Instituto de Investigaciones Biotecnológicas, Universidad Nacional de San Martín-Consejo Nacional de Investigaciones Científicas y Técnicas, General San Martín, Prov. de Buenos Aires, Argentina.
Escuela de Bio y Nanotecnologías (EByN), Universidad Nacional de San Martín, General San Martín, Prov. de Buenos Aires, Argentina.
PLoS Negl Trop Dis. 2024 May 17;18(5):e0012179. doi: 10.1371/journal.pntd.0012179. eCollection 2024 May.
During its life cycle, the human pathogen Trypanosoma cruzi must quickly adapt to different environments, in which the variation in the gene expression of the regulatory U-rich RNA-binding protein 1 (TcUBP1) plays a crucial role. We have previously demonstrated that the overexpression of TcUBP1 in insect-dwelling epimastigotes orchestrates an RNA regulon to promote differentiation to infective forms.
In an attempt to generate TcUBP1 knockout parasites by using CRISPR-Cas9 technology, in the present study, we obtained a variant transcript that encodes a protein with 95% overall identity and a modified N-terminal sequence. The expression of this mutant protein, named TcUBP1mut, was notably reduced compared to that of the endogenous form found in normal cells. TcUBP1mut-knockdown epimastigotes exhibited normal growth and differentiation into infective metacyclic trypomastigotes and were capable of infecting mammalian cells.
We analyzed the RNA-Seq expression profiles of these parasites and identified 276 up- and 426 downregulated genes with respect to the wildtype control sample. RNA-Seq comparison across distinct developmental stages revealed that the transcriptomic profile of these TcUBP1mut-knockdown epimastigotes significantly differs not only from that of epimastigotes in the stationary phase but also from the gene expression landscape characteristic of infective forms. This is both contrary to and consistent with the results of our recent study involving TcUBP1-overexpressing cells.
Together, our findings demonstrate that the genes exhibiting opposite changes under overexpression and knockdown conditions unveil key mRNA targets regulated by TcUBP1. These mostly encompass transcripts that encode for trypomastigote-specific surface glycoproteins and ribosomal proteins, supporting a role for TcUBP1 in determining the molecular characteristics of the infective stage.
在生命周期中，人体病原体克氏锥虫必须快速适应不同的环境，在这些环境中，调节 U 丰富 RNA 结合蛋白 1（TcUBP1）的基因表达变化起着至关重要的作用。我们之前已经证明，在昆虫栖居的前鞭毛体中转录本过表达 TcUBP1 可协调一个 RNA 调控因子，促进向感染性形式的分化。
在尝试使用 CRISPR-Cas9 技术生成 TcUBP1 敲除寄生虫的过程中，本研究获得了一种变体转录本，该转录本编码一种具有 95%整体同一性和修饰的 N 端序列的蛋白质。与在正常细胞中发现的内源性形式相比，这种突变蛋白 TcUBP1mut 的表达显著降低。TcUBP1mut 敲低的前鞭毛体表现出正常的生长和分化为感染性的循环体，并能够感染哺乳动物细胞。
我们分析了这些寄生虫的 RNA-Seq 表达谱，并鉴定出相对于野生型对照样本，有 276 个上调和 426 个下调基因。在不同发育阶段的 RNA-Seq 比较表明，这些 TcUBP1mut 敲低的前鞭毛体的转录组谱不仅与静止期前鞭毛体的转录组谱显著不同，而且与感染性形式的基因表达图谱也显著不同。这与我们最近涉及 TcUBP1 过表达细胞的研究结果既相反又一致。
总之，我们的研究结果表明，在过表达和敲低条件下表现出相反变化的基因揭示了 TcUBP1 调节的关键 mRNA 靶标。这些基因主要包括编码循环体特异性表面糖蛋白和核糖体蛋白的转录本，支持 TcUBP1 在确定感染阶段的分子特征方面的作用。
