PUF family RNA-binding proteins Puf1 and Puf2 promote transcript degradation in Toxoplasma gondii

Toxoplasma gondii, a highly successful apicomplexan parasite, primarily relies on post-transcriptional mechanisms to regulate mRNA stability and translation during rapid life-cycle stage transitions and to adapt to diverse host environments. While RNA-binding proteins (RBPs) are crucial for these regulatory processes, their specific roles in mRNA translation, storage, and degradation during life-stage transitions and under physiological stress in Toxoplasma remain poorly understood. Here, we identified the PUF family of RBPs and characterized two conserved members, TgPuf1 and TgPuf2. We examined their expression, localization, RNA-binding activity, essentiality during asexual stages in cell culture and mouse host, responses to stress conditions, and roles in transcript regulation. Gene-knockout studies in cell-culture showed that TgPuf1 modestly supports parasite fitness under both normal and stress conditions, while TgPuf2 appears largely dispensable. Mice infected with Puf1-deleted tachyzoites showed delayed mortality compared with wild-type, whereas neither Puf1 nor Puf2 deletion affected bradyzoite development. Both TgPuf proteins bind to a conserved RNA sequence known as PUF Recognition Elements (PREs), associate with ribonucleoprotein complexes, and interact with the deadenylase enzyme TgPop2. Using synthetic RNA reporter systems, we demonstrated that, upon interaction with TgPop2, TgPuf proteins stimulate the removal of the poly(A) tail from RNA targets, thereby promoting RNA degradation. The inability to generate the double knockout is adequately addressed using TgPuf1-mAID in the delta TgPuf2 background, indicating that individual Puf proteins are dispensable; however, the lack of both results in severe growth defects. Overall, these findings suggest that PUF proteins regulate transcript levels in Toxoplasma through a deadenylation-dependent mechanism.