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Epitranscriptomic Research

m1A, m5C, ac4C, m7G, and pseudouridine epitranscriptomic modifications in mRNA and lncRNA

 

In addition to m6A as the most studied prominent internal epitranscriptomic modification in mRNA and lncRNAs, other epitranscriptomic modifications, such as m1A, m5C, ac4C, m7G, and Ψ, have been increasingly discovered to play novel biological or clinical roles. Arraystar now provides Epitranscriptomic microarray and Epitranscriptomic sequencing services to profile these modifications transcriptome-wide.

m1A

m1A (1-methyladenosine) is a newly discovered modification in mRNA and lncRNA[1-3]. The modification can affect RNA secondary structures and protein-RNA interactions via its Watson-Crick disruptive nature[1, 2]. Enzymatically, a small proportion of m1A on mRNA is known to be installed by the TRM6–TRM61 complex[1, 3]. YTHDF2 is a potential m1A reader that destabilizes m1A-modified RNAs[4]. YTHDF3 also binds certain m1A-methylated transcripts, especially insulin-like growth factor 1 receptor (IGF1R), consequently inhibiting the invasion of trophoblasts[5]. ALKBH3 is the only known eraser of m1A on mRNA [6]. m1A dysregulation is involved in many diseases. For example, stabilization of colony-stimulating factor 1 (CSF1) mRNA by m1A demethylation can sustain breast and ovarian cancer invasiveness[6], whereas m1A demethylation of Aurora A mRNA by ALKBH3 inhibits ciliogenesis[7]. The ErbB and mTOR pathways are regulated by m1A via ErbB2, mTOR, and AKT1S1 hub genes in gastrointestinal cancer[8]. m1A also has a protective role during stress-induced granulation[9].
 

m5C

While m5C (5-methylcytidine) has long been established in abundant small noncoding RNAs, its presence in coding mRNA RNAs has only recently been shown[10, 11]. NSUN2[12] and NSUN6[13, 14] are the m5C writers on mRNA, and TET2 acts as an eraser to oxidize m5C into hm5C for eventual removal[15]. m5C has two proposed reader proteins: ALYREF, which facilitates export of methylated mRNAs from the nucleus to the cytoplasm[12], and YBX1, which stabilizes m5C-modified mRNAs[16, 17]. m5C mRNA modification has multiple important biological functions, including serving as DNA damage codes to regulate DNA repair[18], participating in stem cell differentiation[11], facilitating the Maternal-to-Zygotic transition [17], as well as promoting adipogenesis by controlling cell cycle progression[19]. m5C is also involved in diseases such as pathogenesis of bladder cancer[16] and pathogen infection-induced myelopoiesis[15].
 

m7G

m7G (7-methylguanosine) is a positively charged, essential modification at the 5’ cap of eukaryotic mRNA[20], which helps to direct mRNA translation, splicing, nuclear export, and degradation prevention. m7G also occurs internally within mRNA [21] and miRNA [22]. These m7G modifications are installed by METTL1-WDR4 heterodimers in mammals. The internal m7G methylation in mRNA can increase the mRNA translation efficiency[21], has remarkable accumulations in the CDS and 3’ UTR regions [23], and is dynamically regulated under both H2O2 and heat shock treatments. The miRNA m7Gs are proposed to augment miRNA processing by disrupting the inhibitory secondary structure of G-quadruplexes found in several pri-miRNA transcripts. m7G modifications in miRNAs are involved in suppressing lung cancer cell migration[22].

tRNAs [24-27] and 18S rRNA [28] are also canonical m7G modification substrates. m7G modification of tRNA, installed by Mettl1/Wdr4, is required for normal translation of mRNA, self-renewal/differentiation of embryonic stem cells[24], and the increased oncogenic mRNA translation in cancers[25-27]. m7G modification of human 18S rRNA at position 1639 is associated with the nuclear pre-rRNA processing and the biosynthesis of 40S ribosomal subunit[29].


ac4C

ac4C (N4-acetylcytidine), a traditional rRNA and tRNA modification, has recently been found also in mRNA [30]. NAT10 is the sole acetyltransferase for ac4C formation in cellular RNA[30]. ac4C peaks show an abundance in the coding sequence (CDS) and 5’UTR clustered near translation start sites but overall depletion within 3’untranslated regions (UTRs)[30]. ac4C acts to stabilize mRNAs and promotes translation[30]. Especially, ac4C in wobble sites stimulates translation efficiency[30]. The biological functions and diseases associations of mRNA acetylation remain largely unknown. ac4C deposition on HIV-1 RNAs enhances viral RNA stability and thus boosts HIV-1 replication [31]. Recently, ac4C has been shown to enhance the replication and pathogenicity enterovirus 71 (EV71) RNA via its selective recruitment of PCBP2 to the IRES to increase RNA stability and binding of RNA-dependent RNA polymerase to viral RNA [32].
 

ψ

ψ (pseudouridine) is the most abundant modification in total RNA from human cells. It is present on most RNA classes, including mRNAs[33, 34]. Pseudouridine on mRNA is installed predominantly by enzymes PUS1[35], PUS7[36], and TRUB1[36]. Besides, TRUB2, and RPUSD3 are involved in pseudouridylating specific residues in mitochondrial mRNAs[37]. ψ in the coding region of an mRNA can alter translation by promoting amino acid substitution at codons containing a ψ [38]. Pseudouridine RNA modifications are installed on pre-mRNAs co-transcriptionally, which can affect alternative pre-mRNA processing for their enrichment in alternatively spliced regions[39]. Biologically, interferon can induce Ψ modifications in interferon-stimulated gene transcripts, suggesting a role for Ψ in IFN signaling pathway and viral defense[40].

 

Arraystar Epitranscriptomic microarrays and Epitranscriptomic Sequencing

To profile and explore these epitranscriptomic modifications, Arraystar offers Epitranscriptomic microarray service for both mRNAs and lncRNAs, and Epitranscriptomic sequencing service for mRNAs. The total RNAs are immunoprecipitated by choice of an antibody specific to the modification of interest for transcript-specific modification detection and quantification. For more details, please see the linked web pages above.

 

References

[1] Safra, M., et al. (2017) "The m1A landscape on cytosolic and mitochondrial mRNA at single-base resolution" Nature 551(7679):251-255 [PMID: 29072297]
[2] Li, X., et al. (2017) "Base-Resolution Mapping Reveals Distinct m(1)A Methylome in Nuclear- and Mitochondrial-Encoded Transcripts" Mol Cell 68(5):993-1005 e9 [PMID: 29107537]
[3] Dominissini, D., et al. (2016) "The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA" Nature 530(7591):441-6 [PMID: 26863196]
[4] Seo, K. W. and Kleiner, R. E. (2020) "YTHDF2 Recognition of N(1)-Methyladenosine (m(1)A)-Modified RNA Is Associated with Transcript Destabilization" ACS Chem Biol 15(1):132-139 [PMID: 31815430]
[5] Zheng, Q., et al. (2020) "Cytoplasmic m(1)A reader YTHDF3 inhibits trophoblast invasion by downregulation of m(1)A-methylated IGF1R" Cell Discov 6:12 [PMID: 32194978]
[6] Woo, H. H. and Chambers, S. K. (2019) "Human ALKBH3-induced m(1)A demethylation increases the CSF-1 mRNA stability in breast and ovarian cancer cells" Biochim Biophys Acta Gene Regul Mech 1862(1):35-46 [PMID: 30342176]
[7] Kuang, W., et al. (2022) "ALKBH3-dependent m(1)A demethylation of Aurora A mRNA inhibits ciliogenesis" Cell Discov 8(1):25 [PMID: 35277482][8] Zhao, Y., et al. (2019) "m1A Regulated Genes Modulate PI3K/AKT/mTOR and ErbB Pathways in Gastrointestinal Cancer" Transl Oncol 12(10):1323-1333 [PMID: 31352195]
[9] Alriquet, M., et al. (2021) "The protective role of m1A during stress-induced granulation" J Mol Cell Biol 12(11):870-880 [PMID: 32462207]
[10] Huber, S. M., et al. (2015) "Formation and abundance of 5-hydroxymethylcytosine in RNA" Chembiochem 16(5):752-5 [PMID: 25676849]
[11] Amort, T., et al. (2017) "Distinct 5-methylcytosine profiles in poly(A) RNA from mouse embryonic stem cells and brain" Genome Biol 18(1):1 [PMID: 28077169]
[12] Yang, X., et al. (2017) "5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m(5)C reader" Cell Res 27(5):606-625 [PMID: 28418038]
[13] Liu, J., et al. (2021) "Sequence- and structure-selective mRNA m(5)C methylation by NSUN6 in animals" Natl Sci Rev 8(6):nwaa273 [PMID: 34691665]
[14] Selmi, T., et al. (2021) "Sequence- and structure-specific cytosine-5 mRNA methylation by NSUN6" Nucleic Acids Res 49(2):1006-1022 [PMID: 33330931]
[15] Shen, Q., et al. (2018) "Tet2 promotes pathogen infection-induced myelopoiesis through mRNA oxidation" Nature 554(7690):123-127 [PMID: 29364877]
[16] Chen, X., et al. (2019) "5-methylcytosine promotes pathogenesis of bladder cancer through stabilizing mRNAs" Nat Cell Biol 21(8):978-990 [PMID: 31358969]
[17] Yang, Y., et al. (2019) "RNA 5-Methylcytosine Facilitates the Maternal-to-Zygotic Transition by Preventing Maternal mRNA Decay" Mol Cell 75(6):1188-1202 e11 [PMID: 31399345]
[18] Chen, H., et al. (2020) "m(5)C modification of mRNA serves a DNA damage code to promote homologous recombination" Nat Commun 11(1):2834 [PMID: 32503981]
[19] Liu, Y., et al. (2021) "mRNA m5C controls adipogenesis by promoting CDKN1A mRNA export and translation" RNA Biol 18(sup2):711-721 [PMID: 34570675]
[20] Ramanathan, A., et al. (2016) "mRNA capping: biological functions and applications" Nucleic Acids Res 44(16):7511-26 [PMID: 27317694]
[21] Zhang, L. S., et al. (2019) "Transcriptome-wide Mapping of Internal N(7)-Methylguanosine Methylome in Mammalian mRNA" Mol Cell 74(6):1304-1316 e8 [PMID: 31031084]
[22] Pandolfini, L., et al. (2019) "METTL1 Promotes let-7 MicroRNA Processing via m7G Methylation" Mol Cell 74(6):1278-1290 e9 [PMID: 31031083]
[23] Malbec, L., et al. (2019) "Dynamic methylome of internal mRNA N(7)-methylguanosine and its regulatory role in translation" Cell Res 29(11):927-941 [PMID: 31520064]
[24] Lin, S., et al. (2018) "Mettl1/Wdr4-Mediated m(7)G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation" Mol Cell 71(2):244-255 e5 [PMID: 29983320]
[25] Orellana, E. A., et al. (2021) "METTL1-mediated m(7)G modification of Arg-TCT tRNA drives oncogenic transformation" Mol Cell 81(16):3323-3338 e14 [PMID: 34352207]
[26] Ma, J., et al. (2021) "METTL1/WDR4-mediated m(7)G tRNA modifications and m(7)G codon usage promote mRNA translation and lung cancer progression" Mol Ther 29(12):3422-3435 [PMID: 34371184]
[27] Dai, Z., et al. (2021) "N(7)-Methylguanosine tRNA modification enhances oncogenic mRNA translation and promotes intrahepatic cholangiocarcinoma progression" Mol Cell 81(16):3339-3355 e8 [PMID: 34352206]
[28] Sloan, K. E., et al. (2017) "Tuning the ribosome: The influence of rRNA modification on eukaryotic ribosome biogenesis and function" RNA Biol 14(9):1138-1152 [PMID: 27911188]
[29] Ounap, K., et al. (2013) "The human WBSCR22 protein is involved in the biogenesis of the 40S ribosomal subunits in mammalian cells" PLoS One 8(9):e75686 [PMID: 24086612]
[30] Arango, D., et al. (2018) "Acetylation of Cytidine in mRNA Promotes Translation Efficiency" Cell 175(7):1872-1886 e24 [PMID: 30449621]
[31] Tsai, K., et al. (2020) "Acetylation of Cytidine Residues Boosts HIV-1 Gene Expression by Increasing Viral RNA Stability" Cell Host Microbe 28(2):306-312 e6 [PMID: 32533923]
[32] Hao, H., et al. (2022) "N4-acetylcytidine regulates the replication and pathogenicity of enterovirus 71" Nucleic Acids Res [PMID: 35971620]
[33] Carlile, T. M., et al. (2014) "Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells" Nature 515(7525):143-6 [PMID: 25192136]
[34] Schwartz, S., et al. (2014) "Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA" Cell 159(1):148-162 [PMID: 25219674]
[35] Carlile, T. M., et al. (2019) "mRNA structure determines modification by pseudouridine synthase 1" Nat Chem Biol 15(10):966-974 [PMID: 31477916]
[36] Safra, M., et al. (2017) "TRUB1 is the predominant pseudouridine synthase acting on mammalian mRNA via a predictable and conserved code" Genome Res 27(3):393-406 [PMID: 28073919]
[37] Antonicka, H., et al. (2017) "A pseudouridine synthase module is essential for mitochondrial protein synthesis and cell viability" EMBO Rep 18(1):28-38 [PMID: 27974379]
[38] Eyler, D. E., et al. (2019) "Pseudouridinylation of mRNA coding sequences alters translation" Proc Natl Acad Sci U S A 116(46):23068-23074 [PMID: 31672910]
[39] Martinez, N. M., et al. (2022) "Pseudouridine synthases modify human pre-mRNA co-transcriptionally and affect pre-mRNA processing" Mol Cell 82(3):645-659 e9 [PMID: 35051350]
[40] Huang, S., et al. (2021) "Interferon inducible pseudouridine modification in human mRNA by quantitative nanopore profiling" Genome Biol 22(1):330 [PMID: 34872593]

 

 

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