Abstract
Over 150 types of RNA modifications are identified in RNA molecules. Transcriptome profiling is one of the key steps in decoding the epitranscriptomic panorama of these chemical modifications and their potential functions. N7-methylguanosine (m7G) is one of the most abundant modifications present in tRNA, rRNA and mRNA 5′cap, and has critical roles in regulating RNA processing, metabolism and function. Besides its presence at the cap position in mRNAs, m7G is also identified in internal mRNA regions. However, its transcriptome-wide distribution and dynamic regulation within internal mRNA regions remain unknown. Here, we have established m7G individual-nucleotide-resolution cross-linking and immunoprecipitation with sequencing (m7G miCLIP-seq) to specifically detect internal mRNA m7G modification. Using this approach, we revealed that m7G is enriched at the 5′UTR region and AG-rich contexts, a feature that is well-conserved across different human/mouse cell lines and mouse tissues. Strikingly, the internal m7G modification is dynamically regulated under both H2O2 and heat shock treatments, with remarkable accumulations in the CDS and 3′UTR regions, and functions in promoting mRNA translation efficiency. Consistently, a PCNA 3′UTR minigene reporter harboring the native m7G modification site displays both enriched m7G modification and increased mRNA translation upon H2O2 treatment compared to the m7G site-mutated minigene reporter (G to A). Taken together, our findings unravel the dynamic profiles of internal mRNA m7G methylome and highlight m7G as a novel epitranscriptomic marker with regulatory roles in translation.
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Acknowledgements
We thank Prof. Hai-Lin Wang and Dr Wei-Yi Lai at Research Center for Eco-Environmental Sciences of CAS for the UHPLC-MRM-MS/MS analysis support. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16010500), the National Natural Science Foundation of China (31625016, 31770872), CAS Key Research Projects of the Frontier Science (QYZDY-SSW-SMC027), the National Key Research and Development Program of China, Stem Cell and Translational Research (2018YFA0109700), the Youth Innovation Promotion Association of CAS (2018133, 2016097), the Ministry of Science and Technology of the People’s Republic of China (MOST2015CB910603), Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), and NSFC consulting grant (91753000). Lionel Malbec is sponsored by CAS-TWAS President’s Fellowship for International Doctoral Students.
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YGY and YY conceived the project, supervised the study and, with LM and YSC, interpreted the data. LM and YSC designed experiments and data analysis. LM performed m7G MeRIP, m7G miCLIP, library construction and other experiments with the help from YY and BYS. TZ and YSC analyzed the data with the assistance from BFS. YZ cultured and provided mESCs. LM, TZ, YSC, YY, BFS, YLZ and YGY wrote the manuscript with input from all authors. All authors read and approved the final manuscript.
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Malbec, L., Zhang, T., Chen, YS. et al. Dynamic methylome of internal mRNA N7-methylguanosine and its regulatory role in translation. Cell Res 29, 927–941 (2019). https://doi.org/10.1038/s41422-019-0230-z
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DOI: https://doi.org/10.1038/s41422-019-0230-z
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