Defining the direct impact of m6A deposition on messenger RNA

Harvard Medical School, Boston MA

Investigators

Abstract

N6-methyladenosine (m6A) is the most common internal mRNA modification and has been suggested to impact the formation and fate of mRNA. METTL3, the enzyme that deposits m6A, is essential for development and has emerged as a promising therapeutic target in various cancers, including acute myeloid leukemia (AML). Despite the dynamic processes m6A is implicated in, our understanding has predominantly been informed by long-term knockdown experiments whereby the cell has had ample time to compensate. However, to understand the direct influence and mechanisms underlying m6A’s role in RNA regulation, this proposal sets out to combine acute perturbations with precise transcriptomic measurements. Surprisingly, despite numerous studies demonstrating that long-term knockdown of METTL3 impacts transcription, short term METTL3 inhibition causes no significant changes in transcription initiation or elongation across gene bodies. Instead, gene upregulation observed by RNA-seq upon METTL3 inhibition reflects increased RNA stability. Although the regulation of RNA stability by cytoplasmic m6A readers is one of the most well-established functions of m6A, these data suggest an alternative mechanism. At the upregulated genes, altered 3’ end processing and a shift towards more distal 3’ Untranslated Regions (UTRs) are observed. Sequences within 3’ UTRs are known to impact mRNA stability, location, and translation, raising the intriguing possibility that regulation of 3’ UTR choice, and the altered RNA fate conferred by shifts in 3’ end sequences is central to m6A function. In this proposal, the hypothesis that m6A deposition impacts AML cell survival by directly regulating 3’ end processing will be tested. First, fast-acting inhibitors of METTL3 in combination with direct measurements of RNA synthesis and transcript 3’ ends will be used to define the consequences of m6A loss in AML cells (Aim 1). The model that an altered 3’ UTR sequence landscape is linked to the upregulation of AML driver genes will be tested. Next, the underlying mechanism by which m6A deposition impacts 3’ end processing will be explored through acute degradation and proteomic techniques (Aim 2). Finally, a cellular reporter assay will be employed to directly assess how 3’ UTR selection influences transcript abundance, stability, and translation of genes that promote AML proliferation (Aim 3). These results have the potential to clarify current models that have centered cytoplasmic m6A readers in regulating mRNA output. In addition, these results should inform therapeutic contexts wherein METTL3 inhibitors would be most beneficial. The proposed work will be conducted in the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School, which provides a rich interdisciplinary training environment and robust infrastructure to facilitate the successful completion of the aims. In addition, the proposed plan will deepen the lead investigator’s technical and conceptual training in cancer biology and transcriptomics. These experiences will support the principal investigator on her path towards leading a team of epigenomics researchers developing cancer therapeutics.