bims-rimeca 2021-10-17 papers

Front Cell Dev Biol. 2021 ;9 737498

The RNA N6-Methyladenosine Methyltransferase METTL3 Promotes the Progression of Kidney Cancer via N6-Methyladenosine-Dependent Translational Enhancement of ABCD1.

Yue Shi, Yanliang Dou, Jianye Zhang, Jie Qi, Zijuan Xin, Mingxin Zhang, Yu Xiao, Weimin Ci.

The role of N6-methyladenosine (m6A)-modifying proteins in cancer progression depends on the cell type and mRNA affected. However, the biological role and underlying mechanism of m6A in kidney cancer is limited. Here, we discovered the variability in m6A methyltransferase METTL3 expression was significantly increased in clear cell renal cell carcinoma (ccRCC) the most common subtype of renal cell carcinoma (RCC), and high METTL3 expression predicts poor prognosis in ccRCC patients using a dataset from The Cancer Genome Atlas (TCGA). Importantly, knockdown of METTL3 in ccRCC cell line impaired both cell migration capacity and tumor spheroid formation in soft fibrin gel, a mechanical method for selecting stem-cell-like tumorigenic cells. Consistently, overexpression of METTL3 but not methyltransferase activity mutant METTL3 can promote cell migration, spheroid formation in cell line and tumor growth in xenograft model. Transcriptional profiling of m6A in ccRCC tissues identified the aberrant m6A transcripts were enriched in cancer-related pathways. Further m6A-sequencing of METTL3 knockdown cells and functional studies confirmed that translation of ABCD1, an ATP-binding cassette (ABC) transporter of fatty acids, was inhibited by METTL3 in m6A-dependent manner. Moreover, knockdown of ABCD1 in ccRCC cells decreased cancer cell migration and spheroid formation, and upregulation of ABCD1 acts as an adverse prognosis factor of kidney cancer patients. In summary, our study identifies that METTL3 promotes ccRCC progression through m6A modification-mediated translation of ABCD1, providing an epitranscriptional insight into the molecular mechanism in kidney cancer.

Keywords: ABCD1; METTL3; cancer progression; kidney cancer; m6A

DOI: https://doi.org/10.3389/fcell.2021.737498

Arterioscler Thromb Vasc Biol. 2021 Oct 14. ATVBAHA121316414

Endothelial METTL3 (Methyltransferase-Like 3) Inhibits Fibrinolysis by Promoting PAI-1 (Plasminogen Activator Inhibitor-1) Expression Through Enhancing Jun Proto-Oncogene N6-Methyladenosine Modification.

Qin Bai, Yao Lu, Yanhua Chen, Han Zhang, Weiwei Zhang, Huang Wu, Aiqing Wen.

OBJECTIVE: METTL3 (methyltransferase-like protein 3)-mediated N6-methyladenosine modification is the most abundant RNA modification on eukaryote mRNAs and plays a crucial role in diverse physiological and pathological processes. However, whether N6-methyladenosine modification has function in thrombosis is unknown. This study aims to determine the role of METTL3 in the endothelial cells-mediated thrombosis. Approach and Results: RNA-sequencing and real-time quantitative PCR revealed that the expression of PAI-1 (plasminogen activator inhibitor-1) was downregulated in METTL3 knockdown human umbilical vein endothelial cells. In vitro experiments showed that METTL3 suppressed fibrinolysis. Mechanically, RNA methylation sequencing and meRIP-quantitative real-time PCR showed that METTL3 catalyzed N6-methyladenosine modification on 3' UTR of JUN mRNA. Western blotting analysis showed that METTL3 promoted JUN protein expression. Chromatin immunoprecipitation analysis demonstrated that JUN bound to the PAI-1 promoter in human umbilical vein endothelial cells. Furthermore, mice challenged with lipopolysaccharide resulted in higher METTL3 expression in vessels. Endothelial-specific knockdown of Mettl3 decreased expression of active PAI-1 in plasma and attenuated fibrin deposition in livers and lungs during endotoxemia.
CONCLUSIONS: Our study reveals that METTL3-mediated N6-methyladenosine modification plays a crucial role in fibrinolysis and is an underlying target for the therapy of thrombotic disorders.

Keywords: downregulation; endothelial cell; fibrinolysis; methylation; thrombosis

DOI: https://doi.org/10.1161/ATVBAHA.121.316414

Front Oncol. 2021 ;11 716921

HNRNPA2B1, as a m6A Reader, Promotes Tumorigenesis and Metastasis of Oral Squamous Cell Carcinoma.

Feiya Zhu, Tianru Yang, Mianfeng Yao, Ting Shen, Changyun Fang.

N6-methyladenosine (m6A) modification is the most prevalent modification on eukaryotic RNA, and the m6A modification regulators were involved in the progression of various cancers. However, the functions of m6A regulators in oral squamous cell carcinoma (OSCC) remain poorly understood. In this study, we demonstrated that 13 of 19 m6A-related genes in OSCC tissues are dysregulated, and HNRNPA2B1 was the most prognostically important locus of the 19 m6A regulatory genes in OSCC. Moreover, HNRNPA2B1 expression is elevated in OSCC, and a high level of HNRNPA2B1 is significantly associated with poor overall survival in OSCC patients. Functional studies, combined with further analysis of the correlation between the expression of HNRNPA2B1 and the EMT-related markers from the TCGA database, reveal that silencing HNRNPA2B1 suppresses the proliferation, migration, and invasion of OSCC via EMT. Collectively, our work shows that HNRNPA2B1 may have the potential to promote carcinogenesis of OSCC by targeting EMT via the LINE-1/TGF-β1/Smad2/Slug signaling pathway and provide insight into the critical roles of HNRNPA2B1 in OSCC.

Keywords: EMT; HNRNPA2B1; Metastasis; N6-methyladenosine; Oral squamous cell carcinoma

DOI: https://doi.org/10.3389/fonc.2021.716921

J Biol Chem. 2021 Oct 08. pii: S0021-9258(21)01099-1. [Epub ahead of print] 101294

Metabolic turnover and dynamics of modified ribonucleosides by 13C labeling.

Paulo A Gameiro, Vesela Encheva, Mariana Silva Dos Santos, James I MacRae, Jernej Ule.

Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications poorly understood. Here, we developed a 13C labeling approach, called 13C-dynamods, to quantify the turnover of base modifications in newly transcribed RNA. This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleoside samples, and showed the distinct kinetics of the N6-methyladenosine (m6A) versus 7-methylguanosine (m7G) modification in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m62A) exhibits distinct turnover in small RNAs and free ribonucleosides when compared to known m62A-modified large rRNAs. Finally, combined measurements of turnover and abundance of these modifications informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, 13C-dynamods enables studies of the origin of modified RNAs at steady-state and subsequent dynamics under non-stationary conditions. These results open new directions to probe the presence and biological regulation of modifications in particular RNAs.

Keywords: 7-methylguanosine; Isotopic labeling; N6,N6-dimethyladenosine; N6-methyladenosine; RNA modifications; RNA turnover; mass spectrometry; metabolic stress; metabolism; methylation dynamics

DOI: https://doi.org/10.1016/j.jbc.2021.101294

Int J Genomics. 2021 ;2021 3803724

Comprehensive Analysis of m5C RNA Methylation Regulator Genes in Clear Cell Renal Cell Carcinoma.

Jiajin Wu, Chao Hou, Yuhao Wang, Zhongyuan Wang, Pu Li, Zengjun Wang.

Background: Recent research found that N5-methylcytosine (m5C) was involved in the development and occurrence of numerous cancers. However, the function and mechanism of m5C RNA methylation regulators in clear cell renal cell carcinoma (ccRCC) remains undiscovered. This study is aimed at investigating the predictive and clinical value of these m5C-related genes in ccRCC.
Methods: Based on The Cancer Genome Atlas (TCGA) database, the expression patterns of twelve m5C regulators and matched clinicopathological characteristics were downloaded and analyzed. To reveal the relationships between the expression levels of m5C-related genes and the prognosis value in ccRCC, consensus clustering analysis was carried out. By univariate Cox analysis and last absolute shrinkage and selection operator (LASSO) Cox regression algorithm, a m5C-related risk signature was constructed in the training group and further validated in the testing group and the entire cohort. Then, the predictive ability of survival of this m5C-related risk signature was analyzed by Cox regression analysis and nomogram. Functional annotation and single-sample Gene Set Enrichment Analysis (ssGSEA) were applied to further explore the biological function and potential signaling pathways. Furthermore, we performed qRT-PCR experiments and measured global m5C RNA methylation level to validate this signature in vitro and tissue samples.
Results: In the TCGA-KIRC cohort, we found significant differences in the expression of m5C RNA methylation-related genes between ccRCC tissues and normal kidney tissues. Consensus cluster analysis was conducted to separate patients into two m5C RNA methylation subtypes. Significantly better outcomes were observed in ccRCC patients in cluster 1 than in cluster 2. m5C RNA methylation-related risk score was calculated to evaluate the prognosis of ccRCC patients by seven screened m5C RNA methylation regulators (NOP2, NSUN2, NSUN3, NSUN4, NSUN5, TET2, and DNMT3B) in the training cohort. The AUC for the 1-, 2-, and 3-year survival in the training cohort were 0.792, 0.675, and 0.709, respectively, indicating that the risk signature had an excellent prognosis prediction in ccRCC. Additionally, univariate and multivariate Cox regression analyses revealed that the risk signature could be an independent prognostic factor in ccRCC. The results of ssGSEA suggested that the immune cells with different infiltration degrees between the high-risk and low-risk groups were T cells including follicular helper T cells, Th1_cells, Th2_cells, and CD8+_T_cells, and the main differences in immune-related functions between the two groups were the interferon response and T cell costimulation. In addition, qRT-PCR experiments confirmed our results in renal cell lines and tissue samples.
Conclusions: According to the seven selected regulatory factors of m5C RNA methylation, a risk signature associated with m5C methylation that can independently predict prognosis in patients with ccRCC was developed and further verified the predictive efficiency.

DOI: https://doi.org/10.1155/2021/3803724