Live-cell single-molecule investigation of localization and fate of m6A-modified mRNA
Biophysics
Cell Biology
Final Report Abstract
Gene expression and cell growth are under stringent and sophisticated control under genetic and epigenetic feedback loops. Over the last decade, RNA modification has gained attention as an additional layer of genetic regulation. Since the discovery of the first posttranscriptional modification of RNA in the 1950s, pseudouridine, >200 posttranscriptional RNA modifications in RNA have been discovered. Recently, the focus has shifted from identification of chemical modifications of RNA towards understanding their biological functions, giving rise to a new field of molecular biology called “epitranscriptomics”. One such dynamic RNA modification is N6-methyladenosine (m6A). On a global scale, m6A influences several essential cellular processes: differentiation in various cell types; contributes to numerous cancer types; controls the circadian clock. The majority of m6A modifications are installed by a multiprotein writer complex (METTL3/METLL14). METTL3 is known to be highly concentrated in the nucleus. m6A’s biological function is achieved and processed by m 6A RNA binding protein domains. m6A modifications are highly enriched near stop codons and in 3’ untranslated regions (3’ UTR) of mRNAs. The 3’ UTR influences mRNA stability, its cellular localization, and translation efficiency. An example for the importance of m6A in a 3’ UTR is that of sex determining region y-box 2 (SOX2) mRNA, which is a high-risk oncogene. SOX2 mRNA has been identified as a target of METTL3, which modifies SOX2’s mRNA transcript stability. This stabilization confers glioblastoma-stem-cell-like characteristics such as enhanced radioresistence. Taken together, the available biochemical, structural, and cellular data provide broad insights into the impact of m6A modifications on RNA and their dynamic nature. Despite the published over 1,300 studies about m 6A modified RNAs, there remain, however, a number fundamental questions unanswered regarding the molecular and mechanistic behavior of 3’ UTR m6A modified mRNAs. The aim of the project is to address these questions by dissecting translation efficiency, kinetic and ribosome load using a 3’ UTR of SOX2 mRNA as a model m 6A system. To achieve this, a combination of cutting-edge single-molecule RNA tracking methods, mRNA translation visualization techniques, and ultrahigh-resolution cellbased single molecule fluorescence microscopy have been employed. It could be show that m6A mRNA results globally in less protein expression. This could be mechanistically assigned to lower ribosome load. Further, for the first time, it could be shown in a cellular context with single molecule resolution that METTL3 itself is participation in mRNA regulation by interaction with mRNA. Furthermore, it could be shown that METTL3 binds both mRNA species (methylated and unmethylated mRNA) with high affinity and is involved in ribosomal load. METTL3 knock down experiments showed that its cellular reduction has a negative effect on overall translational output. All together, these results suggest that METTL3 acts as a ribosome load regulator and is selective for single mRNA translation sites. Further, the data indicates that methylated mRNA undergoes a faster mRNA decay. Overall, the data suggests that METTL3 is not limited to its nuclear function to, but also is implicated in modulation of cytosolic mRNA stability and protein concentration. Fox SOX2 biology, METTL3 represents a crucial factor of glioblastoma therapeutic target.
Publications
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Probing Transient Riboswitch Structures via Single Molecule Accessibility Analysis. Methods in Molecular Biology, 37-51. Springer US.
Welty, Robb; Schmidt, Andreas & Walter, Nils G.
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Spontaneous Confinement of mRNA Molecules at Biomolecular Condensate Boundaries. Cells, 12(18), 2250.
Perelman, Rebecca T.; Schmidt, Andreas; Khan, Umar & Walter, Nils G.
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Utilizing functional cell‐free extracts to dissect ribonucleoprotein complex biology at single‐molecule resolution. WIREs RNA, 14(5).
Duran, Elizabeth; Schmidt, Andreas; Welty, Robb; Jalihal, Ameya P.; Pitchiaya, Sethuramasundaram & Walter, Nils G.
