METTL3-NUP93 interaction facilitates the nuclear export of m6A-modified mRNAs

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PROJECT ABSTRACT The nucleus-cytoplasm shuttling of messenger RNAs (mRNAs) is a key determinant in the spatiotemporal articulation of gene expression. This process is finely tuned by multiple factors, one of which is the addition of a methyl moiety onto the adenosine base at the nitrogen-6 position (m6A). Emerging evidence suggests that m6A enhances the nuclear export of decorated mRNAs. Current theories explaining m6A-assisted mRNA export focus on the crosstalk between the protein enzymes involved in m6A modification and the typical RNA- binding adaptor proteins that hand the mRNA cargoes over to the classical export receptors. In this study, we have found a noncanonical transport axis for m6A-marked mRNAs. Our preliminary data have shown that METTL3, an integral subunit of the m6A methyltransferase complex, directly interacts with NUP93, a component of the nuclear pore complex (NPC). RNA-binding capacity of METTL3 helps link the m6A-modified mRNAs to NUP93 on the nuclear envelope, which accelerates the passage of mRNA cargoes through the NPCs with the assistance of the principal export factor NXF1-NXT1. Integrative analysis of the m6A profile and fractionation transcriptome has identified a large group of m6A- harboring mRNAs whose nuclear export is dependent on the METTL3-NUP93 complex. Interestingly, these mRNAs are distinguished from other transcripts, as they tend to have longer 3' untranslated regions (3'-UTR), which is associated with more m6A sites and stronger methylation intensities. In addition, Gene Ontology analysis has revealed important functions of these transcripts in cellular physiology. In this regard, we found that the genetic mutations on NUP93, which have been reported to cause steroid-resistant nephrotic syndrome (SRNS) in humans, disrupts its interaction with METTL3, implying a potential association between dysregulation of METTL3-NUP93 axis and development of certain pathological conditions. Being guided by these preliminary data, we have developed biological systems to monitor the m6A status of test mRNA species and will employ cutting-edge technologies, such as single-point edge-excitation subdiffraction microscopy and epitranscriptome-editing tools, to define the role of METTL3-NUP93 axis in the nuclear export of m6A- conjugated mRNAs. Our Specific Aims are: (1). To examine how the METTL3-NUP93 interaction facilitates the nuclear export of m6A-marked mRNAs; (2). To determine which transcripts are specifically regulated by the METTL3-NUP93 axis; (3). To elucidate why this new mRNA transport pathway is biologically important. Our proposed research will impact upon the fields of RNA biology and epigenetics. Findings from this study will help delineate a novel mechanism of m6A-assisted mRNA export and offer new therapeutic targets for certain human diseases, such as kidney abnormalities that are caused by NUP93 mutations.