Role of mRNA internal 2′-O methylation in post-transcriptional regulation

Title: Role of mRNA internal 2′-O methylation in post-transcriptional regulation Project Summary/Abstract: Emerging evidence has proved the pivotal roles of RNA modifications in the control of various fundamental bioprocesses as well as diseases. As a prevalent modification type in most RNA species, 2′-O methylation (Nm) could present at all four ribonucleosides to affect RNA structure, stability, and interactions. In sharp contrast to the extensively studied rRNA Nm and 5′-cap mRNA Nm, Nm occurred at mRNA internal sites were only reported until recently, while its biological functions and underlying mechanisms remain underexplored. By combining Nanopore direct RNA-seq (Nanopore-seq) with our newly developed machine learning method, we recently identified thousands of novel mRNA Nm sites in human cells at single-base resolution and characterized fibrillarin (FBL) as a major 2′-O methyltransferase (MTase) to install this modification. Despite these advances, little is known about the regulatory network by which Nm governs mRNA functions and metabolism, and how Nm is manipulated to ensure the precise and synchronous regulation of gene expression. In our current studies, we propose three independent yet complementary projects to resolve this knowledge gap. By utilizing comprehensive proteomic, molecular, and multi-omics approaches, the first project will identify HNRNPF/H1 as distinct Nm downstream effector proteins and elucidate how this Nm&effector signaling remodels the splicing outcome of Nm-modified mRNAs globally. In addition, our Nanopore-seq results have revealed the evident co-occurrence of Nm with N6-methyladenosine (m6A), the most abundant RNA modifications in mammalian cells. Therefore, our second project will uncover the mechanism underlying the crosstalk between Nm and m6A modification and elucidate the impact of Nm&m6A dual methylation on mRNA fate. Dysregulation of Nm is frequently linked to multiple neurodevelopmental disorders. Hence, our last project will dedicate to discover the functional relevance of mRNA Nm on pluripotency maintenance and neuronal fate decision. Overall, our studies will gain novel insights into the biological functions of mRNA internal Nm and pioneer the Nm-targeting technological innovations to benefit human health. Project Number: 1R35GM162118-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Yang Yi | Institution: NORTHWESTERN UNIVERSITY, CHICAGO, IL | Award Amount: $440,000 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award A Study Section[MRAA] View on NIH RePORTER: https://reporter.nih.gov/project-details/11270366