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 previous studies revealed that pseudouridine is a prevalent modification of nascent pre-messenger RNA. These results, together with quantitative in vitro studies showing that pseudouridine can affect both RNA-protein and RNA-RNA interactions, lead to our central hypothesis that pre-mRNA pseudouridylation controls human gene expression at the level of pre-mRNA splicing. In support of this hypothesis, we have demonstrated that loss of one pre- mRNA modifying pseudouridine synthase, PUS1, causes widespread changes to pre-mRNA splicing in human cells, with more than 3,000 PUS1-sensitive alternative splicing events identified.
Our Specific Aims are to (1) Define the splicing-relevant pre-mRNA targets of the predominant pre-mRNA pseudouridylating enzymes: PUS1, PUS7, and RPUSD4; and (2) Elucidate the molecular mechanisms of pseudouridine-sensitive splicing. The proposed work will provide key insight into the molecular functions of pseudouridines in pre-mRNAs and may reveal novel modes of eukaryotic gene regulation. By establishing pre-mRNAs as a broad new class of substrates for PUS enzymes, our work implicates defective splicing as a plausible but understudied mechanism connecting loss of PUS activity to numerous human diseases.

Public Health Relevance

Correct gene expression is fundamental to all aspects of healthy biology. This proposal seeks a better understanding of the functions of the RNA modification pseudouridine, which has significant but poorly understood effects on human gene expression. This research will have the potential to reveal new therapeutic targets to treat diseases caused by mutations in the enzymes that modify RNAs with pseudouridine.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Brown, Anissa F
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Yale University
Schools of Medicine
New Haven
United States
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Martinez, Nicole M; Gilbert, Wendy V (2018) Pre-mRNA modifications and their role in nuclear processing. Quant Biol 6:210-227
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Carlile, Thomas M; Rojas-Duran, Maria F; Zinshteyn, Boris et al. (2014) Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature 515:143-6