Attenuation of Nonsense-Mediated mRNA Decay Enhances In Vivo Nonsense Suppression

Kim M Keeling,Jeremy Clark,Dan Wang,Yanying Dai,Sadanandan E Velu,Timor Baasov,Jeyakumar Kandasamy,Valery Belakhov,David M Bedwell,Balachandra Chenna,Srinivasan Murugesan,David J Reiner

doi:10.1371/journal.pone.0060478

Abstract

Nonsense suppression therapy is an approach to treat genetic diseases caused by nonsense mutations. This therapeutic strategy pharmacologically suppresses translation termination at Premature Termination Codons (PTCs) in order to restore expression of functional protein. However, the process of Nonsense-Mediated mRNA Decay (NMD), which reduces the abundance of mRNAs containing PTCs, frequently limits this approach. Here, we used a mouse model of the lysosomal storage disease mucopolysaccharidosis I-Hurler (MPS I-H) that carries a PTC in the Idua locus to test whether NMD attenuation can enhance PTC suppression in vivo. Idua encodes alpha-L-iduronidase, an enzyme required for degradation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. We found that the NMD attenuator NMDI-1 increased the abundance of the PTC-containing Idua transcript. Furthermore, co-administration of NMDI-1 with the PTC suppression drug gentamicin enhanced alpha-L-iduronidase activity compared to gentamicin alone, leading to a greater reduction of GAG storage in mouse tissues, including the brain. These results demonstrate that NMD attenuation significantly enhances suppression therapy in vivo.

Highlights

Nonsense suppression drugs reduce the efficiency of translation termination at in-frame premature termination codons (PTCs; nonsense mutations), thereby allowing ribosomes to resume translation elongation and generate a full-length protein [1,2]
In agreement with our hypothesis, we found that coadministration of the nonsense-mediated mRNA decay (NMD) attenuator NMDI-1 with a subset of PTC suppression drugs alleviated mucopolysaccharidosis I-Hurler (MPS I-H) biochemical defects to a greater extent than suppression therapy alone both in vitro and in vivo
Caffeine inhibits the SMG1 kinase that phosphorylates UPF1 [23,31], while NMDI-1 blocks the interaction between UPF1 and SMG5, which prevents the recruitment of the PP2A phosphatase to dephosphorylate UPF1 [24]

Summary

Introduction

Nonsense suppression drugs reduce the efficiency of translation termination at in-frame premature termination codons (PTCs; nonsense mutations), thereby allowing ribosomes to resume translation elongation and generate a full-length protein [1,2]. We hypothesize that attenuating NMD to increase the abundance of PTC-containing mRNAs will restore higher levels of functional protein produced by PTC suppression, providing a greater therapeutic benefit. The UPF1 phosphorylation cycle represents a potential pharmacological target for NMD attenuation [16,17]. The kinase SMG1, UPF1, and the release factor complex that mediates translation termination form the SURF complex at PTC-bound ribosomes to initiate NMD [18,19,20]. If a PTC-bound SURF complex interacts with a downstream exon junction complex, SMG1 phosphorylates UPF1 at multiple residues, which marks the transcript for decay. The SMG5/7 complex recruits the PP2A phosphatase to dephosphorylate and recycle UPF1, while the mRNA decay machinery subsequently degrades the PTC-containing transcript [21,22]. NMDI-1 blocks UPF1 dephosphorylation by disrupting the interaction between SMG5 and phospho-UPF1 [24]

Methods

Results

Conclusion