1. Bioinformatics-driven refinement of the commonly used TPI nonsense-mediated decay reporter system.
- Author
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Peter L, Walotka L, Ptok J, Meyer C, Schüller D, Schaal H, and Müller L
- Subjects
- Humans, Introns genetics, RNA, Messenger genetics, RNA, Messenger metabolism, Codon, Nonsense, Nonsense Mediated mRNA Decay, Computational Biology methods, Genes, Reporter, Exons genetics, RNA Splice Sites, Triose-Phosphate Isomerase genetics, Triose-Phosphate Isomerase metabolism
- Abstract
The cellular nonsense-mediated decay (NMD) pathway recognizes and degrades mRNAs with unusual structural features, such as long 3' UTRs or overlapping reading frames, and therefore serves as a transcript quality control mechanism. A broad spectrum of today's knowledge about the nonsense-mediated mRNA decay pathway has been discovered using NMD reporter systems, mostly consisting of multiple exons, with a wild-type and a premature termination codon-containing variant. In a preliminary NMD study, we used the seven-exon triose phosphate isomerase (TPI) reporter and observed that in this well-known NMD reporter, surprisingly, not all splice sites are used constitutively, but additional cryptic splice sites are used. As this is more frequently observed in the construction of minigenes, especially when unknown splicing regulatory elements (SREs) are removed, for example, by shortening introns, this may affect the reliability of such reporters. To demonstrate how such minigenes can be improved in general with respect to constitutive splice site recognition, we restored an intron length in the TPI reporter or made bioinformatic adjustments to SREs or intrinsic strength of the splice sites themselves. As a result, this NMD reporter could be made more robust and specific for the evaluation of NMD sensitivity within a single transcript. The modifications of the TPI reporter shown here as examples can generally be used for the transfer of cellular multiexon transcripts to minigenes., (© 2025 Peter et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.) more...
- Published
- 2024
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