bims-rimeca 2021-04-18 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2021–04–18
eight papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

The evolving landscape of N6-methyladenosine modification in the tumor microenvironment.
Autophagy of the m6A mRNA demethylase FTO is impaired by low-level arsenic exposure to promote tumorigenesis.
A methionine-Mettl3-N6-methyladenosine axis promotes polycystic kidney disease.
Comprehensive analyses of m6A regulators and interactive coding and non-coding RNAs across 32 cancer types.
CLK1/SRSF5 pathway induces aberrant exon skipping of METTL14 and Cyclin L2 and promotes growth and metastasis of pancreatic cancer.
Mettl14-mediated m6A Modification Facilitates Liver Regeneration by Maintaining Endoplasmic Reticulum Homeostasis.
CircPTPRA blocks the recognition of RNA N6-methyladenosine through interacting with IGF2BP1 to suppress bladder cancer progression.
[Establishment and application of HPLC-MS/MS method for detecting m6A level in RNA].

Mol Ther. 2021 Apr 09. pii: S1525-0016(21)00196-9. [Epub ahead of print]

The evolving landscape of N6-methyladenosine modification in the tumor microenvironment.

Yunru Gu, Xi Wu, Jingxin Zhang, Yuan Fang, Yutian Pan, Yongqian Shu, Pei Ma.

  The tumor microenvironment (TME), controlled by intrinsic mechanisms of carcinogenesis and epigenetic modifications, has, in recent years, become a heavily researched topic. The TME can be described in terms of hypoxia, metabolic dysregulation, immune escape, and chronic inflammation. RNA methylation, an epigenetic modification, has recently been found to have a pivotal role in shaping the TME. The N6-methylation of adenosine (m6A) modification is the most common type of RNA methylation that occurs in the N6-position of adenosine, which is the primary internal modification of eukaryotic mRNA. Compelling evidence has demonstrated that m6A regulates transcriptional and protein expression through splicing, translation, degradation, and export, thereby mediating the biological processes of cancer cells and/or stromal cells and characterizing the TME. The TME also has a crucial role in the complicated regulatory network of m6A modifications and, subsequently, influences tumor initiation, progression, and therapy responses. In this review, we describe the features of the TME and how the m6A modification modulates and interacts with it. We also focus on various factors and pathways involved in m6A methylation. Finally, we discuss potential therapeutic strategies and prognostic biomarkers with respect to the TME and m6A modification.

Keywords:  RNA methylation; chronic inflammation; hypoxia; immune escape; m(6)A; metabolic dysregulation; tumor microenvironment

DOI:  https://doi.org/10.1016/j.ymthe.2021.04.009
Nat Commun. 2021 04 12. 12(1): 2183

Autophagy of the m6A mRNA demethylase FTO is impaired by low-level arsenic exposure to promote tumorigenesis.

Yan-Hong Cui, Seungwon Yang, Jiangbo Wei, Christopher R Shea, Wen Zhong, Fang Wang, Palak Shah, Muhammad G Kibriya, Xiaolong Cui, Habibul Ahsan, Chuan He, Yu-Ying He.

Here we show that FTO as an N6-methyladenosine (m6A) RNA demethylase is degraded by selective autophagy, which is impaired by low-level arsenic exposure to promote tumorigenesis. We found that in arsenic-associated human skin lesions, FTO is upregulated, while m6A RNA methylation is downregulated. In keratinocytes, chronic relevant low-level arsenic exposure upregulated FTO, downregulated m6A RNA methylation, and induced malignant transformation and tumorigenesis. FTO deletion inhibited arsenic-induced tumorigenesis. Moreover, in mice, epidermis-specific FTO deletion prevented skin tumorigenesis induced by arsenic and UVB irradiation. Targeting FTO genetically or pharmacologically inhibits the tumorigenicity of arsenic-transformed tumor cells. We identified NEDD4L as the m6A-modified gene target of FTO. Finally, arsenic stabilizes FTO protein through inhibiting p62-mediated selective autophagy. FTO upregulation can in turn inhibit autophagy, leading to a positive feedback loop to maintain FTO accumulation. Our study reveals FTO-mediated dysregulation of mRNA m6A methylation as an epitranscriptomic mechanism to promote arsenic tumorigenicity.

DOI: https://doi.org/10.1038/s41467-021-22469-6
Cell Metab. 2021 Apr 09. pii: S1550-4131(21)00131-5. [Epub ahead of print]

A methionine-Mettl3-N6-methyladenosine axis promotes polycystic kidney disease.

Harini Ramalingam, Sonu Kashyap, Patricia Cobo-Stark, Andrea Flaten, Chun-Mien Chang, Sachin Hajarnis, Kyaw Zaw Hein, Jorgo Lika, Gina M Warner, Jair M Espindola-Netto, Ashwani Kumar, Mohammed Kanchwala, Chao Xing, Eduardo N Chini, Vishal Patel.

  Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic disorder marked by numerous progressively enlarging kidney cysts. Mettl3, a methyltransferase that catalyzes the abundant N6-methyladenosine (m6A) RNA modification, is implicated in development, but its role in most diseases is unknown. Here, we show that Mettl3 and m6A levels are increased in mouse and human ADPKD samples and that kidney-specific transgenic Mettl3 expression produces tubular cysts. Conversely, Mettl3 deletion in three orthologous ADPKD mouse models slows cyst growth. Interestingly, methionine and S-adenosylmethionine (SAM) levels are also elevated in ADPKD models. Moreover, methionine and SAM induce Mettl3 expression and aggravate ex vivo cyst growth, whereas dietary methionine restriction attenuates mouse ADPKD. Finally, Mettl3 activates the cyst-promoting c-Myc and cAMP pathways through enhanced c-Myc and Avpr2 mRNA m6A modification and translation. Thus, Mettl3 promotes ADPKD and links methionine utilization to epitranscriptomic activation of proliferation and cyst growth.

Keywords:  AVPR2; METTL3; N(6)-methyladenosine; S-adenosylmethionine; c-Myc; m6A mRNA methylation; mRNA translation; methionine; polycystic kidney disease

DOI:  https://doi.org/10.1016/j.cmet.2021.03.024
Mol Cancer. 2021 Apr 13. 20(1): 67

Comprehensive analyses of m6A regulators and interactive coding and non-coding RNAs across 32 cancer types.

Sipeng Shen, Ruyang Zhang, Yue Jiang, Yi Li, Lijuan Lin, Zhonghua Liu, Yang Zhao, Hongbing Shen, Zhibin Hu, Yongyue Wei, Feng Chen.

  N6-Methyladenosine (m6A) is an RNA modification that interacts with numerous coding and non-coding RNAs and plays important roles in the development of cancers. Nonetheless, the clinical impacts of m6A interactive genes on these cancers largely remain unclear since most studies focus only on a single cancer type. We comprehensively evaluated m6A modification patterns, including 23 m6A regulators and 83 interactive coding and non-coding RNAs among 9,804 pan-cancer samples. We used clustering analysis to identify m6A subtypes and constructed the m6A signature based on an unsupervised approach. We used the signatures to identify potential m6A modification targets across the genome. The prognostic value of one target was further validated in 3,444 samples from six external datasets. We developed three distinct m6A modification subtypes with different tumor microenvironment cell infiltration degrees: immunological, intermediate, and tumor proliferative. They were significantly associated with overall survival in 24 of 27 cancer types. Our constructed individual-level m6A signature was associated with survival, tumor mutation burden, and classical pathways. With the signature, we identified 114 novel genes as potential m6A targets. The gene shared most commonly between cancer types, BCL9L, is an oncogene and interacts with m6A patterns in the Wnt signaling pathway. In conclusion, m6A regulators and their interactive genes impact the outcome of various cancers. Evaluating the m6A subtype and the signature of individual tumors may inform the design of adjuvant treatments.

Keywords:  Multi-omics; N6-Methyladenosine; Pan-cancer; Survival outcome

DOI:  https://doi.org/10.1186/s12943-021-01362-2
J Hematol Oncol. 2021 Apr 13. 14(1): 60

CLK1/SRSF5 pathway induces aberrant exon skipping of METTL14 and Cyclin L2 and promotes growth and metastasis of pancreatic cancer.

Shi Chen, Can Yang, Zu-Wei Wang, Jian-Fei Hu, Jing-Jing Pan, Cheng-Yu Liao, Jia-Qiang Zhang, Jiang-Zhi Chen, Yi Huang, Long Huang, Qian Zhan, Yi-Feng Tian, Bai-Yong Shen, Yao-Dong Wang.

   BACKGROUND: Both aberrant alternative splicing and m6A methylation play complicated roles in the development of pancreatic cancer (PC), while the relationship between these two RNA modifications remains unclear.
METHODS: RNA sequencing (RNA-seq) was performed using 15 pairs of pancreatic ductal adenocarcinoma (PDAC) tissues and corresponding normal tissues, and Cdc2-like kinases 1 (CLK1) was identified as a significantly upregulated alternative splicing related gene. Real-time quantitative PCR (qPCR) and western blotting were applied to determine the CLK1 levels. The prognostic value of CLK1 was elucidated by Immunohistochemistry (IHC) analyses in two independent PDAC cohorts. The functional characterizations and mechanistic insights of CLK1 in PDAC growth and metastasis were evaluated with PDAC cell lines and nude mice. SR-like splicing factors5250-Ser (SRSF5250-Ser) was identified as an important target phosphorylation site by phosphorylation mass spectrometry. Through transcriptome sequencing, Methyltransferase-like 14exon10 (METTL14exon10) and Cyclin L2exon6.3 skipping were identified as key alternative splicing events regulated by the CLK1-SRSF5 axis. RIP assays, RNA-pulldown and CLIP-qPCR were performed to confirm molecular interactions and the precise binding sites. The roles of the shift of METTL14exon 10 and Cyclin L2exon6.3 skipping were surveyed.
RESULTS: CLK1 expression was significantly increased in PDAC tissues at both the mRNA and protein levels. High CLK1 expression was associated with poor prognosis. Elevated CLK1 expression promoted growth and metastasis of PC cells in vitro and in vivo. Mechanistically, CLK1 enhanced phosphorylation on SRSF5250-Ser, which inhibited METTL14exon10 skipping while promoted Cyclin L2exon6.3 skipping. In addition, aberrant METTL14exon 10 skipping enhanced the N6-methyladenosine modification level and metastasis, while aberrant Cyclin L2exon6.3 promoted proliferation of PDAC cells.
CONCLUSIONS: The CLK1/SRSF5 pathway induces aberrant exon skipping of METTL14 and Cyclin L2, which promotes growth and metastasis and regulates m6A methylation of PDAC cells. This study suggests the potential prognostic value and therapeutic targeting of this pathway in PDAC patients.

Keywords:  Alternative splicing; CLK1; Cyclin L2; M6A Modification; METTL14; Pancreatic cancer; SRSF5

DOI:  https://doi.org/10.1186/s13045-021-01072-8
Cell Mol Gastroenterol Hepatol. 2021 Apr 10. pii: S2352-345X(21)00072-2. [Epub ahead of print]

Mettl14-mediated m6A Modification Facilitates Liver Regeneration by Maintaining Endoplasmic Reticulum Homeostasis.

Xiaoyue Cao, Yuke Shu, Yuwei Chen, Qing Xu, Gang Guo, Zhenru Wu, Mingyang Shao, Yongjie Zhou, Menglin Chen, Yuping Gong, Chuan Li, Yujun Shi, Hong Bu.

   BACKGROUND & AIMS: N6-methyladenosine (m6A), the most prevalent and dynamic posttranscriptional methylation modification of mammalian mRNA, is involved in various biological processes, but its role in liver regeneration has not been characterized.
METHODS: We first conducted transcriptome-wide m6A mRNA sequencing (m6A-seq) and characterized the expression pattern of m6A in regenerating mouse liver. Next, we generated hepatocyte-specific Mettl3- or Mettl14-deficient mice and investigated their role in liver regeneration. A series of biochemical experiments in vitro and in vivo was further performed to investigate potential mechanisms.
RESULTS: We identified an overwhelming proportion of m6A-modified genes with initially upregulated and subsequently downregulated m6A levels as liver regeneration progressed. Loss of Mettl14 but not of Mettl3 resulted in markedly disrupted liver regeneration, and Mettl14-ablated hepatocytes were arrested in the G1 phase of the cell cycle. Most strikingly, the Mettl14-ablated regenerating liver exhibited extensive parenchymal necrosis. mRNA transcripts, such as Hsp90b1, Erp29, Stt3a, P4hb, and Lman1, encoding proteins involved in polypeptide processing and the endoplasmic reticulum (ER) stress response, were m6A-hypomethylated, and their mRNA and protein levels were subsequently decreased, resulting in unresolved ER stress, hepatocyte death and inhibited proliferation.
CONCLUSION: We demonstrate the essential role of Mettl14 in facilitating liver regeneration by modulating polypeptide-processing proteins in the ER in an m6A-dependent manner.

Keywords:  Mettl14; N(6)-methyladenosine; endoplasmic reticulum stress; liver regeneration

DOI:  https://doi.org/10.1016/j.jcmgh.2021.04.001
Mol Cancer. 2021 Apr 14. 20(1): 68

CircPTPRA blocks the recognition of RNA N6-methyladenosine through interacting with IGF2BP1 to suppress bladder cancer progression.

Fei Xie, Chao Huang, Feng Liu, Hui Zhang, Xingyuan Xiao, Jiayin Sun, Xiaoping Zhang, Guosong Jiang.

   BACKGROUND: Circular RNAs (circRNAs) have been found to have significant impacts on bladder cancer (BC) progression through various mechanisms. In this study, we aimed to identify novel circRNAs that regulate the function of IGF2BP1, a key m6A reader, and explore the regulatory mechanisms and clinical significances in BC.
METHODS: Firstly, the clinical role of IGF2BP1 in BC was studied. Then, RNA immunoprecipitation sequencing (RIP-seq) analysis was performed to identify the circRNAs interacted with IGF2BP1 in BC cells. The overall biological roles of IGF2BP1 and the candidate circPTPRA were investigated in both BC cell lines and animal xenograft studies. Subsequently, we evaluated the regulation effects of circPTPRA on IGF2BP1 and screened out its target genes through RNA sequencing. Finally, we explored the underlying molecular mechanisms that circPTPRA might act as a blocker in recognition of m6A.
RESULTS: We demonstrated that IGF2BP1 was predominantly binded with circPTPRA in the cytoplasm in BC cells. Ectopic expression of circPTPRA abolished the promotion of cell proliferation, migration and invasion of BC cells induced by IGF2BP1. Importantly, circPTPRA downregulated IGF2BP1-regulation of MYC and FSCN1 expression via interacting with IGF2BP1. Moreover, the recognition of m6A-modified RNAs mediated by IGF2BP1 was partly disturbed by circPTPRA through its interaction with KH domains of IGF2BP1.
CONCLUSIONS: This study identifies exonic circular circPTPRA as a new tumor suppressor that inhibits cancer progression through endogenous blocking the recognition of IGF2BP1 to m6A-modified RNAs, indicating that circPTPRA may serve as an exploitable therapeutic target for patients with BC.

Keywords:  Bladder cancer; IGF2BP1; N 6-methyladenosine; circPTPRA

DOI:  https://doi.org/10.1186/s12943-021-01359-x
Zhonghua Yu Fang Yi Xue Za Zhi. 2021 Apr 06. 55(4): 528-534

[Establishment and application of HPLC-MS/MS method for detecting m6A level in RNA].

M M Zhang, S S Zhao, N Huang, M Ding, F Y Sun.

Objective: Explore the establishment of a fast, stable and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for detecting the level of m6A modification in RNA and its application. Methods: The degree of m6A in RNA can be expressed as the ratio of m6A and adenosine (A) in concentration, which can be determined by ESI source positive ion multiple reaction monitoring (MRM) mode. The established method was verified by analyzing three quality control samples (m6A: 4, 40, 400 nmol/L; A: 40, 400, 4 000 nmol/L) with three different concentrations of low, medium, and high. The method was used to detect the degree of m6A in RNA from mouse spleen T cells treated in different ways. The t test was used to compare the differences between the two groups of data. Results: The established method had a good Linearity (R2>0.99) in a range of 1-500 nmol/L for m6A and 10-5 000 nmol/L for A. The limit of detection (LOD) was 1 nmol/L for m6A and 10 nmol/L for A. The recoveries were between 98.9% and 116.5%. The intra-day (n=5) RSDs and the inter-day (n=15, 5 days) RSDs were 2.4%-9.5% and 4.4%-9.6%, respectively. And this method was used to detect the degree of m6A in the RNA from mouse spleen T cells cultured in different conditions. The results showed that the m6A modification level in the RNA of primary CD8+T cell was 0.271 5±0.017 9, and the m6A modification level in the RNA of primary CD8+T cell with IL-27 was 0.251 7±0.015 0, indicating that primary CD8+T cells have a higher level of RNA methylation. Conclusion: This research has established a fast, simplemethylation degree in RNA with HPLC-MS/MS. This method is easy to be popularized and is suitable for the detection of large quantity of samples, and of great significance in analyzing the relationship between methylation and diseases.

DOI: https://doi.org/10.3760/cma.j.cn112150-20200824-01145