Influence of mRNA sequences downstream of a premature stop codon on nonsense suppression.

Abstract

Premature stop codons (PSCs) arrest translation of full-length protein and trigger nonsense-mediated decay (NMD) of mRNA. Nonsense suppression by translational readthrough inducing drugs (TRIDs) like ataluren can restore protein function by inhibiting translation termination at PSCs catalyzed by release factor complex (RFC, eRF1.eRF3.GTP). We seek to understand what determines readthrough efficiency and apply our results to improve treatment of PSC diseases such as cystic fibrosis (CF). For this purpose, we utilize an in vitro translation system to examine the effects on RFC enzymatic activity of varying PSCs and downstream mRNA sequences in pretermination complexes, as measured with a high throughput fluorescence anisotropy assay and single-molecule TIRF microscopy. We have so far studied a total of seven sequences downstream from stop codons. Within this limited set, we find much lower EC50RFC values for sequences with stop codons UAG or UAA than for sequences having a UGA stop codon. These sequences display a ∼170-fold difference in measured EC50RFC values, a much larger range than the very limited range of RFC binding rates, 1.7-fold. We speculate that this difference is due, at least in part, to differences in the rate of reversible RFC dissociation. Additional sequences are currently under study. Our preliminary results explain, at least in part, why readthrough frequency is generally much higher for the UGA stop codon than for the UAG or UAA stop codons. They further suggest that combinations of drugs acting orthogonally to stimulate readthrough may be required to treat some PSC diseases, and that consideration of the mRNA sequence context flanking a nonsense codon might be critical for developing patient-specific therapeutic regimens.