Puf3 participates in ribosomal biogenesis in malaria parasites

Abstract

In this study, we characterized the Puf family gene member Puf3 in the malaria parasites Plasmodium falciparum and Plasmodium yoelii Secondary structure prediction suggested that the RNA-binding domains of the Puf3 proteins consisted of 11 pumilio repeats that were similar to those in the human Puf-A (also known as PUM3) and Saccharomyces cerevisiae Puf6 proteins, which are involved in ribosome biogenesis. Neither P. falciparum (Pf)Puf3 nor P. yoelii (Py)Puf3 could be genetically disrupted, suggesting they may be essential for the intraerythrocytic developmental cycle. Cellular fractionation of PfPuf3 in the asexual stages revealed preferential partitioning to the nuclear fraction, consistent with nuclear localization of PfPuf3::GFP and PyPuf3::GFP as detected by immunofluorescence. Furthermore, PfPuf3 colocalized with the nucleolar marker PfNop1, demonstrating that PfPuf3 is a nucleolar protein in the asexual stages. We found, however, that PyPuf3 changed its localization from being nucleolar to being present in cytosolic puncta in the mosquito and liver stages, which may reflect alternative functions in these stages. Affinity purification of molecules that associated with a PTP-tagged variant of PfPuf3 revealed 31 proteins associated with the 60S ribosome, and an enrichment of 28S rRNA and internal transcribed spacer 2 sequences. Taken together, these results suggest an essential function for PfPuf3 in ribosomal biogenesis.

Keywords: Plasmodium; Puf protein; RNA-binding protein; Ribosomal biogenesis; Translational regulation.

Fig. 1.

Prediction of Puf3 structure and functions based on sequence alignments and a homology model. (A) A phylogenetic tree showing the relationship between amino acid sequences of Puf members including proteins from Plasmodium species and representative members from human, Drosophila, Arabidopsis, S. cerevisiae, C. elegans and T. brucei. The three Puf subfamilies are indicated. (B) Homology-based structural model of PfPuf3. Left panel: crystal structure of Human Puf-A (Qiu et al., 2014), which was used as the template (PDB ID: 4WZR) for homology-based modeling. Highlighted in yellow is the longest connecting loop (aa 540–553) in the structure. Middle panel: a homologous model of PfPuf3 (aa 156–774) was generated by the template-based protein structure prediction server (PS)²-v2 and is illustrated here (Chen et al., 2009). The long loop (aa 573–687) in PfPuf3 corresponding to the yellow region in the left panel could not be reliably modeled and is omitted. Right panel: superposition of the PfPuf3 model onto the HsPuf-A structure. The non-modeled loop is shown as a dashed line. (C) Modeling report generated by the (PS)²-v2 server, which shows the model is reliable. (D) Conservation of Puf3 RNA-recognition motifs. The sequence logos were generated by WebLogo using the sequences from Plasmodium Puf3s, HsPuf-A and ScPuf6 in A. R indicates each repeat. R9–R11 show less conservation.

Fig. 2.

PfPuf3 protein expression during asexual erythrocytic development. (A) Western blotting of PfPuf3 and PfAldolase proteins showed their expression levels in ring (R), early trophozoite (ET), late trophozoite (LT) and schizont (S) stages. Image is representative of three experiments. Equal amounts of protein (∼50 µg) from synchronized parasites at designated stages were separated by 10% SDS-PAGE and probed with anti-protein C antibodies (upper panel). Protein loading was monitored with anti-PfAldolase antibodies (lower panel). Numbers underneath the protein bands indicate the density values of the bands quantified by means of GIMP2 software. (B) The ratios of density values of PfPuf3 and PfAldolase bands in A normalized to those in the ring stage. (C) Western blotting of the parasite nuclear (Nuc) and cytoplasmic (Cyto) fractions. Proteins were separated by 10% SDS-PAGE, and probed with anti-Protein C antibodies (upper panel), anti-PfAldolase antibodies (middle panel) and anti-H3 antibodies (lower panel).

Fig. 3.

Expression and localization of Puf3 in P. falciparum and P. yoelii. (A) Colocalization of PfPuf3 (green) and the nucleolar marker PfNop1 (red) was observed in ring (R), early trophozoite (ET), late trophozoite (LT) and schizont (S) stages. Nuclei were stained with Hoechst 33342. (B) PyPuf3::GFP expression was observed in asexual blood stage parasites by using antibodies against ACP (an apicoplast marker) to identify ring (R), trophozoite (T) and schizont (S) stage parasites. Localization patterns of PyPuf3 match those of PfPuf3. (C) PyPuf3::GFP expression was monitored in day 7 oocysts by live fluorescence and in sporozoites by means of an IFA using anti-CSP to provide the location of the sporozoite plasma membrane. (D) PyPuf3::GFP expression was monitored in mid-liver stage parasites (24 h) and late liver stage parasites (48 h) by using anti-CSP and anti-MSP as counterstains, respectively. DIC, differential interference contrast images. Scale bars: 5 μm (B, C middle and lower panels; D, upper panels); 10 μm (C, upper panel); 20 μm (D, lower panels).

Fig. 4.

Comparison of Illumina sequencing counts of selected genes. (A) Counts of mature rRNA species sequenced from the immunoprecipitated (IP) and input RNAs obtained from the PfPuf3-PTP parasites. (B) Counts of ITS sequences from the immunoprecipitated (IP) and input RNAs obtained from PfPuf3-PTP parasites. Note that rRNAs transcribed from the chromosomes 5 and 7 cluster, as does the 5S rRNA from chromosome 14, and are included for comparison. (C) IP:input ratios from A and B showing the fold enrichment of the rRNA and ITS sequences. (D) Schematic drawing of the rRNA gene clusters on chromosomes 5 and 7, and 5S rRNA from chromosome 14. The fold enrichment of the rRNA and ITS fragments is shown beneath the scheme.