Single-base mapping of m6A by an antibody-independent method

N6-methyladenosine (m6A) is one of the most abundant messenger RNA modifications in eukaryotes involved in various pivotal processes of RNA metabolism. The most popular high-throughput m6A identification method depends on the anti-m6A antibody but suffers from poor reproducibility and limited resolution. Exact location information is of great value for understanding the dynamics, machinery, and functions of m6A. Here, we developed a precise and high-throughput antibody-independent m6A identification method based on the m6A-sensitive RNA endoribonuclease recognizing ACA motif (m6A-sensitive RNA-Endoribonuclease–Facilitated sequencing or m6A-REF-seq). Whole-transcriptomic, single-base m6A maps generated by m6A-REF-seq quantitatively displayed an explicit distribution pattern with enrichment near stop codons. We used independent methods to validate methylation status and abundance of individual m6A sites, confirming the high reliability and accuracy of m6A-REF-seq. We applied this method on five tissues from human, mouse, and rat, showing that m6A sites are conserved with single-nucleotide specificity and tend to cluster among species.

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Single-base mapping of m6A by an antibody-independent method

10.1101/575555 ◽

2019 ◽

Cited By ~ 2

Author(s):

Zhang Zhang ◽

Li-Qian Chen ◽

Yu-Li Zhao ◽

Cai-Guang Yang ◽

Ian A Roundtree ◽

...

Keyword(s):

High Throughput ◽

High Reliability ◽

Methylation Status ◽

Independent Method ◽

Location Information ◽

Single Nucleotide ◽

Single Base ◽

Identification Method ◽

Nucleotide Specificity ◽

Base Mapping

AbstractN6-methyladenosine (m6A) is one of the most abundant mRNA modifications in eukaryotes, involved in various pivotal processes of RNA metabolism. The most popular high-throughput m6A identification method depends on the anti-m6A antibody but suffers from poor reproducibility and limited resolution. Exact location information is of great value for understanding the dynamics, machinery and functions of m6A. Here we developed a precise and high-throughput antibody-independent m6A identification method based on the m6A-sensitive RNA endoribonuclease (m6A-sensitive RNA-Endoribonuclease-Facilitated sequencing or m6A-REF-seq). Whole-transcriptomic, single-base m6A maps generated by m6A-REF-seq quantitatively displayed an explicit distribution pattern with enrichment on stop codons. Independent methods were used to validate the methylation status and abundance of individual m6A sites, confirming the high reliability and accuracy of m6A-REF-seq. We applied this method on five tissues from human, mouse and rat, showing that m6A sites were conserved with single nucleotide specificity and tend to cluster among species.

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Mapping messenger RNA methylations at single base resolution

Current Opinion in Chemical Biology ◽

10.1016/j.cbpa.2021.02.001 ◽

2021 ◽

Vol 63 ◽

pp. 28-37

Author(s):

Jie Cao ◽

Xiao Shu ◽

Xin-Hua Feng ◽

Jianzhao Liu

Keyword(s):

Messenger Rna ◽

Single Base ◽

Single Base Resolution

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High-throughput genotyping of single-nucleotide polymorphisms inace-1gene of mosquitoes using MALDI-TOF mass spectrometry

Insect Science ◽

10.1111/j.1744-7917.2012.01520.x ◽

2012 ◽

Vol 20 (2) ◽

pp. 167-174 ◽

Cited By ~ 3

Author(s):

Yun Mao ◽

Feng Tan ◽

Shuai-Guo Yan ◽

Guo-Xing Wu ◽

Chuan-Ling Qiao ◽

...

Keyword(s):

Mass Spectrometry ◽

Single Nucleotide Polymorphisms ◽

High Throughput ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Maldi Tof Mass Spectrometry ◽

Maldi Tof

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Hepatic IGF1 DNA methylation is influenced by gender but not by intrauterine growth restriction in the young lamb

Journal of Developmental Origins of Health and Disease ◽

10.1017/s2040174415001415 ◽

2015 ◽

Vol 6 (6) ◽

pp. 558-572 ◽

Cited By ~ 4

Author(s):

D. J. Carr ◽

J. S. Milne ◽

R. P. Aitken ◽

C. L. Adam ◽

J. M. Wallace

Keyword(s):

Dna Methylation ◽

Growth Hormone ◽

Sexual Dimorphism ◽

Mrna Expression ◽

Intrauterine Growth Restriction ◽

Messenger Rna ◽

Methylation Status ◽

Growth Restriction ◽

Intrauterine Growth ◽

Increased Risk

Intrauterine growth restriction (IUGR) and postnatal catch-up growth confer an increased risk of adult-onset disease. Overnourishment of adolescent ewes generates IUGR in ∼50% of lambs, which subsequently exhibit increased fractional growth rates. We investigated putative epigenetic changes underlying this early postnatal phenotype by quantifying gene-specific methylation at cytosine:guanine (CpG) dinucleotides. Hepatic DNA/RNA was extracted from IUGR [eight male (M)/nine female (F)] and normal birth weight (12 M/9 F) lambs. Polymerase chain reaction was performed using primers targeting CpG islands in 10 genes: insulin, growth hormone, insulin-like growth factor (IGF)1, IGF2, H19, insulin receptor, growth hormone receptor, IGF receptors 1 and 2, and the glucocorticoid receptor. Using pyrosequencing, methylation status was determined by quantifying cytosine:thymine ratios at 57 CpG sites. Messenger RNA (mRNA) expression of IGF system genes and plasma IGF1/insulin were determined. DNA methylation was independent of IUGR status but sexual dimorphism in IGF1 methylation was evident (M<F, P=0.008). IGF1 mRNA:18S and plasma IGF1 were M>F (both P<0.001). IGF1 mRNA expression correlated negatively with IGF1 methylation (r=−0.507, P=0.002) and positively with plasma IGF1 (r=0.884, P<0.001). Carcass and empty body weights were greater in males (P=0.002–0.014) and this gender difference in early body conformation was mirrored by sexual dimorphism in hepatic IGF1 DNA methylation, mRNA expression and plasma IGF1 concentrations.

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The development and validation of EpiComet-Chip, a modified high-throughput comet assay for the assessment of DNA methylation status

Environmental and Molecular Mutagenesis ◽

10.1002/em.22101 ◽

2017 ◽

Vol 58 (7) ◽

pp. 508-521 ◽

Cited By ~ 17

Author(s):

Todd A. Townsend ◽

Marcus C. Parrish ◽

Bevin P. Engelward ◽

Mugimane G. Manjanatha

Keyword(s):

Dna Methylation ◽

Comet Assay ◽

High Throughput ◽

Methylation Status ◽

Development And Validation

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Precision of High-Throughput Single-Nucleotide Polymorphism Genotyping with Fingernail DNA: Comparison with Blood DNA

Clinical Chemistry ◽

10.1373/clinchem.2008.108225 ◽

2008 ◽

Vol 54 (10) ◽

pp. 1746-1748 ◽

Cited By ~ 5

Author(s):

Mitsuko Nakashima ◽

Masayoshi Tsuda ◽

Akira Kinoshita ◽

Tatsuya Kishino ◽

Shinji Kondo ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

High Throughput ◽

Single Nucleotide Polymorphism Genotyping ◽

Nucleotide Polymorphism ◽

Single Nucleotide

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ANALYSIS OF THE ERROR IN DETERMINING THE EQUIVALENT SIZE OF A DEFECT BY THE STANDARDLESS METHOD DURING ULTRASONIC TESTING

Kontrol Diagnostika ◽

10.14489/td.2021.04.pp.050-057 ◽

2021 ◽

pp. 50-57

Author(s):

A. N. Kireev ◽

M. A. Kireeva

Keyword(s):

Relative Error ◽

High Reliability ◽

Ultrasonic Method ◽

Experimental Studies ◽

Standard Samples ◽

Defect Identification ◽

Identification Method ◽

Maximum Value ◽

Software Product ◽

The Relationship

The article provides a review and analysis of the defect identification method for determining the size of discontinuities when diagnosing various machine parts and units by the manual ultrasonic method. This method makes it possible to determine the equivalent size of discontinuities of various types without using standard samples of an enterprise: point planar and volumetric; extended planar and volumetric. The method is based on the use of the relationship between the amplitude and time characteristics of the echo signal from the discontinuity and the backside signal in the object being diagnosed and the equivalent size of the discontinuity. The article presents the mathematical apparatus for the implementation of this method. Also presented is a software product that allows you to automate calculations when using this defect identification method. The article contains experimental studies of the method for determining the equivalent dimensions of discontinuities of various types, which have shown its high reliability. The maximum value of the relative error in determining the equivalent size of a point planar discontinuity was 2.867 %. The maximum value of the relative error in determining the equivalent size of a point volumetric discontinuity was 1.986 %. The maximum value of the relative error in determining the transverse equivalent size of an extended planar discontinuity was 0.667 %. The maximum value of the relative error in determining the transverse equivalent size of an extended volumetric discontinuity was 1.95 %.

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