Mechanism of the RNA helicase UPF1 regulatory loop in RNA decay target selection

The National Institute of General Medical Sciences awarded Villanova University $396,941 on August 12, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to investigate the mechanism of the RNA helicase UPF1 regulatory loop in RNA decay target selection.

The research focuses on nonsense-mediated decay (NMD), an RNA quality control pathway responsible for eliminating defective transcripts with premature termination codons and regulating turnover of 5–10 percent of normal messenger RNAs. One-third of human genetic diseases are affected by NMD, yet no FDA-approved strategies exist to modulate this pathway. The project seeks to understand how a conserved structural element called the regulatory loop in UPF1 controls target selection for NMD, with the long-term goal of designing systems to precisely modulate UPF1 function to control the fate of specific RNAs in human cells. The work will generate a library of regulatory loop mutants and characterize effects on UPF1 RNA binding and NMD substrate selection using methods ranging from in vitro helicase assays to genome-wide RNA-sequencing investigations.

The award is classified as a Project Grant with performance in Villanova, Pennsylvania. The period of performance runs from August 12, 2026, through July 31, 2029.

Abstract

Nonsense-mediated decay (NMD) is an RNA quality control pathway responsible for the elimination of defective transcripts that harbor premature termination codons as well as the regulated turnover of 5-10% of normal messenger rnas. An estimated one third of human genetic diseases are affected by NMD, yet there are no FDA-approved strategies to modulate this RNA decay pathway. This unmet need is due to our poor understanding of the basic mechanisms by which transcripts are selected for NMD and the step-by- step process by which substrates are decayed. Our long-term goal is to understand mechanisms of RNA target selection in NMD to modulate clinical outcomes associated with this RNA decay pathway. The objective of this proposal is to understand the mechanism by which a conserved structural element known as the regulatory loop in the core of the RNA helicase UPF1 controls target selection for the NMD pathway. Our central hypothesis is that the sequence, length, and structure of the regulatory loop are exquisitely tuned for modulating the association of UPF1 with RNA. The rationale behind this research objective is that, once IT is known how the UPF1 regulatory loop controls RNA decay target selection, we will use this understanding to design a system in which the function of UPF1 can be precisely modulated to control the fate of specific rnas for the NMD pathway in human cells. The central hypothesis will be tested by generating a library of regulatory loop mutants and characterizing the effects on UPF1 RNA binding and NMD substrate selection using methods that span from in vitro helicase assays to genome-wide investigations of RNA abundance by RNA-sequencing. In aim 1, the importance of regulatory loop architecture will be understood by determining how sequence, length, and structure modulate the binding of UPF1 to RNA in the presence of ATP. In aim 2, the function of the regulatory loop in controlling NMD target selection in response to a complex cellular environment will be determined. In aim 3, the extent to which the regulatory loop modulates activation of UPF1 by its cofactor UPF2 will be elucidated. The proposed research is significant because IT is expected to provide a mechanism for UPF1 target selection in NMD and offer new horizons for how this knowledge can be used to modulate the fate of specific rnas for this decay pathway in human cells. This work is broadly applicable for understanding many mechanisms by which RNA helicases govern gene regulatory pathways on which to continue future research.