Functions of mRNA Pseudouridylation

点击翻译按钮获取中文摘要

PROJECT SUMMARY Multiple pseudouridine synthases (PUS) are implicated in human disease, but the mechanisms that connect loss of PUS activity to mitochondrial myopathy, digestive disorders, intellectual disability, resistance to viral infection, dyskeratosis congenita, and diverse cancers remain largely unknown. There are several critical gaps in our current knowledge of the functions of PUS proteins. Although the basic biochemical activity of PUS proteins in catalyzing the isomerization of uridine to pseudouridine is well understood, the specific RNA targets of most human PUS proteins are unknown or incompletely known. Our long-term goals include identifying the targets of all PUS proteins and determining the molecular consequences of modification of specific RNAs with pseudouridine in disease-relevant cellular contexts. This will be critical for understanding the etiology of diseases caused by PUS deficiency and may reveal new therapeutic targets for treatment. Our recent work (Martinez et al. Mol Cell 2022) established that pseudouridine (Ψ) is deposited co-transcriptionally in nascent human pre-mRNA at thousands of sites where it is poised to affect every step of mRNA metabolism from birth to death. This renewal application will rigorously investigate the biochemical basis for mRNA regulation by pseudouridylation. The biological effects of Ψ must originate in chemical differences between U and Ψ, which directly affect RNA backbone conformation, the stability of base pairs, and the binding of proteins. Our preliminary data establish that hundreds of pseudouridines are present within the experimentally determined binding sites of regulatory RNA-binding proteins (RBPs) and micro RNAs (miRNAs) in human HepG2 cells. Widespread occurrence of Ψ in RBP and miRNA binding sites—together with biochemical studies of artificially pseudouridylated RNAs that demonstrate the capacity of Ψ to determine whether, and to what extent, an mRNA will be bound—establish a strong premise for the proposed work. We will (1) Determine impact of mRNA Ψ on functional interactions with regulatory RNA-binding proteins, (2) Identify the roles of pseudouridylation in miRNA-mediated gene regulation, and (3) Define the contributions of regulated RNA pseudouridylation to the control of gene expression. Together, the proposed work will elucidate the molecular consequences of mRNA pseudouridylation in human cells and define functionally relevant mRNA targets of human pseudouridine synthases. This will fill a critical knowledge gap for understanding how RNA modifying enzymes that are essential for human health control gene expression.