Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne , Parkville, Victoria, Australia.
Department of Physiology, Anatomy and Microbiology, La Trobe University , Bundoora, Victoria, Australia.
mSphere. 2023 Aug 24;8(4):e0023323. doi: 10.1128/msphere.00233-23. Epub 2023 Jun 27.
Nonsense-mediated decay (NMD) is a conserved mRNA quality control process that eliminates transcripts bearing a premature termination codon. In addition to its role in removing erroneous transcripts, NMD is involved in post-transcriptional regulation of gene expression via programmed intron retention in metazoans. The apicomplexan parasite shows relatively high levels of intron retention, but it is unclear whether these variant transcripts are functional targets of NMD. In this study, we use CRISPR-Cas9 to disrupt and epitope-tag the orthologs of two core NMD components: UPF1 (PF3D7_1005500) and UPF2 (PF3D7_0925800). We localize both UPF1 and UPF2 to puncta within the parasite cytoplasm and show that these proteins interact with each other and other mRNA-binding proteins. Using RNA-seq, we find that although these core NMD orthologs are expressed and interact in , they are not required for degradation of nonsense transcripts. Furthermore, our work suggests that the majority of intron retention in has no functional role and that NMD is not required for parasite growth . IMPORTANCE In many organisms, the process of destroying nonsense transcripts is dependent on a small set of highly conserved proteins. We show that in the malaria parasite, these proteins do not impact the abundance of nonsense transcripts. Furthermore, we demonstrate efficient CRISPR-Cas9 editing of the malaria parasite using commercial Cas9 nuclease and synthetic guide RNA, streamlining genomic modifications in this genetically intractable organism.
无意义介导的衰变 (NMD) 是一种保守的 mRNA 质量控制过程，可消除携带过早终止密码子的转录本。除了在去除错误转录本中的作用外，NMD 还通过真核生物中的程序性内含子保留参与基因表达的转录后调控。顶复门寄生虫 表现出相对较高的内含子保留水平，但尚不清楚这些变体转录本是否是 NMD 的功能靶标。在这项研究中，我们使用 CRISPR-Cas9 破坏和表位标记两个核心 NMD 成分的 直系同源物：UPF1（PF3D7_1005500）和 UPF2（PF3D7_0925800）。我们将 UPF1 和 UPF2 定位到寄生虫细胞质内的斑点中，并表明这些蛋白质相互作用以及与其他 mRNA 结合蛋白相互作用。使用 RNA-seq，我们发现尽管这些核心 NMD 直系同源物在 中表达并相互作用，但它们不是降解无意义转录本所必需的。此外，我们的工作表明， 中大多数内含子保留没有功能作用，并且 NMD 不是寄生虫生长所必需的。重要性 在许多生物体中，破坏无意义转录本的过程依赖于一小部分高度保守的蛋白质。我们表明，在疟原虫中，这些蛋白质不会影响无意义转录本的丰度。此外，我们使用商业 Cas9 核酸酶和合成向导 RNA 展示了在疟原虫中进行有效的 CRISPR-Cas9 编辑，简化了这个遗传上难以处理的生物体中的基因组修饰。
