NcPuf1 Is a Key Virulence Factor in Neospora caninum

Background: Neospora caninum is an apicomplexan parasite that infects many mammals and particularly causes abortion in cattle. The key factors in its wide distribution are its virulence and ability to transform between tachyzoite and bradyzoite forms. However, the factors are not well understood. Although Puf protein (named after Pumilio from Drosophila melanogaster and fem-3 binding factor from Caenorhabditis elegans) have a functionally conserved role in promoting proliferation and inhibiting differentiation in many eukaryotes, the function of the Puf proteins in N. caninum is poorly understood. Methods: The CRISPR/CAS9 system was used to identify and study the function of the Puf protein in N. caninum. Results: We showed that N. caninum encodes a Puf protein, which was designated NcPuf1. NcPuf1 is found in the cytoplasm in intracellular parasites and in processing bodies (P-bodies), which are reported for the first time in N. caninum in extracellular parasites. NcPuf1 is not needed for the formation of P-bodies in N. caninum. The deletion of NcPuf1 (ΔNcPuf1) does not affect the differentiation in vitro and tissue cysts formation in the mouse brain. However, ΔNcPuf1 resulted in decreases in the proliferative capacity of N. caninum in vitro and virulence in mice. Conclusions: Altogether, the disruption of NcPuf1 does not affect bradyzoites differentiation, but seriously impairs tachyzoite proliferation in vitro and virulence in mice. These results can provide a theoretical basis for the development of attenuated vaccines to prevent the infection of N. caninum.

Effective RNA Regulation by Combination of Multiple Programmable RNA-Binding Proteins

RNAs play important roles in gene expression through translation and RNA splicing. Regulation of specific RNAs is useful to understand and manipulate specific transcripts. Pumilio and fem-3 mRNA-binding factor (PUF) proteins, programmable RNA-binding proteins, are promising tools for regulating specific RNAs by fusing them with various functional domains. The key question is: How can PUF-based molecular tools efficiently regulate RNA functions? Here, we show that the combination of multiple PUF proteins, compared to using a single PUF protein, targeting independent RNA sequences at the 3′ untranslated region (UTR) of a target transcript caused cooperative effects to regulate the function of the target RNA by luciferase reporter assays. It is worth noting that a higher efficacy was achieved with smaller amounts of each PUF expression vector introduced into the cells compared to using a single PUF protein. This strategy not only efficiently regulates target RNA functions but would also be effective in reducing off-target effects due to the low doses of each expression vector.

Puf4 Regulates both Splicing and Decay of HXL1 mRNA Encoding the Unfolded Protein Response Transcription Factor in Cryptococcus neoformans

ABSTRACT The endoplasmic reticulum (ER) responds to errors in protein folding or processing by induction of the unfolded protein response (UPR). During conditions of ER stress, unconventional splicing of an mRNA encoding the UPR-responsive transcription factor occurs at the ER surface, resulting in activation of the UPR. UPR activation is necessary for adaptation to ER stress and for the pathogenic fungus Cryptococcus neoformans is an absolute requirement for temperature adaptation and virulence. In this study, we have determined that C. neoformans has co-opted a conserved PUF RNA binding protein to regulate the posttranscriptional processing of the HXL1 mRNA encoding the UPR transcription factor. PUF elements were identified in both the 5′ and 3′ untranslated regions of the HXL1 transcript, and both elements bound Puf4. Deletion of PUF4 resulted in delayed unconventional splicing of HXL1 mRNA and delayed induction of Hxl1 target genes. In addition, the HXL1 transcript was stabilized in the absence of Puf4. The puf4 Δ mutant exhibited temperature sensitivity but was as virulent as the wild type, despite a reduction in fungal burden in the brains of infected mice. Our results reveal a novel regulatory role in which a PUF protein influences the unconventional splicing of the mRNA encoding the UPR-responsive transcription factor. These data suggest a unique role for a PUF protein in controlling UPR kinetics via the posttranscriptional regulation of the mRNA encoding the UPR transcription factor Hxl1.