Translational regulation of gene expression plays an important role in the development of diverse eukaryotes. In many cases, post-transcriptional regulation requires cis-acting sequences located in either the 3′ or 5′ UTRs of the transcript. We have shown here that T.gondii possesses two distinct Puf members, which share limited sequence similarity, suggesting they might regulate different RNA repertoires and have different functions. Interestingly, TgPuf1 and TgPuf2 are more homologous to their respective Puf1 and Puf2 genes in Plasmodium, suggesting that the duplication of Puf genes in these two Apicomplexan parasites occurred earlier before the divergence of these parasite taxa. In the malaria parasite P. berghei, only PbPuf2 are found to regulate the stage-transition in sporozoites, whereas deletion of PbPuf1 had no effects on this process[1, 2, 19, 28]. In P. falciparum both Puf proteins are abundantly expressed in gametocytes and Puf2 plays an important role during gametocytogenesis. In Toxoplasma, TgPuf1 appeared to be more abundantly expressed in bradyzoites at the protein level, suggesting that TgPuf1 protein may also function during the tachyzoite-bradyzoite transformation. Future study will be directed to decipher the potential role of TgPuf1 in regulating stage transition through gene disruption analysis.
The PUF domain contains eight PUM repeats, each containing three α-helices packed together in a curved structure. RNA is bound as an extended strand to the concave surface of the PUF domain with the bases contacted by protein side chains. Specifically, the eight bases of the target RNA, 1–8, are contacted by protein repeats 8–1, with the critical UGU sequence recognized by repeats 8, 7 and 6, respectively. Here we showed that the rTgPuf1 PUM domain has the conserved RNA binding activity to canonical target RNAs and the binding depends on the presence of the essential UGU motif. In line with other reports, mutations in the UGUR sequence abolishes or significantly interferes with the binding[30, 31, 34–38]. A search of the Toxoplasma genome for the presence of the PBE sequence identified 130,571 UGUX3UA motifs, which remain to be determined as Puf binding targets.
In accordance with the primary role of Puf proteins, Puf proteins are predominantly localized within the cytoplasm of cells. Two exceptions are T. brucei Puf7, which is localized in the nucleolus, and S. cerevisiae Puf6p, which is present in both the cytoplasm and nucleus. TgPuf1 is localized in the cytoplasm and it forms punctate cytoplasmic structures in bradyzoites. These structures are reminiscent of “stress granules” or “processing bodies (p-bodies)” formed upon exposure of the cells to stress conditions[41, 42]. Stress granules are large cytoplasmic aggregates, where mRNAs stalled at translation initiation are stored. They contain numerous RBPs, mRNA, the 40S ribosomal subunit and a number of initiation factors[41, 42]. Whereas stress granules are rarely found in growing cells, they are induced rapidly after exposure to many types of stress. P-bodies are typically found in growing cells, however, they become larger and more numerous upon exposure to stress, and can be observed to physically interact with stress granules[41, 42]. Stress granules and P-bodies have been found to contain a large number of RBPs, including Puf proteins[41–43]. Given the localization of human PUM1 and PUM1 to cytoplasmic stress granules, the punctate staining patterns of TgPuf1 suggest that they might be localized to similar granules, although it still requires co-localization confirmation with a marker for the stress granules or P-bodies[10, 44]. Interestingly, several target mRNAs protected from translation and degradation in the P. berghei gametocytes are bound to the DOZI RNA helicase complex, which is apparently devoid of Puf proteins[45, 46]. Future work is necessary to elucidate the biological roles, spatial and temporal regulation, interaction partners, and regulated biological pathways of TgPuf proteins.