PUF3 RNA binding protein of Trypanosoma cruzi regulates mitochondrial morphology and function

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.