Live-cell single-molecule investigation of localization and fate of m6A-modified mRNA

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Post-transcriptional RNA modifications are crucial for cell viability. Today over 170 posttranscriptional RNA modifications in protein coding as well as non-coding RNA are known. One such RNA modification is methylation on the sixth position of the purine ring of adenine in both mRNA (N6-methyladenosine, m6A) and non-coding RNA. m6A represents the most abundant internal mRNA modification in eukaryotes. Its importance is highlighted by the fact that m6A modification is crucial for many aspects of mRNA metabolism from birth to death that includes nuclear pre mRNA processing, cytosolic export of mRNA, mRNA translation, mRNA decay and interaction with RNA granule. RNA sequencing studies discovered that m6A modifications are highly enriched near stop codons and in 3’ untranslated regions (3’ UTR) of mRNAs. The 3’ UTR influences mRNA stability, translation efficiency, and its subcellular localization. One example of biologically important and functional methylation target 3’ UTR is given by the sex determining region y-box 2 (SOX2) mRNA which encodes for a key transcription factor and represents an oncogene. Its methylation status substantially shapes the fate of mRNA and viability of cells, respectively. However, a number of fundamental questions with respect to 3’ UTR m6A modified mRNAs and its influence on translation efficiency, kinetic and ribosome load, and RNA granules interactions are still unanswered. Utilizing 3’ UTR of SOX2 mRNA as model m6A system, a combination of cutting-edge microinjection‐based single-molecule RNA tracking methods, mRNA translation visualization techniques, and ultrahigh-resolution live cell-based single molecule fluorescence microscopy the objective of this project is to characterize the influence of 3’ UTR methylation in mRNA with respect to translation efficiency, translation kinetics, and partitioning kinetics into RNA granules (P bodies/stress granules) as well as its enrichment in RNA granules under normal and stress conditions. All this will provide unprecedented insights into understanding of m6A modification in mRNA and open a novel perspective and concept on the dynamics of the gene regulation processes collectively referred to as epitranscriptomics.