Ionis Pharmaceuticals, Inc., Carlsbad, CA, USA.
Cystic Fibrosis Foundation Therapeutics Lab, Cystic Fibrosis Foundation, Lexington, MA, USA.
Nat Commun. 2022 Apr 29;13(1):2344. doi: 10.1038/s41467-022-29935-9.
Approximately 10% of cystic fibrosis patients harbor nonsense mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene which can generate nonsense codons in the CFTR mRNA and subsequently activate the nonsense-mediated decay (NMD) pathway resulting in rapid mRNA degradation. However, it is not known which NMD branches govern the decay of CFTR mRNAs containing nonsense codons. Here we utilize antisense oligonucleotides targeting NMD factors to evaluate the regulation of nonsense codon-containing CFTR mRNAs by the NMD pathway. We observe that CFTR mRNAs with nonsense codons G542X, R1162X, and W1282X, but not Y122X, require UPF2 and UPF3 for NMD. Furthermore, we demonstrate that all evaluated CFTR mRNAs harboring nonsense codons are degraded by the SMG6-mediated endonucleolytic pathway rather than the SMG5-SMG7-mediated exonucleolytic pathway. Finally, we show that upregulation of all evaluated CFTR mRNAs with nonsense codons by NMD pathway inhibition improves outcomes of translational readthrough therapy.
约 10%的囊性纤维化（CF）患者的囊性纤维化跨膜电导调节因子（CFTR）基因存在无义突变，这些突变会在 CFTR mRNA 中产生无义密码子，进而激活无义介导的降解（NMD）途径，导致 mRNA 迅速降解。然而，目前尚不清楚哪些 NMD 分支负责无义密码子 CFTR mRNA 的降解。本研究利用靶向 NMD 因子的反义寡核苷酸来评估 NMD 途径对含无义密码子的 CFTR mRNA 的调控。结果发现，虽然 CFTR 基因的 G542X、R1162X 和 W1282X 等无义突变的 mRNA 需要 UPF2 和 UPF3 才能进行 NMD，但 Y122X 无义突变的 mRNA 不需要。此外，本研究表明，所有评估的 CFTR 无义突变 mRNA 均通过 SMG6 介导的内切酶途径降解，而不是通过 SMG5-SMG7 介导的外切酶途径。最后，研究结果显示，通过抑制 NMD 途径上调所有评估的含无义密码子的 CFTR mRNA 可改善翻译通读治疗的效果。
