Insight into m
            <sup>6</sup>
            A methylation from occurrence to functions

Ru, Wenxiu; Zhang, Xiaoyan; Yue, Binglin; Qi, Ao; Shen, Xuemei; Huang, Yongzhen; Lan, Xianyong; Lei, Chuzhao

doi:10.1098/rsob.200091

Cited by 33 publications

(30 citation statements)

References 139 publications

(343 reference statements)

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“…The use of pseudouridine is a basic modification of foreign mRNA that is necessary for effective protein translation [ 20 , 29 ]. The use of methylated adenosine and methylated cytosine also provides stabilization and additional regulation to mRNA translations [ 30 , 31 , 32 ].…”

Section: Resultsmentioning

confidence: 99%

“…Overall, together with capping and polyadenylation, introducing modified nucleotides makes it possible to regulate translation efficiency, increase RNA stability, and evade RNA-dependent cascades of inherent immunity aimed at recognizing native and foreign RNA molecules [ 20 , 30 , 31 , 32 ]. The basic requirement for the wide applicability of mRNA-based vaccines is the presence of all components necessary for their production at the GMP level.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, we tried to include m5C and m6A in mRNA vaccines. However, most of their inclusion in mRNA composition may slow down translation [ 30 , 31 , 32 ]. Therefore, we used a relatively low share of their inclusion—up to 50% of m5C and 20% of m6A.…”

Section: Discussionmentioning

confidence: 99%

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Construction and Immunogenicity of Modified mRNA-Vaccine Variants Encoding Influenza Virus Antigens

et al. 2021

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Nucleic acid-based influenza vaccines are a promising platform that have recently and rapidly developed. We previously demonstrated the immunogenicity of DNA vaccines encoding artificial immunogens AgH1, AgH3, and AgM2, which contained conserved fragments of the hemagglutinin stem of two subtypes of influenza A—H1N1 and H3N2—and conserved protein M2. Thus, the aim of this study was to design and characterize modified mRNA obtained using the above plasmid DNA vaccines as a template. To select the most promising protocol for creating highly immunogenic mRNA vaccines, we performed a comparative analysis of mRNA modifications aimed at increasing its translational activity and decreasing toxicity We used mRNA encoding a green fluorescent protein (GFP) as a model. Eight mRNA-GFP variants with different modifications (M0–M7) were obtained using the classic cap(1), its chemical analog ARCA (anti-reverse cap analog), pseudouridine (Ψ), N6-methyladenosine (m6A), and 5-methylcytosine (m5C) in different ratios. Modifications M2, M6, and M7, which provided the most intensive fluorescence of transfected HEK293FT cells were used for template synthesis when mRNA encoded influenza immunogens AgH1, AgH3, and AgM2. Virus specific antibodies were registered in groups of animals immunized with a mix of mRNAs encoding AgH1, AgH3, and AgM2, which contained either ARCA (with inclusions of 100% Ψ and 20% m6A (M6)) or a classic cap(1) (with 100% substitution of U with Ψ (M7)). M6 modification was the least toxic when compared with other mRNA variants. M6 and M7 RNA modifications can therefore be considered as promising protocols for designing mRNA vaccines.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Construction and Immunogenicity of Modified mRNA-Vaccine Variants Encoding Influenza Virus Antigens

et al. 2021

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“…Recent technological improvements in nucleic acid sequencing methods have now revealed these and many other modifications such as N1‐methyladenosine (m1A) and acetylcytidine (ac4C) within mRNAs throughout phylogeny, and there are indications that RNA pseudouridylation, methylation, and acetylation are dynamically regulated through development, differentiation, and environmental adaptation in many organisms (Frye & Blanco, 2016; Kadumuri & Janga, 2018; Roundtree et al., 2017; Roundtree & He, 2016; Seo & Kleiner, 2020; Zhao et al., 2017). Furthermore, the modifications m6A and ac4C can promote translation and stability of modified mRNAs (Arango et al., 2018; Boo & Kim, 2020; Ru et al., 2020; Wang et al., 2015). For example, interactions between the eukaryotic translation factor eIF3 and the m1A and m6A‐modified mRNA‐binding protein YTHDF1 increase the translational efficiency of modified transcripts (Wang et al., 2015).…”

Section: Introductionmentioning

confidence: 99%

Epitranscriptome machinery in Trypanosomatids: New players on the table?

Maran

Pitta

Vasconcelos

et al. 2021

Molecular Microbiology

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Trypanosoma and Leishmania parasites cause devastating tropical diseases resulting in serious global health consequences. These organisms have complex life cycles with mammalian hosts and insect vectors. The parasites must, therefore, survive in different environments, demanding rapid physiological and metabolic changes. These responses depend upon regulation of gene expression, which primarily occurs posttranscriptionally. Altering the composition or conformation of RNA through nucleotide modifications is one posttranscriptional mechanism of regulating RNA fate and function, and modifications including N6‐methyladenosine (m6A), N1‐methyladenosine (m1A), N5‐methylcytidine (m5C), N4‐acetylcytidine (ac4C), and pseudouridine (Ψ), dynamically regulate RNA stability and translation in diverse organisms. Little is known about RNA modifications and their machinery in Trypanosomatids, but we hypothesize that they regulate parasite gene expression and are vital for survival. Here, we identified Trypanosomatid homologs for writers of m1A, m5C, ac4C, and Ψ and analyze their evolutionary relationships. We systematically review the evidence for their functions and assess their potential use as therapeutic targets. This work provides new insights into the roles of these proteins in Trypanosomatid parasite biology and treatment of the diseases they cause and illustrates that Trypanosomatids provide an excellent model system to study RNA modifications, their molecular, cellular, and biological consequences, and their regulation and interplay.

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“…In contrast, the RNA methylation generating N6mA has been found in most eukaryotic RNA molecules including mRNA, tRNA, rRNA, noncoding RNA, and chromosome-associated regulatory RNA (17)(18)(19)(20)(21). Here, we chose three recently identified human RNA adenine MTases: PCIF1, acting on mRNA, MettL5 on rRNA, and MettL16 on snRNA.…”

mentioning

confidence: 99%

Enzymatic characterization of three human RNA adenosine methyltransferases reveals diverse substrate affinities and reaction optima

Kaur

Blumenthal

et al. 2021

Journal of Biological Chemistry

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RNA methylations of varied RNA species (mRNA, tRNA, rRNA, non-coding RNA) generate a range of modified nucleotides, including N6-methyladenosine. Here we study the enzymology of three human RNA methyltransferases that methylate the adenosine amino group in diverse contexts, when it is: the first transcribed nucleotide after the mRNA cap (PCIF1), at position 1832 of 18S rRNA (MettL5-Trm112 complex), and within a hairpin in the 3′ UTR of the S-adenosyl- l -methionine synthetase (MettL16). Among these three enzymes, the catalytic efficiency ranges from PCIF1, with the fastest turnover rate of >230 h −1 μM −1 on mRNA cap analog, down to MettL16, which has the lowest rate of ∼3 h −1 μM −1 acting on an RNA hairpin. Both PCIF1 and MettL5 have a binding affinity ( K m ) of ∼1 μM or less for both substrates of SAM and RNA, whereas MettL16 has significantly lower binding affinities for both ( K m >0.4 mM for SAM and ∼10 μM for RNA). The three enzymes are active over a wide pH range (∼5.4–9.4) and have different preferences for ionic strength. Sodium chloride at 200 mM markedly diminished methylation activity of MettL5-Trm112 complex, whereas MettL16 had higher activity in the range of 200 to 500 mM NaCl. Zinc ion inhibited activities of all three enzymes. Together, these results illustrate the diversity of RNA adenosine methyltransferases in their enzymatic mechanisms and substrate specificities and underline the need for assay optimization in their study.

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Insight into m ⁶ A methylation from occurrence to functions

Cited by 33 publications

References 139 publications

Construction and Immunogenicity of Modified mRNA-Vaccine Variants Encoding Influenza Virus Antigens

Construction and Immunogenicity of Modified mRNA-Vaccine Variants Encoding Influenza Virus Antigens

Epitranscriptome machinery in Trypanosomatids: New players on the table?

Enzymatic characterization of three human RNA adenosine methyltransferases reveals diverse substrate affinities and reaction optima

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Insight into m 6 A methylation from occurrence to functions

Cited by 33 publications

References 139 publications

Construction and Immunogenicity of Modified mRNA-Vaccine Variants Encoding Influenza Virus Antigens

Construction and Immunogenicity of Modified mRNA-Vaccine Variants Encoding Influenza Virus Antigens

Epitranscriptome machinery in Trypanosomatids: New players on the table?

Enzymatic characterization of three human RNA adenosine methyltransferases reveals diverse substrate affinities and reaction optima

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Insight into m ⁶ A methylation from occurrence to functions