Signatures and Molecular Mechanisms of N6-Methyladenosine circRNAs in Human Rheumatoid Arthritis from Microarray Data
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ABSTRACT: We used the m6A-circRNA epitranscriptomic microarray to measure the m6A methylated quantities and levels of circRNAs in the synovial tissues of human rheumatoid arthritis and osteoarthritis.
Project description:Rheumatoid arthritis (RA), a chronic and systemic disease of unknown etiology, is characterized by hyperplasia of synovial cells, which ultimately lead to the destruction of cartilage and bone. To elucidate the molecular mechanisms that lead to RA, we analyzed synovial cells established from patient with RA by oligonucleotide microarrays. Gene expression profiles reveal a novel pathophysiologic function of RA synovial cells as a generator of oxidative stress, and a self-defense mechanism against self-generated oxidative stress. Experiment Overall Design: We isolated synovial cell culture from patients with rheumatoid arthritis and osteoarthritis. Fibroblast from patient with osteoarthritis was used for the reference.
Project description:N⁶-methyladenosine (m6A) and its reader, writer, and eraser (RWE) proteins assume crucial roles in regulating the splicing, stability, and translation of mRNA. To our knowledge, no systematic investigations have been conducted about the crosstalk between m6A and other modified nucleosides in RNA. Herein, we modified our recently established liquid chromatography-parallel-reaction monitoring (LC-PRM) method by incorporating stable isotope-labeled (SIL) peptides as internal or surrogate standards for profiling epitranscriptomic RWE proteins. We were able to detect reproducibly a total of 114 RWE proteins in HEK293T cells with the genes encoding m6A eraser proteins (i.e., ALKBH5, FTO) and the catalytic subunit of the major m6A writer complex (i.e., METTL3) being individually ablated. Notably, eight proteins were altered by more than 1.5-fold in the opposite directions in HEK293T cells depleted of METTL3 and ALKBH5. Analysis of published m6A mapping results revealed the presence of m6A in the corresponding mRNAs of four of these proteins. Together, we integrated SIL peptides into our LC-PRM method for quantifying epitranscriptomic RWE proteins, and our work revealed potential crosstalks between m6A and other epitranscriptomic modifications. Our modified LC-PRM method with the use of SIL peptides should be applicable for high-throughput profiling of epitranscriptomic RWE proteins in other cell types and in tissues.
Project description:Rheumatoid arthritis (RA) is a chronic, inflammatory joint disease of unknown etiology and pronounced inter-patient heterogeneity. To characterize RA at the molecular level and to uncover key pathomechanisms, we performed whole-genome gene expression analyses. Synovial tissues from rheumatoid arthritis patients were compared to those from osteoarthritis patients and to normal donors. Keywords: disease state analysis
Project description:Transcriptomics of distinct subpopulations of synovial fibroblasts from osteoarthritis and rheumatoid arthritis arthroplasty tissues.
Project description:Transcriptomics of distinct subpopulations of synovial fibroblasts from osteoarthritis and rheumatoid arthritis arthroplasty tissues.
Project description:Transcriptomics of distinct subpopulations of synovial fibroblasts from osteoarthritis and rheumatoid arthritis arthroplasty tissues.
Project description:mRNA expression levels in synovial fibroblasts in 6 rheumatoid arthritis patients versus 6 osteoarthritis patients. Experiment Overall Design: Synovial tissue was obtained from open joint replacement surgery or Experiment Overall Design: arthroscopic synovectomy. Patients with RA or OA (n = 6 each for gene expression analysis and further patients for validation experiments) were classified according to the ACR criteria.
Project description:Gene expression microarray was applied to discover novel rheumatoid arthritis (RA)-specific gene expressions by comparing the expression profiles of synovial membranes from patients with RA, osteoarthritis (OA) and ankylosing spondylitis (AS). We performed a gene expression microarray analysis of RA synovial membranes and simultaneously compared the expression profile with the profiles of AS and OA synovial membranes.
Project description:Gene expression microarray was applied to discover novel rheumatoid arthritis (RA)-specific gene expressions by comparing the expression profiles of synovial membranes from patients with RA, osteoarthritis (OA) and ankylosing spondylitis (AS). We performed a gene expression microarray analysis of RA synovial membranes and simultaneously compared the expression profile with the profiles of AS and OA synovial membranes. This study was undertaken to investigate the global gene expression profiles in synovial tissues from RA (n=10), OA (n=7), and AS patients (n=5). The Illumina HumanHT-12 v4 Expression BeadChip were used for a complete genome-wide transcript profiling.
Project description:N6-methyladenosine (m6A) is the most abundant internal messenger (mRNA) modification in mammalian mRNA. This modification is reversible and non-stoichiometric, which potentially adds an additional layer of variety and dynamic control of mRNA metabolism. The m6A-modified mRNA can be selectively recognized by the YTH family “reader” proteins. The preferential binding of m6A-containing mRNA by YTHDF2 is known to reduce the stability of the target transcripts; however, the exact effects of m6A on translation has yet to be elucidated. Here we show that another m6A reader protein, YTHDF1, promotes ribosome loading of its target transcripts. YTHDF1 forms a complex with translation initiation factors to elevate the translation efficiency of its bound mRNA. In a unified mechanism of translation control through m6A, the YTHDF2-mediated decay controls the lifetime of target transcripts; whereas, the YTHDF1-based translation promotion increases the translation efficiency to ensure effective protein production from relatively short-lived transcripts that are marked by m6A. PAR-CLIP and RIP was used to identify YTHDF1 binding sites followed by ribosome profling and RNA seq to assess the consequences of YTHDF1 siRNA knock-down