UPF1 mutants with intact ATPase but deficient helicase activities promote efficient nonsense-mediated mRNA decay

Abstract

The conserved RNA helicase UPF1 coordinates nonsense-mediated mRNA decay (NMD) by engaging with mRNAs, RNA decay machinery and the terminating ribosome. UPF1 ATPase activity is implicated in mRNA target discrimination and completion of decay, but the mechanisms through which UPF1 enzymatic activities such as helicase, translocase, RNP remodeling, and ATPase-stimulated dissociation influence NMD remain poorly defined. Using high-throughput biochemical assays to quantify UPF1 enzymatic activities, we show that UPF1 is only moderately processive (<200 nt) in physiological contexts and undergoes ATPase-stimulated dissociation from RNA. We combine an in silico screen with these assays to identify and characterize known and novel UPF1 mutants with altered helicase, ATPase, and RNA binding properties. We find that UPF1 mutants with substantially impaired processivity (E797R, G619K/A546H), faster (G619K) or slower (K547P, E797R, G619K/A546H) unwinding rates, and/or reduced mechanochemical coupling (i.e. the ability to harness ATP hydrolysis for work; K547P, R549S, G619K, G619K/A546H) can still support efficient NMD of well-characterized targets in human cells. These data are consistent with a central role for UPF1 ATPase activity in driving cycles of RNA binding and dissociation to ensure accurate NMD target selection.

Two Single Nucleotide Deletions in the ABCD1 Gene Causing Distinct Phenotypes of X-Linked Adrenoleukodystrophy

“…Although both pathogenic variants reported here were predicted to cause a complete absence of the ABCD1 protein, the variant c.253delC in exon 1 led to the most severe cerebral X-ALD, while c.1275delA in exon 4 caused moderate AMN phenotype. The culprit seems to be multifactorial and besides toxicity of the accumulated VLCFA, modifier genes and environmental/epigenetic factors might contribute [ 21 , 22 , 23 ].…”

Nonsense-Mediated mRNA Decay Factor Functions in Human Health and Disease

“…A wide range of conformations have been observed in structural studies, shedding light on the molecular dynamics linking UPF1’s RNA-associated catalytic functions to ATP-binding and hydrolysis (reviewed in [ 29 ]). ATP binding hinders the binding of RNA by UPF1 [ 81 ], and UPF1’s ATPase activity is essential to NMD [ 38 , 83 , 84 ]. The ATPase activity has been shown to be required for UPF1 to remodel ribonucleoprotein complexes (RNPs) by fuelling its RNA translocase and unwinding activities [ 34 , 85 , 86 , 87 ], promoting ribosome recycling at PTCs [ 34 , 86 ], displacing protective RNA-binding proteins [ 78 , 85 ] and disassembling NMD complexes for efficient RNA degradation [ 83 ].…”

“…The ATPase activity has been shown to be required for UPF1 to remodel ribonucleoprotein complexes (RNPs) by fuelling its RNA translocase and unwinding activities [ 34 , 85 , 86 , 87 ], promoting ribosome recycling at PTCs [ 34 , 86 ], displacing protective RNA-binding proteins [ 78 , 85 ] and disassembling NMD complexes for efficient RNA degradation [ 83 ]. A recent study using a range of UPF1 ATPase and helicase mutants has shown that ATP-hydrolysis stimulates dissociation from the RNA in a helicase-decoupled manner [ 84 ]. Intriguingly, UPF1 mutants with impaired helicase activity but intact ATPase function still support NMD [ 84 ].…”

“…A recent study using a range of UPF1 ATPase and helicase mutants has shown that ATP-hydrolysis stimulates dissociation from the RNA in a helicase-decoupled manner [ 84 ]. Intriguingly, UPF1 mutants with impaired helicase activity but intact ATPase function still support NMD [ 84 ]. In contrast, mutants deficient in the ATPase activity result in compromised NMD substrate selectivity [ 88 , 89 ].…”

“…In contrast, mutants deficient in the ATPase activity result in compromised NMD substrate selectivity [ 88 , 89 ]. Together, these results suggest that UPF1’s ATPase activity is essential for NMD, due to stimulation of RNA dissociation and concomitant release of non-NMD substrates, while UPF1’s helicase activity, which unwinds RNA and remodels RNPs, is not crucial for NMD [ 84 ].…”