Authors

Discussion

The RNA-binding PUF proteins are post-transcriptional regulators found throughout the eukaryotic domain that control the stability and translation of transcripts through the binding to specific recognition sequences in the 3´untranslated regions (3´-UTRs) of mRNAs. Few PUF proteins have been characterized in Trypanosoma cruzi. Considering that the control of gene expression in this parasite is mainly at the posttranscriptional level, further studies are needed to determine the functional depiction of the PUF family. Here, 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 analysis in both genotypes showed significant changes in the number of regulated transcripts compared with wild-type parasites. Thus, the differentially expressed genes analysis in the knockout (ΔTcPuf3) and the overexpressor (pTEXTcPuf3) were 238 and 187, respectively. However, the crucial distinction lies in the direction of the change in expression. 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 (166 out of 187). Additionally, when we predicted the subcellular location of the differentially expressed genes, the results revealed a notable overrepresentation of nuclear genes encoding mitochondrial proteins. To ascertain the direct targets of PUF3 among the identified genes, we searched for the PUF3 UGUAYAUW binding motif in the 3'-UTR. Our analysis reveals that 25% of differentially expressed genes in the knockout parasites possess at least one binding site for PUF3 in their 3'-UTR. Interestingly, a parallel proportion of 18.82% was observed in the overexpressor. Since our transcriptomic result suggests an overrepresentation of mitochondrial genes, we wanted to determine whether overexpression or knock-out of TcPuf3 could lead to changes in both mitochondrial structure and cellular respiration. When mitochondria fromΔTcPuf3 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 theΔTcPuf3 and pTEXTcPuf3 had lower ATP-linked respiration and a diminished bioenergetic reserve, which suggests an impaired electron transport system capacity. Likewise, the mitochondria from overexpressing parasites showed hyperpolarization. Additionally, several biological features were altered, such as growth, cell cycle, cell infection, metacyclogenesis, ROS production, and response to benznidazole, depending on energy obtained from mitochondria. In conclusion, our results suggest that although PUF3 is not an essential protein in T. cruzi, it strongly influences mitochondrial transcripts, affecting mitochondrial morphology and function.