PDF(Pubmed)
Abstract：
The RNA-binding PUF proteins are post-transcriptional regulators found throughout the eukaryotic domain. In Trypanosoma cruzi, ten Puf genes termed Puf1 to Puf10 have been identified. Considering that the control of gene expression in this parasite is mainly at the post-transcriptional level, we characterized the PUF3 protein by knocking out and overexpressing the gene in T. cruzi epimastigotes and studied different genetic and biological features. The RNA-seq analyses in both genotypes showed significant changes in the number of regulated transcripts compared with wild-type parasites. Thus, the number of differentially expressed genes in the knockout (ΔTcPuf3) and the overexpressor (pTEXTcPuf3) were 238 and 187, respectively. In the knockout, a more significant proportion of genes was negatively regulated (166 out of 238). In contrast, in the overexpressor, positively regulated genes were predominant (149 out of 170). Additionally, when we predicted the subcellular location of the differentially expressed genes, the results revealed an important representation of nuclear genes encoding mitochondrial proteins. Therefore, we determined whether overexpression or knockout of TcPuf3 could lead to changes in both mitochondrial structure and cellular respiration. When mitochondria from ΔTcPuf3 and pTEXTcPuf3 parasites were analyzed by transmission electron microscopy (TEM), it was observed that the overexpressor had an abnormal mitochondrial morphology, evidenced by swelling. The results associated with cellular respiration showed that both the ΔTcPuf3 and pTEXTcPuf3 had a lower efficiency in routine respiration and the electron transport system capacity. Likewise, the mitochondria from overexpressing parasites showed a slight hyperpolarization. Additionally, several biological features, depending on the function of the mitochondria, were altered, such as growth, cell division, metacyclogenesis, ROS production, and response to benznidazole. In conclusion, our results suggest that although PUF3 is not an essential protein in T. cruzi, it influences mitochondrial transcripts, affecting mitochondrial morphology and function.
摘要：
RNA结合PUF蛋白是在整个真核结构域中发现的转录后调节因子。在克氏锥虫中，已经鉴定了10个称为Puf1至Puf10的Puf基因。考虑到该寄生虫中基因表达的控制主要在转录后水平，我们通过敲除和过表达T.cruziepimastigotes中的基因来表征PUF3蛋白，并研究了不同的遗传和生物学特征。两种基因型的RNA-seq分析显示，与野生型寄生虫相比，受调控的转录物数量发生了显着变化。因此，敲除基因（ΔTcPuf3）和过表达基因（pTEXTcPuf3）中差异表达基因的数量分别为238和187。在淘汰赛中，更显著比例的基因受到负调控(238个基因中的166个).相比之下,在过压缩中，正调控基因占优势（170个中的149个）。此外,当我们预测差异表达基因的亚细胞位置时，结果揭示了编码线粒体蛋白的核基因的重要代表。因此,我们确定了TcPuf3的过表达或敲除是否会导致线粒体结构和细胞呼吸的变化.当通过透射电子显微镜（TEM）分析ΔTcPuf3和pTEXTcPuf3寄生虫的线粒体时，观察到过表达具有异常的线粒体形态，肿胀证明。与细胞呼吸相关的结果表明，ΔTcPuf3和pTEXTcPuf3在常规呼吸和电子传递系统容量中均具有较低的效率。同样，来自过表达寄生虫的线粒体显示出轻微的超极化。此外,几个生物学特征，取决于线粒体的功能，被改变了，比如增长，细胞分裂,元环发生，ROS生产，和对苯并咪唑的反应。总之,我们的结果表明，尽管PUF3不是克氏毛虫的必需蛋白，它影响线粒体转录本，影响线粒体形态和功能。
