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1.
Anal Chim Acta ; 1098: 56-65, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31948587

RESUMO

RNA molecules carry diverse modifications that exert important influences in many cellular processes. In addition to the single modification occurring in either nucleobase or 2' hydroxyl of ribose in RNA, some dual modifications occur in both the nucleobase and 2' hydroxyl of ribose in RNA. 2'-O-methyl-5-methylcytidine (m5Cm), the dual modifications of cytidine, was first discovered from the tRNA of archaea. Recent studies identified that 2'-O-methyl-5-hydroxymethylcytidine (hm5Cm) and 2'-O-methyl-5-formylcytidine (f5Cm) were present in the anticodon of cytoplasmic tRNA of mammals. Similar to the series of single modification of cytidines of 5-methylcytosine (m5C), 5-hydroxymethylcytidine (hm5C), 5-formylcytidine (f5C), and 5-carboxylcytidine (ca5C) in nucleic acids, the dual modifications of m5Cm, hm5Cm, f5Cm and 2'-O-methyl-5-carboxylcytidine (ca5Cm) may also constitute the series of cytidine modifications in mammals. However, it is normally challenging to detect these modifications because of their low endogenous levels. Here, we established a method by chemical labeling-assisted liquid chromatography - electrospray ionization - tandem mass spectrometry (LC-ESI-MS/MS) analysis for the sensitive and simultaneous determination of all these four cytidine dual modifications, i.e., m5Cm, hm5Cm, f5Cm and ca5Cm. Three different labeling reagents (2-bromo-1-(3,4-dimeth oxyphenyl)-ethanone, BDMOPE; 2-bromo-1-(4-methoxyphenyl)-ethanone, BMOPE; 2-bromo-1-(4-diethylaminophenyl)-ethanone, BDEPE) were used for the chemical labeling. The results showed that the detection sensitivities of m5Cm, hm5Cm, f5Cm and ca5Cm increased up to 462 folds after chemical labeling. With the developed method, we achieved the simultaneous detection of m5Cm, hm5Cm and f5Cm in RNA of mammals. In addition, we found these cytidine dual modifications mainly exist in small RNA (<200 nt) and barely detected in other types of RNA. Moreover, we found that the levels of m5Cm in RNA of human lung carcinoma tissues significantly increased, while hm5Cm and f5Cm significantly decreased compared to tumor adjacent normal tissues. The significant changes of m5Cm, hm5Cm and f5Cm levels may serve as indicator for the detection and prognosis of lung cancer.

2.
Anal Chem ; 92(3): 2612-2619, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-31948230

RESUMO

RNA modification, such as N1-methyladenosine (m1A), affects the secondary structure of RNA and its ability to recognize specific reader proteins. Methods for detecting site-specific m1A are in demand. We report here a ligation-assisted differentiation approach for quantitative detection of m1A in mRNA with single-base resolution. The methyl group in m1A disrupts the Watson-Crick base pairing with uridine, resulting in a lower ligation efficiency of certain ligases and lower amounts of ligation products. Detection of the ligation products using quantitative real-time PCR provided site-specific evaluation of m1A. We first screened appropriate ligase and found that T3 DNA ligase offered the best discrimination between m1A and adenosine. We successfully detected and quantified m1A at position 1674 of bromodomain containing 2 (BRD2) mRNA from HEK293T cells. In lung carcinoma tissues, the level of m1A at position 1674 of BRD2 mRNA was significantly decreased compared to the tumor-adjacent normal tissues, suggesting that site-specific m1A may be involved in carcinogenesis.

3.
Anal Chem ; 91(16): 10477-10483, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31318193

RESUMO

RNA molecules harbor diverse chemical modifications that play important regulatory roles in a variety of biological processes. Up to date, more than 150 modifications have been identified in various RNA species. Most of these modifications occurring in nucleic acids are the methylation of nucleic acids. It has been demonstrated that many of these methylation are reversible and undergo dynamic demethylation. Previous studies established that the demethylation of the two most important and prevalent modifications of 5-methylcytidine (m5C) and N6-methyladenosine (m6A) in nucleic acids is through the hydroxylation of m5C and m6A, forming 5-hydroxymethylcytidine (hm5C) and N6-hydroxymethyladenosine (hm6A), respectively. This indicates the hydroxylation of the methylated nucleosides may be a general pathway for the demethylation of nucleic acid methylation. However, few other hydroxylmethylation modifications have yet to be reported in existence in mammals. In the current study, we developed a neutral enzymatic digestion method for the mild digestion of nucleic acids, followed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. With the established method, we reported the existence of a new hydroxylmethylated nucleosides, N2-hydroxymethylguanosine (hm2G), in mammalian RNA. In addition, we found that the contents of hm2G, as well as N2-methylguanosine (m2G), showed significant differences between thyroid carcinoma tissues and tumor-adjacent normal tissues, indicating that m2G and hm2G in RNA may play certain roles in the carcinogenesis of thyroid carcinoma. Collectively, our study suggests that RNA hydroxylmethylation may be a new prevalent group of modifications existing in RNA, which expands the diversity of nucleic acid modifications and should exert regulatory functions in living organisms.

4.
ACS Chem Biol ; 14(7): 1418-1425, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31188562

RESUMO

RNA contains diverse modifications that exert important influences in a variety of cellular processes. So far more than 150 modifications have been identified in various RNA species, mainly in rRNA and tRNA. Recent research advances in RNA modifications have been sparked by the discovery of dynamic and reversible modifications in mRNA. Moving beyond the abundant tRNA and rRNA to mRNA is opening new directions in understanding RNA modification-mediated regulation of gene expression. Recently, it was reported that N3-methylcytidine (m3C) existed in mRNA of mammalian cells, and methyltransferase-like 8 (METTL8) was identified to be the writer enzyme of m3C. However, little is known about the eraser enzyme of m3C in mRNA. In the current study, we found that the AlkB homologue 1 (ALKBH1) was capable of demethylating m3C in mRNA of mammalian cells in vitro. Overexpression and knockdown of ALKBH1 in cultured human cells can induce decrease and increase of the level of m3C in mRNA, respectively, revealing the eraser enzyme property of ALKBH1 on m3C in mRNA. In addition, we observed significant decrease of the level of m3C in mRNA in hepatocellular carcinoma (HCC) tissues compared to tumor-adjacent normal tissues, which could be attributed to the increased expression of ALKBH1 as well as the decreased expression of METTL8 in HCC tissues. These results indicated that m3C in mRNA may play certain roles in tumorigenesis. Our study shed light on understanding the demethylation of m3C in mRNA.


Assuntos
Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Citidina/análogos & derivados , RNA Mensageiro/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Citidina/metabolismo , Desmetilação , Células HEK293 , Humanos , Neoplasias Hepáticas/metabolismo , Mamíferos
5.
Nucleic Acids Res ; 47(3): 1268-1277, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30517733

RESUMO

In addition to DNA cytosine methylation (5-methyl-2'-deoxycytidine, m5dC), DNA adenine methylation (N6-methyl-2'-deoxyadenosine, m6dA) is another DNA modification that has been discovered in eukaryotes. Recent studies demonstrated that the content and distribution of m6dA in genomic DNA of vertebrates and mammals exhibit dynamic regulation, indicating m6dA may function as a potential epigenetic mark in DNA of eukaryotes besides m5dC. Whether m6dA undergoes the further oxidation in a similar way to m5dC remains elusive. Here, we reported the existence of a new DNA modification, N6-hydroxymethyl-2'-deoxyadenosine (hm6dA), in genomic DNA of mammalian cells and tissues. We found that hm6dA can be formed from the hydroxylation of m6dA by the Fe2+- and 2-oxoglutarate-dependent ALKBH1 protein in genomic DNA of mammals. In addition, the content of hm6dA exhibited significant increase in lung carcinoma tissues. The increased expression of ALKBH1 in lung carcinoma tissues may contribute to the increase of hm6dA in DNA. Taken together, our study reported the existence and formation of hm6dA in genomic DNA of mammals.


Assuntos
Adenina/metabolismo , Metilação de DNA/genética , DNA/genética , Epigênese Genética , Adenina/análogos & derivados , Adenina/síntese química , Adenina/farmacologia , Animais , DNA/efeitos dos fármacos , DNA/metabolismo , Genoma/efeitos dos fármacos , Células HeLa , Humanos , Hidroxilação/efeitos dos fármacos , Mamíferos
6.
Chem Sci ; 9(17): 4160-4167, 2018 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-29780546

RESUMO

DNA and RNA contain diverse chemical modifications that exert important influences in a variety of cellular processes. In addition to enzyme-mediated modifications of DNA and RNA, previous in vitro studies showed that pre-modified nucleoside triphosphates (NTPs) can be incorporated into DNA and RNA during replication and transcription. Herein, we established a chemical labeling method in combination with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis for the determination of endogenous NTPs in the mammalian cells and tissues. We synthesized 8-(diazomethyl)quinoline (8-DMQ) that could efficiently react with the phosphate group under mild condition to label NTPs. The developed method allowed sensitive detection of NTPs, with the detection limits improved by 56-137 folds. The results showed that 12 types of endogenous modified NTPs were distinctly determined in the mammalian cells and tissues. In addition, the majority of these modified NTPs exhibited significantly decreased contents in human hepatocellular carcinoma (HCC) tissues compared to tumor-adjacent normal tissues. Taken together, our study revealed the widespread existence of various modified NTPs in eukaryotes.

7.
ACS Chem Biol ; 13(12): 3243-3250, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29313662

RESUMO

The recent discovery of reversible chemical modifications on mRNA has opened a new era of post-transcriptional gene regulation in eukaryotes. Among the 15 types of modifications identified in mRNA of eukaryotes, N7-methylguanosine (m7G) is unique owing to its presence in the 5' cap structure. It remains unknown whether m7G is also present internally in mRNA, and this is largely attributed to the lack of an appropriate analytical method to differentiate internal m7G in mRNA from that in the 5' cap. To address this analytical challenge, we developed a novel strategy of combining differential enzymatic digestion with liquid chromatography-tandem mass spectrometry analysis to quantify the levels of these two types of m7G modifications in mRNA. In particular, we found that S1 nuclease and phosphodiesterase I exhibit differential activities toward internal and 5'-terminal m7G. By using this method, we found that internal m7G was present in mRNA of cultured human cells as well as plants and rat tissue. In addition, our results showed that plants contain higher levels of internal m7G in mRNA than mammals. We also observed that exposure of rice to cadmium (Cd) stimulated marked diminution in the levels of m7G at both the 5' cap and internal positions of mRNA, which was correlated with the Cd-induced elevated expression of m7G-decapping enzymes. Taken together, we reported here a strategy to distinguish internal and 5'-terminal m7G in mRNA, and by using this method, we demonstrated the prevalence of internal m7G modification in mRNA, which we believe will stimulate future functional studies of m7G on post-transcriptional gene regulation in eukaryotes.


Assuntos
Endorribonucleases/química , Guanina/análogos & derivados , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Animais , Cádmio/farmacologia , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Guanina/química , Humanos , Masculino , Espectrometria de Massas/métodos , Oryza/enzimologia , Processamento Pós-Transcricional do RNA/efeitos dos fármacos , RNA Mensageiro/síntese química , RNA Mensageiro/genética , Ratos Sprague-Dawley
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