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. 2017 Mar 21;12(3):e0173980.
doi: 10.1371/journal.pone.0173980. eCollection 2017.

A System for Coordinated Analysis of Translational Readthrough and Nonsense-Mediated mRNA Decay

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Free PMC article

A System for Coordinated Analysis of Translational Readthrough and Nonsense-Mediated mRNA Decay

Stacey L Baker et al. PLoS One. .
Free PMC article

Abstract

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing premature termination codons, limiting the expression of potentially deleterious truncated proteins. This activity positions the pathway as a regulator of the severity of genetic diseases caused by nonsense mutations. Because many genetic diseases result from nonsense alleles, therapeutics inducing readthrough of premature termination codons and/or inhibition of NMD have been of great interest. Several means of enhancing translational readthrough have been reported to concomitantly inhibit NMD efficiency, but tools for systematic analysis of mammalian NMD inhibition by translational readthrough are lacking. Here, we introduce a system that allows concurrent analysis of translational readthrough and mRNA decay. We use this system to show that diverse readthrough-promoting RNA elements have similar capacities to inhibit NMD. Further, we provide evidence that the level of translational readthrough required for protection from NMD depends on the distance of the suppressed termination codon from the end of the mRNA.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Unified system for assays of recoding, mRNA decay, and mRNP composition.
(a) Schematic of constructs used to express PP7-tagged mRNAs encoding dual-fluorescent protein reporters. Stop signs indicate possible positions of termination codons; pA refers to bovine growth hormone polyadenylation signal. (b) Immunoblot of UPF1 and PABPC1 present in input extracts (left) or PP7-based affinity purifications containing mRNAs with (UAA) or without (CAA) a stop codon between the GFP and mCherry ORFs. Bottom, mRNA recovery was monitored by northern blot. Mock purifications lacking tagged RNAs were performed in parallel to assess background recovery of proteins. (c) Constructs containing an efficient UAA TC or no TC (CAA) between the GFP and mCherry ORFs were used for pulse-chase mRNA decay assays. Northern blotting with a probe against GFP sequence was used to quantify abundance of tetracycline-regulated experimental mRNAs (pcTET2 2FP) and constitutively expressed control mRNAs (pcGFP-bGH). RNA half-lives corresponding to best-fit lines to semi-log plots of RNA abundance data and 95% confidence intervals (CI) are listed (n = 3). (d) Semi-log plot of mRNA decay assays shown in C. Error bars indicate +/- SD (n = 3); p-value derived from ANCOVA analysis comparing CAA and UAA. (e) Decay assay of mRNAs containing a UAA TC in cells treated with non-silencing control siRNAs or anti-UPF1 siRNAs. RNA half-lives corresponding to best-fit lines to semi-log plots of RNA abundance data and 95% confidence intervals (CI) are listed (n = 3). (f) Semi-log plot of mRNA decay assays shown in E. Error bars indicate +/- SD (n = 3); p-value derived from ANCOVA analysis comparing siNT and siUPF1. (g) Schematic of MLVPK RNA secondary structure, indicating the position of the wild-type UAG TC [39]. (h) Schematic of mRNAs (top) containing a single upstream TC regulated by a MLVPK variant, (middle) containing the MLVPK sequence but lacking a TC, and (bottom) containing the MLVPK sequence and an additional in-frame UAA TC. Positions of the pseudoknot sequences are indicated by PK. (i) Relative readthrough efficiencies calculated from GFP and mCherry levels produced by mRNAs containing the indicated MLVPK variants. Error bars indicate ± SD (n = 3); ****: p<0.0001 in one-way ANOVA analysis comparing the indicated samples to UAG. See Materials and methods for details of readthrough efficiency calculations.
Fig 2
Fig 2. Relationship between readthrough-mediated stabilization and downstream ORF length.
(a) NanoLuc or HIV RNase H sequences were added to dual-fluorescent reporter mRNAs in the indicated positions. (b) Decay assays of mRNAs in which UGA or UAA TCs following the GFP ORF were modulated by the MLVPK and NanoLuc (Nluc) or HIV RNase H (HIV RH) sequences were added as indicated. RNA half-lives corresponding to best-fit lines to semi-log plots of RNA abundance data and 95% confidence intervals are listed (n = 3). Readthrough efficiencies (mean +/- SD; n = 3) for each construct are indicated below the blot panels. (c) Semi-log plot of mRNA decay assays shown in B. Error bars indicate +/- SD (n = 3); p-values derived from ANCOVA analysis. (d) Decay assays of mCherry-NanoLuc reporter mRNAs as in A. Cells were treated with control siRNAs (siNS) or UPF1 siRNAs (siUPF1) as indicated. Half-lives are represented as in B. (e) Quantification of mRNA decay assays shown in D; note reduced duration of timecourse vs. C (360 min.). Error bars indicate +/- SD (n = 3); p-values derived from ANCOVA analysis.
Fig 3
Fig 3. Distinct readthrough-promoting elements inhibit NMD.
(a) Schematic of the proposed CTFV readthrough-promoting RNA hairpin structure (HP) [36]. (b) CTFV HP variants were inserted into dual-flourescent reporters. As a “no-readthrough” control, an additional TC was inserted downstream of the HP sequence (CTFV HP +TC). (c) Decay assays of mRNAs containing the indicated CTFV HP variants. RNA half-lives corresponding to best-fit lines to semi-log plots of RNA abundance data and 95% confidence intervals are listed (n = 3). Readthrough efficiencies (mean +/- SD; n = 3) for each construct are indicated below the blot panels. (d) Semi-log plot of mRNA decay assays shown in C. Error bars indicate +/- SD (n = 3); p-values derived from ANCOVA analysis. (e) Decay assays of CTFV +TC reporter mRNAs. Cells were treated with control siRNAs (siNS) or UPF1 siRNAs (siUPF1) as indicated. Half-lives are represented as in C. (f) Quantification of mRNA decay assays shown in E; note reduced duration of timecourse vs. D (360 min.). Error bars indicate +/- SD (n = 4); p-values derived from ANCOVA analysis.
Fig 4
Fig 4. mRNA stabilization by readthrough of a pathogenic nonsense allele.
(a) Sequence of the LAMA3 R943X allele [37]. (b) Decay assays of mRNAs containing the indicated LAMA3-derived sequence variants. Readthrough efficiencies (mean +/- SD; n = 3) for each construct are indicated below the blot panels. (c) Semi-log plot of mRNA decay assays shown in C. Error bars indicate +/- SD (n = 3); p-value derived from ANCOVA analysis.

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Grant support

This work was supported by the Intramural Research Program, National Institutes of Health, National Heart, Lung, and Blood Institute. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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