Deciphering the regulatory programs of RNA binding proteins in rheumatoid arthritis through single-cell transcriptome analysis

Abstract

Single-cell RNA sequencing (scRNA-seq) data from published datasets were obtained to investigate the expression and dysregulation of RNA-binding proteins (RBPs), which are critical for alternative mRNA splicing and translational control in rheumatoid arthritis (RA). How RBP regulation differs between RA and osteoarthritis (OA) was examined using RBP for single-cell sub-clustering. Quantitative polymerase chain reactions (PCRs) were performed to confirm differentially expressed RBPs in RA fibroblast-like synoviocytes (FLSs) and OA-FLSs, as well as in mice with collagen-induced arthritis (CIA) and control mice. Additionally, bulk RNA-seq data were collected and RBP-alternative splicing event (ASE) co-expression analyses were performed to reveal the potential regulatory role of RA-related RBPs on ASEs. Significant variations in relative proportions of cell subtypes were demonstrations between RA and OA with downregulated RBPs outnumbering upregulated RBPs in each cell type and showing high specificity for particular subsets. One hundred five upregulated and 133 downregulated RBPs were identified in fibroblasts. Y-Box binding protein 3 (YBX3) and splicing factor 3b subunit 6 (SF3B6) were confirmed to be upregulated in RA-FLS and CIA mice, while eukaryotic translation initiation factor 4A1 (EIF4A1) and U2 small nuclear RNA auxiliary factor 1 (U2AF1) were downregulated in RA-FLS. The RA group displayed stronger cell type interactions compared to the OA group with enhanced signaling pathways, such as fibronectin 1-cluster of differentiation 44 (FN1-CD44) and C-X-C motif chemokine ligand 12-C-X-C motif chemokine receptor 4 (CXCL12-CXCR4). Furthermore, three upregulated genes (spectrin repeat containing nuclear envelope protein 2 [SYNE2], S100 calcium binding protein A9 [S100A9], and interferon induced protein with tetratricopeptide repeats 3 [IFIT3]) and four downregulated genes (ribonuclease 1 [RNASE1], granulin [GRN], FN1, and sorbin and SH3 domain containing 2 [SORBS2]) were co-expressed in RA-associated RBPs and ASEs. These findings suggest that dysregulation of RBPs may contribute to the development of RA and provide potential targets for therapeutic interventions.

Keywords

scRNA-seq / RNA binding proteins / rheumatoid arthritis / cell-to-cell communication / fibroblasts

References

[1] Smolen JS, Aletaha D, McInnes IB: Rheumatoid Arthritis. Lancet (London, England) 2016, 388: 2023-2038.

[2] Radu AF, Bungau SG: Management of Rheumatoid Arthritis: An Overview. Cells 2021, 10: 2857.

[3] Orr C, Vieira-Sousa E, Boyle DL, Buch MH, Buckley CD, Cañete JD, et al.: Synovial Tissue Research: A State-of-the-Art Review. Nature Reviews Rheumatology 2017, 13: 463-475.

[4] Glisovic T, Bachorik JL, Yong J, Dreyfuss G: RNA-binding Proteins and Post-transcriptional Gene Regulation. FEBS Letters 2008, 582: 1977-1986.

[5] Meyer A, Zack SR, Nijim W, Burgos A, Patel V, Zanotti B, et al.: Metabolic Reprogramming by Syntenin-1 Directs RA FLS and Endothelial Cell-mediated Inflammation and Angiogenesis. Cellular and Molecular Immunology 2024, 21: 33-46.

[6] Luo J, Yao Z, Ye C, Liu Y: Genome-wide Analysis of RNA-binding Protein Co-expression with Alternative Splicing Events in Acute Respiratory Distress Syndrome Following Hematopoietic Stem Cell Transplantation. Medicine (Baltimore) 2023, 102: e34599.

[7] Gebauer F, Schwarzl T, Valcárcel J, Hentze MW: RNA-binding Proteins in Human Genetic Disease. Nature Reviews Genetics 2021, 22: 185-198.

[8] Hashimoto S, Kishimoto T: Roles of RNA-binding Proteins in Immune Diseases and Cancer. Seminars in Cancer Biology 2022, 86: 310-324.

[9] Jeltsch KM, Heissmeyer V: Regulation of T Cell Signaling and Autoimmunity by RNA-binding Proteins. Current Opinion in Immunology 2016, 39: 127-135.

[10] Guan Y, Zhang Y, Zhao X, Wang Y: Comprehensive Analysis Revealed the Immunoinflammatory Targets of Rheumatoid Arthritis based on Intestinal Flora, miRNA, Transcription Factors, and RNA-binding Proteins Databases, GSEA and GSVA Pathway Observations, and Immunoinfiltration Typing. Hereditas 2024, 161: 6.

[11] Yoshinaga M, Takeuchi O: RNA Binding Proteins in the Control of Autoimmune Diseases. Immunological Medicine 2019, 42: 53-64.

[12] Yoon DS, Lee KM, Choi Y, Ko EA, Lee NH, Cho S, et al.: Correction: TLR4 Downregulation by the RNA-binding Protein PUM1 Alleviates Cellular Aging and Osteoarthritis. Cell Death Differentiation 2022, 29: 1077.

[13] Arandel L, Matloka M, Klein AF, Rau F, Sureau A, Ney M, et al.: Reversal of RNA Toxicity in Myotonic Dystrophy via a Decoy RNA-binding Protein with High Affinity for Expanded CUG Repeats. Nature Biomedical Engineering 2022, 6: 207-220.

[14] Zhou B, Liu J, Kang R, Klionsky DJ, Kroemer G, Tang D: Ferroptosis is a Type of Autophagy-dependent Cell Death. Seminars in Cancer Biology 2020, 66: 89-100.

[15] Feldstein O, Ben-Hamo R, Bashari D, Efroni S, Ginsberg D: RBM38 is a Direct Transcriptional Target of E2F1 that Limits E2F1-induced Proliferation. Molecular Cancer Research 2012, 10: 1169-1177.

[16] Chin K, DeVries S, Fridlyand J, Spellman PT, Roydasgupta R, Kuo WL, et al.: Genomic Andtranscriptional Aberrations Linked to Breast Cancer Pathophysiologies. Cancer Cell 2006, 10: 529-541.

[17] Li J, Yu T, Sun J, Ma M, Zheng Z, Kang W, et al.: Comprehensive Integration of Single-cell RNA and Transcriptome RNA Sequencing to Establish a Pyroptosis-related Signature for Improving Prognostic Prediction of Gastric Cancer. Computational and Structural Biotechnology Journal 2024, 23: 990-1004.

[18] Potter SS: Single-cell RNA Sequencing for the Study of Development, Physiology and Disease. Nature Reviews Nephrology 2018, 14: 479-492.

[19] Seumois G, Vijayanand P: Single-cell Analysis to Understand the Diversity of Immune Cell Types that Drive Disease Pathogenesis. Journal of Allergy and Clinical Immunology 2019, 144: 1150-1153.

[20] Argyriou A, Wadsworth MH, Lendvai A, Christensen SM, Hensvold AH, Gerstner C, et al.: Single Cell Sequencing Identifies Clonally Expanded Synovial CD4(+) T(PH) Cells Expressing GPR56 in Rheumatoid Arthritis. Nature Communications 2022, 13: 4046.

[21] Armaka M, Konstantopoulos D, Tzaferis C, Lavigne MD, Sakkou M, Liakos A, et al.: Single-cell Multimodal Analysis Identifies Common Regulatory Programs in Synovial Fibroblasts of Rheumatoid Arthritis Patients and Modeled TNF-driven Arthritis. Genome Medicine 2022, 14: 78.

[22] Cai S, Ming B, Ye C, Lin S, Hu P, Tang J, et al.: Similar Transition Processes in Synovial Fibroblasts from Rheumatoid Arthritis and Osteoarthritis: A Single-Cell Study. Journal of Immunology Research 2019, 2019: 4080735.

[23] Butler A, Hoffman P, Smibert P, Papalexi E, Satija R: Integrating Single-cell Transcriptomic Data Across Different Conditions, Technologies, and Species. Nature Biotechnology 2018, 36: 411-420.

[24] Ianevski A, Giri AK, Aittokallio T: Fully-automated and Ultra-fast Cell-type Identification Using Specific Marker Combinations from Single-cell Transcriptomic Data. Nature Communications 2022, 13: 1246.

[25] Gagnon J, Pi L, Ryals M, Wan Q, Hu W, Ouyang Z, et al.: Recommendations of scRNA-seq Differential Gene Expression Analysis Based on Comprehensive Benchmarking. Life (Basel) 2022, 12: 850.

[26] Castello A, Fischer B, Frese CK, Horos R, Alleaume AM, Foehr S, et al.: Comprehensive Identification of RNA-Binding Domains in Human Cells. Molecular Cell 2016, 63: 696-710.

[27] He S, Valkov E, Cheloufi S, Murn J: The Nexus Between RNA-binding Proteins and their Effectors. Nature Reviews Genetics 2023, 24: 276-294.

[28] Lin W, Wang J, Ge J, Zhou R, Hu Y, Xiao L, et al.: The Activity of Cuproptosis Pathway Calculated by AUCell Algorithm was Employed to Construct Cuproptosis Landscape in Lung Adenocarcinoma. Discover Oncology 2023, 14: 135.

[29] Xie C, Mao X, Huang J, Ding Y, Wu J, Dong S, et al.: KOBAS 2.0: A Web Server for Annotation and Identification of Enriched Pathways and Diseases. Nucleic Acids Research 2011, 39: W316-W322.

[30] Jin S, Guerrero-Juarez CF, Zhang L, Chang I, Ramos R, Kuan CH, et al.: Inference and Analysis of Cell-cell Communication Using CellChat. Nature Communications 2021, 12: 1088.

[31] Kim D, Langmead B, Salzberg SL: HISAT: A Fast Spliced Aligner with Low Memory Requirements. Nature Methods 2015, 12: 357-360.

[32] Elahimanesh M, Najafi M: Differentially Expressed Genes of RNA-seq Data are Suggested on the Intersections of Normalization Techniques. Biochemistry and Biophysics Reports 2023, 37: 101618.

[33] Cheng C, Liu L, Bao Y, Yi J, Quan W, Xue Y, et al.: SUVA: Splicing Site Usage Variation Analysis from RNA-seq Data Reveals Highly Conserved Complex Splicing Biomarkers in Liver Cancer. RNA Biology 2021, 18: 157-171.

[34] Phipson B, Sim CB, Porrello ER, Hewitt AW, Powell J, Oshlack A: Propeller: Testing for Differences in Cell Type Proportions in Single Cell Data. Bioinformatics (Oxford, England) 2022, 38: 4720-4726.

[35] Lin D, Lai W, Zheng N, Luo H, Chen X, Que W, et al.: Novel Mechanistic Study of HDAC6 Regulation of Rheumatoid Arthritis via CMA: Exploring Potential Therapeutic Targets. Frontiers in Pharmacology 2024, 15: 1383663.

[36] Yang X, Chen B, Zhang M, Xu S, Shuai Z: Tristetraprolin Gene Single-nucleotide Polymorphisms and mRNA Level in Patients with Rheumatoid Arthritis. Frontiers in Pharmacology 2021, 12: 728015.

[37] Shi JX, Li JS, Hu R, Zhao XC, Liang CC, Li XM, et al.: CNOT1 is Involved in TTP-mediated ICAM-1 and IL-8 mRNA Decay. Molecular Medicine Reports 2018, 18: 2321-2327.

[38] Liu Y, Wei W, Wang Y, Wan C, Bai Y, Sun X, et al.: TNF-α/Calreticulin Dual Signaling Induced NLRP3 Inflammasome Activation Associated with HuR Nucleocytoplasmic Shuttling in Rheumatoid Arthritis. Inflammation Research: Official Journal of the European Histamine Research Society … [et al.] 2019, 68: 597-611.

[39] Van Aelst LN, Voss S, Carai P, Van Leeuwen R, Vanhoutte D, Sanders-van Wijk S, et al.: Osteoglycin Prevents Cardiac Dilatation and Dysfunction after Myocardial Infarction Through Infarct Collagen Strengthening. Circulation Research 2015, 116: 425-436.

[40] Funderburgh JL, Corpuz LM, Roth MR, Funderburgh ML, Tasheva ES, Conrad GW: Mimecan, the 25-kDa Corneal Keratan Sulfate Proteoglycan, is a Product of the Gene Producing Osteoglycin. The Journal of Biological Chemistry 1997, 272: 28089-28095.

[41] Dunlevy JR, Beales MP, Berryhill BL, Cornuet PK, Hassell JR: Expression of the Keratan Sulfate Proteoglycans Lumican, Keratocan and Osteoglycin/Mimecan During Chick Corneal Development. Experimental Eye Research 2000, 70: 349-362.

[42] Starup-Linde J, Viggers R, Handberg A: Osteoglycin and Bone - A Systematic Review. Current Osteoporosis Reports 2019, 17: 250-255.

[43] Rienks M, Papageorgiou A, Wouters K, Verhesen W, Leeuwen RV, Carai P, et al.: A Novel 72-kDa Leukocyte-derived Osteoglycin Enhances the Activation of Toll-like Receptor 4 and Exacerbates Cardiac Inflammation During Viral Myocarditis. Cellular and Molecular Life Sciences 2017, 74: 1511-1525.

[44] Lomnytska MI, Becker S, Hellman K, Hellström AC, Souchelnytskyi S, Mints M, et al.: Diagnostic Protein Marker Patterns in Squamous Cervical Cancer. Proteomics. Clinical Applications 2010, 4: 17-31.

[45] Xu T, Zhang R, Dong M, Zhang Z, Li H, Zhan C, et al.: Osteoglycin (OGN) Inhibits Cell Proliferation and Invasiveness in Breast Cancer via PI3K/Akt/mTOR Signaling Pathway. OncoTargets and Therapy 2019, 12: 10639-10650.

[46] Ge G, Seo NS, Liang X, Hopkins DR, Höök M, Greenspan DS: Bone Morphogenetic Protein-1/Tolloid-related Metalloproteinases Process Osteoglycin and Enhance its Ability to Regulate Collagen Fibrillogenesis. The Journal of Biological Chemistry 2004, 279: 41626-41633.

[47] Wu S, Romfo CM, Nilsen TW, Green MR: Functional Recognition of the 3′ Splice Site AG by the Splicing Factor U2AF35. Nature 1999, 402: 832-835.

[48] Nie M, Balda MS, Matter K: Stress- and Rho-activated ZO-1-associated Nucleic Acid Binding Protein Binding to p21 mRNA Mediates Stabilization, Translation, and Cell Survival. Proceedings of the National Academy of Sciences of the United States of America 2012, 109: 10897-10902.

[49] Cooke A, Schwarzl T, Huppertz I, Kramer G, Mantas P, Alleaume AM, et al.: The RNA-Binding Protein YBX3 Controls Amino Acid Levels by Regulating SLC mRNA Abundance. Cell Reports 2019, 27: 3097-3106.e5.

[50] Schmidt T, Dabrowska A, Waldron JA, Hodge K, Koulouras G, Gabrielsen M, et al.: eIF4A1-dependent mRNAs Employ Purine-rich 5′UTR Sequences to Activate Localised eIF4A1-unwinding Through eIF4A1-multimerisation to Facilitate Translation. Nucleic Acids Research 2023, 51: 1859-1879.

[51] Qin H, Ni H, Liu Y, Yuan Y, Xi T, Li X, et al.: RNA-binding Proteins in Tumor Progression. Journal of Hematology and Oncology 2020, 13: 90.

[52] Rybak-Wolf A, Plass M: RNA Dynamics in Alzheimer’s Disease. Molecules (Basel, Switzerland) 2021, 26: 5113.

[53] Rau F, Freyermuth F, Fugier C, Villemin JP, Fischer MC, Jost B, et al.: Misregulation of miR-1 Processing is Associated with Heart Defects in Myotonic Dystrophy. Nature Structural and Molecular Biology 2011, 18: 840-845.

[54] Charizanis K, Lee KY, Batra R, Goodwin M, Zhang C, Yuan Y, et al.: Muscleblind-like 2-mediated Alternative Splicing in the Developing Brain and Dysregulation in Myotonic Dystrophy. Neuron 2012, 75: 437-450.

[55] Wang ET, Cody NA, Jog S, Biancolella M, Wang TT, Treacy DJ, et al.: Transcriptome-wide Regulation of Pre-mRNA Splicing and mRNA Localization by Muscleblind Proteins. Cell 2012, 150: 710-724.

[56] Graubert TA, Shen D, Ding L, Okeyo-Owuor T, Lunn CL, Shao J, et al.: Recurrent Mutations in the U2AF1 Splicing Factor in Myelodysplastic Syndromes. Nature Genetics 2011, 44: 53-57.

[57] Yip BH, Steeples V, Repapi E, Armstrong RN, Llorian M, Roy S, et al.: The U2AF1S34F Mutation Induces Lineage-specific Splicing Alterations in Myelodysplastic Syndromes. The Journal of Clinical Investigation 2017, 127: 2206-2221.

[58] Chen C, Zhou P, Zhang Z, Liu Y: U2AF1 Mutation Connects DNA Damage to the Alternative Splicing of RAD51 in Lung Adenocarcinomas. Clinical and Experimental Pharmacology and Physiology 2022, 49: 740-747.

[59] Zhang Z, Will CL, Bertram K, Dybkov O, Hartmuth K, Agafonov DE, et al.: Molecular Architecture of the Human 17S U2 snRNP. Nature 2020, 583: 310-313.

[60] Will CL, Schneider C, MacMillan AM, Katopodis NF, Neubauer G, Wilm M, et al.: A Novel U2 and U11/U12 snRNP Protein that Associates with the Pre-mRNA Branch Site. The EMBO Journal 2001, 20: 4536-4546.

[61] Perea W, Schroeder KT, Bryant AN, Greenbaum NL: Interaction Between the Spliceosomal Pre-mRNA Branch Site and U2 snRNP Protein p14. Biochemistry 2016, 55: 629-632.

[62] Schellenberg MJ, Dul EL, MacMillan AM: Structural Model of the p14/SF3b155 Branch Duplex Complex. RNA (New York, NY) 2011, 17: 155-165.

[63] Ni Y, Zhang H, Xian Q, Qin W, Su H, Wang L, et al.: RfxCas13d-mediated Inhibition of Circ1647 Alleviates Renal Fibrosis via PI3K/AKT Signaling Pathway . Renal Failure 2024, 46: 2331612.

[64] Tang F, Gong H, Ke T, Yang W, Yang Y, Liu Z: USP7 Promotes TGF-β1 Signaling by De-Ubiquitinating Smad2/Smad3 in Pulmonary Fibrosis. Discovery Medicine 2024, 36: 1616-1626.

[65] Yang J, Zhang Y, Liang J, Yang X, Liu L, Zhao H: Fibronectin-1 is a Dominant Mechanism for Rheumatoid Arthritis via the Mediation of Synovial Fibroblasts Activity. Frontiers in Cell and Development Biology 2022, 10: 1010114.

[66] Xu J, Zhang MY, Jiao W, Hu CQ, Wu DB, Yu JH, et al.: Identification of Candidate Genes Related to Synovial Macrophages in Rheumatoid Arthritis by Bioinformatics Analysis. International Journal of General Medicine 2021, 14: 7687-7697.

[67] Peng L, Zhu N, Mao J, Huang L, Yang Y, Zhou Z, et al.: Expression Levels of CXCR4 and CXCL12 in Patients with Rheumatoid Arthritis and its Correlation with Disease Activity. Experimental and Therapeutic Medicine 2020, 20: 1925-1934.

[68] Sucur A, Jajic Z, Artukovic M, Matijasevic MI, Anic B, Flegar D, et al.: Chemokine Signals are Crucial for Enhanced Homing and Differentiation of Circulating Osteoclast Progenitor Cells. Arthritis Research and Therapy 2017, 19: 142.

[69] Inoue K, Yuasa H: Molecular Basis for Pharmacokinetics and Pharmacodynamics of Methotrexate in Rheumatoid Arthritis Therapy. Drug Metabolism and Pharmacokinetics 2014, 29: 12-19.

[70] Farrugia M, Baron B: The Role of TNF-α in Rheumatoid Arthritis: A Focus on Regulatory T Cells. Journal of Clinical and Translational Research 2016, 2: 84-90.

[71] Zhou SY, Shu C, Korth-Bradley J, Raible D, Palmisano M, Wadjula J, et al.: Integrated Population Pharmacokinetics of Etanercept in Healthy Subjects and in Patients with Rheumatoid Arthritis and Ankylosing Spondylitis. Journal of Clinical Pharmacology 2011, 51: 864-875.

[72] Wong M, Ziring D, Korin Y, Desai S, Kim S, Lin J, et al.: TNF-Alpha Blockade in Human Diseases: Mechanisms and Future Directions. Clinical Immunology (Orlando, Fla) 2008, 126: 121-136.

[73] Klotz U, Teml A, Schwab M: Clinical Pharmacokinetics and use of Infliximab. Clinical Pharmacokinetics 2007, 46: 645-660.

[74] Abdallah H, Hsu JC, Lu P, Fettner S, Zhang X, Douglass W, et al.: Pharmacokinetic and Pharmacodynamic Analysis of Subcutaneous Tocilizumab in Patients with Rheumatoid Arthritis From 2 Randomized, Controlled Trials: SUMMACTA and BREVACTA. Journal of Clinical Pharmacology 2017, 57: 459-468.

[75] Weisman MH, Durez P, Hallegua D, Aranda R, Becker JC, Nuamah I, et al: Reduction of Inflammatory Biomarker Response by Abatacept in Treatment of Rheumatoid Arthritis. The Journal of Rheumatology 2006, 33: 2162-2166.

[76] Edwards JC, Szczepanski L, Szechinski J, Filipowicz-Sosnowska A, Emery P, Close DR, et al.: Efficacy of B-Cell-Targeted Therapy with Rituximab in Patients with Rheumatoid Arthritis. The New England Journal of Medicine 2004, 350: 2572-2581.

[77] Fleischmann RM, Schechtman J, Bennett R, Handel ML, Burmester GR, Tesser J, et al.: Anakinra, a Recombinant Human Interleukin-1 Receptor Antagonist (r-metHuIL-1ra), in Patients with Rheumatoid Arthritis: A Large, International, Multicenter, Placebo-Controlled Trial. Arthritis and Rheumatism 2003, 48: 927-934.

[78] Khodadust F, Philippon EML, Steinz MM, van Hamburg JP, van Meerloo J, van Beijnum JR, et al.: Unveiling the Anti-Angiogenic Potential of Small-Molecule (Kinase) Inhibitors for Application in Rheumatoid Arthritis. Cells 2025, 14: 102.

[79] Kuriya B, Cohen MD, Keystone E: Baricitinib in Rheumatoid Arthritis: Evidence-to-Date and Clinical Potential. Therapeutic Advances in Musculoskeletal Disease 2017, 9: 37-44.

[80] Klünder B, Mittapalli RK, Mohamed MF, Friedel A, Noertersheuser P, Othman AA: Population Pharmacokinetics of Upadacitinib Using the Immediate-Release and Extended-Release Formulations in Healthy Subjects and Subjects with Rheumatoid Arthritis: Analyses of Phase I-III Clinical Trials. Clinical Pharmacokinetics 2019, 58: 1045-1058.

[81] Huizinga TW, Fleischmann RM, Jasson M, Radin AR, van Adelsberg J, Fiore S, et al.: Sarilumab, a Fully Human Monoclonal Antibody against IL-6Rα in Patients with Rheumatoid Arthritis and an Inadequate Response to Methotrexate: Efficacy and Safety Results from the Randomised SARIL-RA-MOBILITY Part A Trial. Annals of the Rheumatic Diseases 2014, 73: 1626-1634.