Peter Laura, Walotka Lara, Ptok Johannes, Meyer Caroline, Schüller Dominik, Schaal Heiner, Müller Lisa
Institute of Virology, University Hospital Düsseldorf, Medical Faculty, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany.
Institute of Virology, University Hospital Düsseldorf, Medical Faculty, Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany
RNA. 2024 Dec 16;31(1):32-42. doi: 10.1261/rna.080134.124.
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.
细胞无义介导的衰变（NMD）途径可识别并降解具有异常结构特征的mRNA，例如长3'非翻译区（UTR）或重叠阅读框，因此它作为一种转录本质量控制机制。目前关于无义介导的mRNA衰变途径的广泛知识是通过NMD报告系统发现的，该系统大多由多个外显子组成，包括一个野生型和一个含有提前终止密码子的变体。在一项初步的NMD研究中，我们使用了七外显子磷酸丙糖异构酶（TPI）报告基因，并观察到在这个著名的NMD报告基因中，令人惊讶的是，并非所有剪接位点都是组成性使用的，而是使用了额外的隐蔽剪接位点。由于这种情况在小基因构建中更频繁地出现，特别是当未知的剪接调节元件（SRE）被去除时，例如通过缩短内含子，这可能会影响此类报告基因的可靠性。为了证明一般情况下如何针对组成性剪接位点识别来改进此类小基因，我们恢复了TPI报告基因中的内含子长度，或者对SRE或剪接位点本身的内在强度进行了生物信息学调整。结果，这个NMD报告基因可以变得更加强健和特异，用于评估单个转录本内的NMD敏感性。此处作为示例展示的TPI报告基因的修饰通常可用于将细胞多外显子转录本转移到小基因中。
