Abstract
Many heritable genetic disorders arise from nonsense mutations, which generate premature termination codons (PTCs) in transcribed mRNA. PTCs ablate protein synthesis by prematurely terminating the translation of mutant mRNA, as well as reducing mutant mRNA quantity through targeted degradation by nonsense-mediated decay (NMD) mechanisms. Therapeutic strategies for nonsense mutations include facilitating ribosomal readthrough of the PTC and/or inhibiting NMD to restore protein function. However, the efficacy of combining readthrough agents and NMD inhibitors has not been thoroughly explored. In this study, we examined combinations of known NMD inhibitors and readthrough agents using functional analysis of the CFTR protein in primary cells from a mouse model carrying a G542X nonsense mutation in Cftr. We observed synergy between an inhibitor of the NMD component SMG-1 (SMG1i) and the readthrough agents G418, gentamicin, and paromomycin, but did not observe synergy with readthrough caused by amikacin, tobramycin, PTC124, escin, or amlexanox. These results indicate that treatment with NMD inhibitors can increase the quantity of functional protein following readthrough, and that combining NMD inhibitors and readthrough agents represents a potential therapeutic option for treating nonsense mutations.
Highlights
IntroductionNonsense mutations are single nucleotide alterations which generate premature UAG, UAA, or UGA termination codons (PTCs) in mRNA transcripts
Ten percent of heritable diseases are caused by nonsense mutations [1,2]
We examined forskolin-induced swelling (FIS) in G542X intestinal organoids following a 24 h incubation range of each drug included doses used in other publications [15] as well as the highest with PTC124, escin, and amlexanox in conjunction with SMG1i treatment
Summary
Nonsense mutations are single nucleotide alterations which generate premature UAG, UAA, or UGA termination codons (PTCs) in mRNA transcripts. ERF1 is preferentially incorporated into the A site of the ribosome, causing hydrolysis of the ester bond linking the elongating polypeptide chain to the P-site tRNA [3]. This results in premature termination of translation and generates truncated proteins which are often nonfunctional or have deleterious dominant-negative properties [4,5]. Expending of energy and resources on producing dysfunctional protein is a wasteful procedure for the cell; nonsensecarrying transcripts are degraded prior to translation by nonsense-mediated decay (NMD). Failure to complete the first round of translation signals the recruitment of NMD machinery which degrades the mutant transcripts, preventing future attempts at translation [6,7,8]
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