Rabbit Anti-Human YTHDF2 (a.a. 289-348) Polyclonal antibody

N-6-Methyladenosine (m(6)A) in messenger RNA (mRNA) regulates its stability, splicing, and translation efficiency. Here, we explored how the expression levels of small GTPase proteins are regulated by m(6)A modulators. We employed a high-throughput scheduled multiple-reaction monitoring (MRM)-based targeted proteomic approach to quantify systemically the changes in expression of small GTPase proteins in cells upon genetic ablation of METTL3 (the catalytic subunit of the major m(6)A methyltransferase complex), m(6)A demethylases (ALKBHS and FTO), or m(6)A reader proteins (YTHDF1, YTHDF2, and YTHDF3). Depletions of METTL3 and ALKBHS resulted in substantially diminished and augmented expression, respectively, of a subset of small GTPase proteins, including RHOB and RHOC. Our results also revealed that the stability of RHOB mRNA is significantly increased in cells depleted of METTL3, suggesting an m(6)A-elicited destabilization of this mRNA. Those small GTPases that are targeted by METTL3 and/or ALKBHS also displayed higher discrepancies between protein and mRNA expression in paired primary/metastatic melanoma or colorectal cancer cells than those that are not. Together, this is the first comprehensive analysis of the alterations in small GTPase proteome regulated by epitranscriptomic modulators of m(6)A, and our study suggests the potential of an alternative therapeutic approach to target the currently "undruggable" small GTPases.

Background: Metastasis is the leading cause of death in patients with osteosarcoma. Some of these patients fail to respond to chemotherapy and die of metastasis within a short period. Therefore, it is important to identify novel biomarkers to improve the diagnosis and treatment of osteosarcoma. TRIM7 is a member of the tripartite motif (TRIM) family protein that is involved in various pathological conditions including cancer; however, its role in osteosarcoma remains elusive. Methods: Cell proliferation, invasion and migration were measured by CCK-8 and Transwell. Immunoprecipitation and mass spectrometry analysis were used to identify candidate proteins associated with TRIM7. Immunoprecipitation, immunofluorescence, pull down and ubiquitination assay were performed to examine the regulation between TRIM7 and its candidate protein. m6A modification of TRIM7 was measured by RNA immunoprecipitation. Findings: TRIM7 expression was upregulated in osteosarcoma tissues and was an independent risk factor in predicting poor prognosis. TRIM7 regulates osteosarcoma cell migration and invasion through ubiquitination of breast cancer metastasis suppressor 1 (BRMS1). Moreover, chemoresistance was readily observed in osteosarcoma cells and in patient-derived xenograft (PDX) mice with higher TRIM7 levels. Loss of TRIM7 m6A modification was observed in osteosarcoma tissues. METTL3 and YTHDF2 were the main factors involved in the aberrant m6A modification of TRIM7. Interpretation: Overall, our findings show that TRIM7 plays a key role in regulating metastasis and chemoresistance in osteosarcoma through ubiquitination of BRMS1. (C) 2020 The Authors. Published by Elsevier B.V.