbance of chondrocytes in Cbfa1-deficient mice. Dev. Dyn., 1999, 214: 279-290.

[51] Qin X, et al. . Runx2 is essential for the transdifferentiation of chondrocytes into osteoblasts. PLoS Genet, 2020, 16: e1009169

[52] Lui JC, et al. . Persistent Sox9 expression in hypertrophic chondrocytes suppresses transdifferentiation into osteoblasts. Bone, 2019, 125: 169-177.

[53] Gubitz AK, Feng W, Dreyfuss G. The SMN complex. Exp. Cell Res., 2004, 296: 51-56.

[54] Nilsen TW. The spliceosome: the most complex macromolecular machine in the cell?. Bioessays, 2003, 25: 1147-1149.

[55] Will CL, Luhrmann R. Spliceosomal UsnRNP biogenesis, structure and function. Curr. Opin. Cell Biol., 2001, 13: 290-301.

[56] Gabanella F, et al. . Ribonucleoprotein assembly defects correlate with spinal muscular atrophy severity and preferentially affect a subset of spliceosomal snRNPs. PLoS One, 2007, 2: e921

[57] Eliseeva IA, Kim ER, Guryanov SG, Ovchinnikov LP, Lyabin DN. Y-box-binding protein 1 (YB-1) and its functions. Biochem. Mosc., 2011, 76: 1402-1433.

[58] Stojanovska, V. et al. YB-1 is altered in pregnancy-associated disorders and affects trophoblast in vitro properties via alternation of multiple molecular traits. Int. J. Mol. Sci.22, 7226 (2021).

[59] Gong H, et al. . Effect and mechanism of YB-1 knockdown on glioma cell growth, migration, and apoptosis. Acta Biochim. Biophys. Sin., 2020, 52: 168-179.

[60] Lyabin DN, Eliseeva IA, Ovchinnikov LP. YB-1 protein: functions and regulation. Wiley Interdiscip. Rev. RNA, 2014, 5: 95-110.

[61] Kumari P, et al. . An essential role for maternal control of Nodal signaling. Elife, 2013, 2e00683

[62] Hussain SA, Venkatesh T. YBX1/lncRNA SBF2-AS1 interaction regulates proliferation and tamoxifen sensitivity via PI3K/AKT/MTOR signaling in breast cancer cells. Mol. Biol. Rep., 2023, 50: 3413-3428.

[63] Xu J, et al. . CircRNA-SORE mediates sorafenib resistance in hepatocellular carcinoma by stabilizing YBX1. Sig.l Transduct. Target Ther., 2020, 5: 298

[64] Chen S, et al. . circNEIL3 inhibits tumor metastasis through recruiting the E3 ubiquitin ligase Nedd4L to degrade YBX1. Proc. Natl. Acad. Sci. USA, 2023, 120e2215132120

[65] Zou ZL, Sun MH, Yin WF, Yang L, Kong LY. Avicularin suppresses cartilage extracellular matrix degradation and inflammation via TRAF6/MAPK activation. Phytomedicine, 2021, 91153657

[66] Garcia, E. L. et al. Dysregulation of innate immune signaling in animal models of Spinal Muscular Atrophy. BMC Biol. 22, 94 (2024).

[67] Hua Y, et al. . Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model. Nature, 2011, 478: 123-126.

[68] Wilm M, et al. . Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry. Nature, 1996, 379: 466-469.

[69] Hao Y, et al. . Dictionary learning for integrative, multimodal and scalable single-cell analysis. Nat. Biotechnol., 2024, 42: 293-304.

[70] Hao Y, et al. . Integrated analysis of multimodal single-cell data. Cell, 2021, 184: 3573-3587 e3529.

[71] Stuart T, et al. . Comprehensive integration of single-cell data. Cell, 2019, 177: 1888-1902 e1821.

[72] Butler A, Hoffman P, Smibert P, Papalexi E, Satija R. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat. Biotechnol., 2018, 36: 411-420.

[73] Satija R, Farrell JA, Gennert D, Schier AF, Regev A. Spatial reconstruction of single-cell gene expression data. Nat. Biotechnol., 2015, 33: 495-502.

[74] Trapnell C, et al. . The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat. Biotechnol., 2014, 32: 381-386.

[75] Qiu X, et al. . Single-cell mRNA quantification and differential analysis with Census. Nat. Methods, 2017, 14: 309-315.

[76] Qiu X, et al. . Reversed graph embedding resolves complex single-cell trajectories. Nat. Methods, 2017, 14: 979-982.

[77] Cao J, et al. . The single-cell transcriptional landscape of mammalian organogenesis. Nature, 2019, 566: 496-502.

Funding

National Natural Science Foundation of China (National Science Foundation of China)(81902179)

China Postdoctoral Science Foundation(2020T130308)

Key Medical Subjects of Jiangsu Province(JSDW202223)

Natural Science Foundation of Jiangsu Province (Jiangsu Provincial Natural Science Foundation)(BK20221241)

Science and Technology Project of Suzhou (SKJY2021094) Gusu Talent Program (GSWS2022046)

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