© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs212597. doi:10.1242/jcs.212597

RESEARCH ARTICLE Puf3 participates in ribosomal biogenesis in malaria parasites Xiaoying Liang1, Kevin J. Hart2, Gang Dong3, Faiza A. Siddiqui1, Aswathy Sebastian4, Xiaolian Li1, Istvan Albert4, Jun Miao1, Scott E. Lindner2 and Liwang Cui1,*

ABSTRACT of Puf-encoding genes varies. Drosophila has only one Puf gene, In this study, we characterized the Puf family gene member Puf3 in the whereas 26 and 19 Puf-like genes have been found in the malaria parasites Plasmodium falciparum and Plasmodium yoelii. Arabidopsis and rice genomes, respectively (Tam et al., 2010). Secondary structure prediction suggested that the RNA-binding Puf members have diverse functions during development of domains of the Puf3 proteins consisted of 11 pumilio repeats that eukaryotic organisms. Puf members share a highly conserved were similar to those in the human Puf-A (also known as PUM3) and RNA-binding domain (RBD), which mostly consists of eight ∼ Saccharomyces cerevisiae Puf6 proteins, which are involved in imperfect tandem repeats of 36 amino acids, termed the Pum ribosome biogenesis. Neither P. falciparum (Pf)Puf3 nor P. yoelii repeats (Zamore et al., 1997; Zhang et al., 1997). (Py)Puf3 could be genetically disrupted, suggesting they may be Recent structural and functional studies have revealed three essential for the intraerythrocytic developmental cycle. Cellular distinct subfamilies within the Puf family proteins. Classical Puf fractionation of PfPuf3 in the asexual stages revealed preferential members contain eight Pum repeats organized in a crescent shape, partitioning to the nuclear fraction, consistent with nuclear localization of with the concave surface recognizing specific single-stranded RNA PfPuf3::GFP and PyPuf3::GFP as detected by immunofluorescence. sequences. These classical Puf proteins are predominantly localized Furthermore, PfPuf3 colocalized with the nucleolar marker PfNop1, within the cytoplasm of the cell, which is consistent with their roles demonstrating that PfPuf3 is a nucleolar protein in the asexual stages. in translational repression or activation of mRNAs (Archer et al., We found, however, that PyPuf3 changed its localization from being 2009; Gu et al., 2004; Lublin and Evans, 2007; Macdonald, 1992; nucleolar to being present in cytosolic puncta in the mosquito and liver Moore et al., 2003; Zhang et al., 1997). Classical Puf members stages, which may reflect alternative functions in these stages. Affinity perform a wide range of functions. In D. melanogaster, binding of purification of molecules that associated with a PTP-tagged variant of Pum to the Nanos response element (NRE) in the maternal PfPuf3 revealed 31 proteins associated with the 60S ribosome, and an hunchback mRNA determines abdominal formation by repressing enrichment of 28S rRNA and internal transcribed spacer 2 sequences. its translation (Murata and Wharton, 1995; Wharton et al., 1998; Taken together, these results suggest an essential function for PfPuf3 in Wharton and Struhl, 1991). In C. elegans, the Puf proteins FBF-1 ribosomal biogenesis. and FBF-2 were originally identified by their ability to bind regulatory elements in the 3′ UTR of fem-3 mRNA and repress its KEY WORDS: Plasmodium, Translational regulation, Puf protein, translation (Zhang et al., 1997), which is required for the switch RNA-binding protein, Ribosomal biogenesis from spermatogenesis to oogenesis in hermaphrodites (Ahringer and Kimble, 1991; Hodgkin, 1986). In addition, the FBF proteins INTRODUCTION were also reported to bind to and activate the translation of the target Translational control of gene expression plays an important role mRNA egl-4, which is important for adaptation to odors (Kaye during the development of eukaryotes. This regulation is often et al., 2009). In the budding yeast Saccharomyces cerevisiae, the mediated by cis-acting elements in the 3′ untranslated region (UTR) Puf protein Puf3p represses expression of the cox17 mRNA, of the mRNA. Binding of specific regulators to these cis elements promoting its deadenylation and subsequent decay (García- activates or represses translation of the mRNAs (Gray and Wickens, Rodríguez et al., 2007; Jackson et al., 2004; Olivas and Parker, 1998; Waters et al., 1989). One of these well-characterized 2000), while Puf5p and Puf6p have been shown to control mating regulators is the Puf family of RNA-binding proteins (RBPs), type switching by regulating the expression of the ho gene which were named after the two founding members, Pumilio (Pum) (Goldstrohm et al., 2006; Gu et al., 2004; Tadauchi et al., 2001). in Drosophila melanogaster and fem-3 binding factor (FBF) in Another subfamily of Puf proteins is represented by the human Caenorhabditis elegans (Zamore et al., 1997; Zhang et al., 1997). Puf-A (also known as PUM3) and S. cerevisiae Puf6 proteins (Li Members from this family represent a highly conserved group of et al., 2009; Qiu et al., 2014). Crystal structures of Puf-A and Puf6 RBPs present in many eukaryotic organisms including animals, revealed 11 Pum repeats that are arranged in an ‘L-like’ shape, with plants, fungi and protists. In different model organisms, the number the concave surfaces of the N- and C-terminal subdomains binding single- or double-stranded nucleic acids without apparent sequence specificity (Qiu et al., 2014). Both proteins primarily adopt a 1Department of Entomology, Pennsylvania State University, University Park, PA 16802, USA. 2Department of Biochemistry and Molecular Biology, Center for nucleolar localization, and are involved in biogenesis of the 60S Malaria Research, Pennsylvania State University, University Park, PA 16802, USA. ribosome (Chang et al., 2011; Li et al., 2009; Qiu et al., 2014). A 3Department of Medical Biochemistry, Max F. Perutz Laboratories, Medical closer look at Puf6 revealed that this protein is required for pre- University of Vienna, 1030 Vienna, Austria. 4Bioinformatics Consulting Center, Pennsylvania State University, University Park, Pennsylvania 16802, USA. rRNA processing and 60S ribosome export, and it also facilitates loading of the ribosome protein Rpl43 (Yang et al., 2016). *Author for correspondence ([email protected]) Moreover, S. cerevisiae Puf6 is also found in the cytoplasm, L.C., 0000-0002-8338-1974 where it acts as a translational repressor of the ash1 mRNA and is required for the asymmetric localization of ash1 to the bud tip of the

Received 26 October 2017; Accepted 16 February 2018 daughter cell (Gu et al., 2004). Whereas human Puf-A appears to be Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212597. doi:10.1242/jcs.212597 important for tumorigenesis (Fan et al., 2013), knockdown of Puf-A is a member of the Puf-A/Puf6 subfamily and may participate in in zebrafish results in abnormal eye development and primordial ribosomal biogenesis in Plasmodium parasites. germ cell migration (Kuo et al., 2009). The third Puf subfamily is represented by S. cerevisiae Nop9, a RESULTS nucleolar protein that is essential for 40S ribosome biogenesis PfPuf3 shows higher similarity to the Puf-A subfamily than (Thomson et al., 2007). Recently, structural and functional analyses the classical Puf subfamily showed that Nop9 contains 11 Pum repeats and forms a ‘C-shaped’ A search of the PlasmoDB database using the Pumilio Puf domain structure (Zhang et al., 2016). Consistent with its role in processing identified another putative Puf gene (PF3D7_0621300) in the the 20S pre-rRNA to produce mature 18S rRNA, Nop9 recognizes P. falciparum genome, which we here call PfPuf3.ThePfPuf3 the base of the internal transcribed spacer (ITS) 1 RNA stem loop, open reading frame is 2382 bp, located on chromosome 6 and which prevents premature cleavage of the 20S pre-rRNA by Nob1 in contains no introns. PfPuf3 encodes a protein of 793 amino acids (aa) the nucleolus (Zhang et al., 2016). In Arabidopsis, APUM23, a with a predicted molecular mass of ∼94.3 kDa. The predicted Puf Nop9 subfamily member, is involved in pre-rRNA processing, and RBD of PfPuf3 is located from aa 166 to 526. Whereas the amino acid disruption of apum23 leads to the accumulation of 35S pre-rRNA sequences of the Puf RBD of PfPuf1 and PfPuf2 are 41 and 58% and unprocessed 18S and 5.8S rRNA precursors without affecting identical to the respective sequences of the Pum RBDs from the steady-state levels of the mature rRNAs (Abbasi et al., 2010, Drosophila melanogaster (Cui et al., 2002), PfPuf3 and Pum RBD 2011). Consistent with its role in ribosomal biogenesis, apum23 only have 15% amino acid identity overall. Like the Puf1 and Puf2 disruption results in an abnormal growth phenotype reminiscent of genes, Puf3 is highly conserved in Plasmodium spp. and a Puf3 that in ribosomal protein gene mutants. In the protozoan parasite ortholog is present in the genome of each sequenced Plasmodium Trypanosoma brucei, two nucleolar Puf proteins, T. brucei species (Fig. 1A). The full-length PfPuf3 sequence shares 45% amino (Tb)PUF7 and TbPUF10, may also be Nop9 subfamily members, acid identity with the orthologous PyPuf3 (PY17X_1121600) and their knockdown (through RNAi) affects rRNA processing, (Fig. S1A). A search for homologous structures based on the leading to slow-growth phenotypes (Droll et al., 2010; Schumann PfPuf3 primary sequence by using NCBI Blastp identified Homo Burkard et al., 2013). These examples have illustrated somewhat sapiens (Hs)Puf-A as the most similar structure in the Protein Data convergent functions for the Puf-A/Puf6 and Nop9 subfamilies of Bank (Qiu et al., 2014). However, sequence alignment showed that the Puf proteins in pre-rRNA processing and ribosomal biogenesis. PfPuf3 shares only 15.1% amino acid identity in the 511-residue (aa It has been increasingly recognized that translational regulation 156–774) fragment that aligned with HsPuf-A (Fig. S1B). Within the plays essential roles in the development of malaria parasites, aligned region, PfPuf3 has a long insertion spanning aa 573–687 especially during developmental stage transitions (Cui et al., 2015). (Fig. 1B) that was predicted to be intrinsically disordered and A large number of RNA-binding proteins have been predicted therefore was left as an unmodeled region. Similar to PfPuf3, PyPuf3 and identified from the Plasmodium falciparum genome by in silico shares only 16.3% amino acid identity in the fragment (aa 129–665) and genome-wide RNA-pulldown analyses (Bunnik et al., 2016; that aligned with HsPuf-A (Fig. S1C). Further phylogenetic analysis Reddy et al., 2015). Among them, the two classical Puf proteins, using the 11 Puf proteins from Plasmodium spp. and ten P. falciparum (Pf)Puf1 and PfPuf2, are differentially expressed and representatives of the three Puf subfamilies from model organisms utilized during development (Cui et al., 2002). Functional studies showed that Plasmodium Puf3 proteins form a subgroup together have shown that PfPuf2 disruption leads to an increase in with the HsPuf-A and S. cerevisiae Puf6 proteins (Fig. 1A). gametocytogenesis, especially for male gametocytes, suggesting a To further predict the structural features of PfPuf3, homology- role for PfPuf2 in repressing gametocytogenesis and differentiation based structural modeling was carried out using the HsPuf-A crystal in the human parasite P. falciparum (Miao et al., 2010). In the structure as the template, based on our preliminary search results. rodent malaria parasites Plasmodium berghei (Pb)Puf2 and Compared with the crescent-shape structure of the classical Puf Plasmodium yoelii (Py)Puf2 proteins play important roles in protein Pumilio and the C-shaped structure of Nop9, the maintenance of the sporozoite infectivity in the salivary glands of homologous model of PfPuf3 more readily adopts an L-like the vectors (Gomes-Santos et al., 2011; Lindner et al., 2013a; shape, similar to that of HsPuf-A (Fig. 1B,C) (Qiu et al., 2014). By Müller et al., 2011). Whereas PbPuf1 appears to be dispensable using a similar modeling approach, we found that the PyPuf3 model throughout the P. berghei life cycle, disruption of PfPuf1 results in a also superimposed well with the HsPuf-A (Fig. S1D,E). Taken significant reduction in the number of mature female gametocytes, together, these sequence comparisons and predicted structures indicating that PfPuf1 is required for the maintenance of female suggest that PfPuf3 and PyPuf3 are likely members of the Puf-A gametocytes (Shrestha et al., 2016). subfamily. Comparison of the predicted RNA base-interacting In this study, we identified and functionally characterized two residues in the Puf-A group showed relatively higher levels of orthologs of a putative Puf protein, PfPuf3 (PF3D7_0621300) in conservation in the first eight repeats, whereas the last three repeats P. falciparum and its syntenic ortholog PyPuf3 (PY17X_1121600) showed little sequence conservation (Fig. 1D). in P. yoelii. Our results showed that both PfPuf3 and PyPuf3 were essential for the asexual parasite erythrocytic cycle. Subcellular Puf3 is essential for asexual development localization studies showed that both proteins were limited to the To study the function of PfPuf3, plasmid pHD22Y/Puf3KO-GFP nucleus and nucleolus in the asexual stages. However, we also was transfected into 3D7 parasites to disrupt PfPuf3. Southern blot observed changes of PyPuf3 localization to cytosolic granules in analysis of resistant parasites that emerged after drug selection oocysts, oocyst sporozoites and early liver stage parasites, as well as showed that none of the six independent transfection experiments its absence in salivary gland sporozoites and in late liver stage led to the disruption of PfPuf3 (Fig. S2A). In P. yoelii, we attempted parasites. Analyses of molecules associated with PfPuf3 identified to genetically delete PyPuf3 by double homologous recombination the majority of associated proteins as being from the large ribosome in two experiments. Similarly, the genotyping PCR results subunit and the associated RNAs as originating from the internal demonstrated that PyPuf3 could not be deleted in either attempt transcribed spacer (ITS) 2 RNA sequence. This suggests that PfPuf3 (Fig. S2B). These results from two parasite species collectively Journal of Cell Science

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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 showsthe 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 inA. R indicateseach repeat. R9 R11 show less conservation. Journal of Cell Science

3 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212597. doi:10.1242/jcs.212597 indicate that Puf3 is likely essential for the intraerythrocytic developmental cycle (IDC).

PfPuf3 is differentially expressed in the IDC Microarray analysis shows that PfPuf3 mRNA is present throughout the IDC, with its mRNA abundance being the lowest in the late trophozoite stage (∼35 h) (PlasmoDB). To detect PfPuf3 protein expression, the endogenous PfPuf3 was tagged with a PTP tag (comprising a protein C epitope, a TEV protease site and a protein A epitope) at the C-terminus. Correct integration of the transfected plasmid at the PfPuf3 locus was verified by integration-specific PCR, which produces the tagged protein (detected by western blotting) when proper insertion occurs. Analysis of three clones from each parasite strain (3D7, NF54) by genotyping PCR revealed bands that were consistent with the expected size of the transgenic locus (1233 bp) (Fig. S2C). Moreover, western blotting of a wild- type 3D7 strain and PfPuf3-PTP clone 1 showed that a protein band of ∼114 kDa was detected with the anti-protein C antibodies only in the PfPuf3–PTP clone, which is consistent with the fusion of the 20 kDa PTP tag to the 94.3 kDa PfPuf3 (Fig. S2C). Further examination of PfPuf3 expression dynamics by western blotting with anti-protein C antibodies revealed PfPuf3 expression during the entire IDC, with the highest and lowest expression in the early and late trophozoites, respectively (Fig. 2A,B), which is similar to the PfPuf3 mRNA expression profile.

PfPuf3 is a nucleolar protein In order to analyze the subcellular distribution and localization of Fig. 2. PfPuf3 protein expression during asexual erythrocytic PfPuf3, asexual blood stage parasites were separated into nuclear development. (A) Western blotting of PfPuf3 and PfAldolase proteins showed their expression levels in ring (R), early trophozoite (ET), late trophozoite (LT) and cytoplasmic fractions. SDS-PAGE and immunoblotting of and schizont (S) stages. Image is representative of three experiments. Equal these fractions showed that PfPuf3–PTP is predominantly present in amounts of protein (∼50 µg) from synchronized parasites at designated stages the nuclear fraction (Fig. 2C), as determined by using histone H3 were separated by 10% SDS-PAGE and probed with anti-protein C antibodies and PfAldolase as markers of the nuclear and cytoplasmic fractions, (upper panel). Protein loading was monitored with anti-PfAldolase antibodies respectively (Thavayogarajah et al., 2015). (lower panel). Numbers underneath the protein bands indicate the density To study the subcellular localization of PfPuf3, we tagged the 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 endogenous PfPuf3 with GFP at its C-terminus. Correct integration ring stage. (C) Western blotting of the parasite nuclear (Nuc) and cytoplasmic of the transfected plasmid at the PfPuf3 locus was verified by (Cyto) fractions. Proteins were separated by 10% SDS-PAGE, and probed with genotyping PCR, which generated a 1249 bp PCR product with anti-Protein C antibodies (upper panel), anti-PfAldolase antibodies (middle primers gF and g-GR (see Table S1) in three clones of each of the panel) and anti-H3 antibodies (lower panel). two transfection attempts using 3D7 and NF54 (Fig. S2C). Fluorescence microscopy analysis of the PfPuf3::GFP parasites PyPuf3::GFP expression in the asexual blood stage was confined detected expression in all asexual stages, with foci that were to the nucleus with a pattern that was also suggestive of nucleolar preferentially located towards the nuclear periphery in a Hoechst localization (Fig. 3B). After transmission of the transgenic parasite 33342-negative area in ring, early trophozoite and early schizont line to mosquitoes, PyPuf3::GFP was observed to be concentrated stages (Fig. 3A). This pattern of localization is reminiscent of the into banded foci in the developing oocysts (Fig. 3C). When nucleolar localization reported for two nucleolar proteins NuProC2 sporozoites were dissected from the developing oocysts on day 10, and NuProC3 (Oehring et al., 2012). To determine whether PfPuf3 PyPuf3::GFP was found in punctate foci of the oocyst sporozoites is indeed localized in the nucleolus, we performed colocalization (Fig. 3C). Some puncta are located adjacent to the DAPI-stained analysis using the known nucleolar marker PfNop1 (Figueiredo nuclear DNA, in agreement with its observed localization in asexual et al., 2005). Using the PfPuf3::GFP line with PfNop1-tdTomato blood stages. However, additional puncta were found in the expressed from a nuclear plasmid, we found excellent overlap of cytoplasm in positions similar to that of PyPuf2 (Fig. 3C; these two tagged proteins in ring, trophozoite and schizont stages Fig. S3A) (Lindner et al., 2013a). Intriguingly, the PyPuf3::GFP (Fig. 3A), indicating that PfPuf3 is a nucleolar protein. foci were not observed in mature salivary gland sporozoites (Fig. 3C, Fig. S3A). During mid-liver stage development (24 h), PyPuf3 displays differential expression and localization large foci containing PyPuf3::GFP were visible, and they were patterns during mosquito and liver stage development adjacent to DAPI-stained DNA but had minimal overlap (Fig. 3D). Similarly, we interrogated the expression and localization of Puf3 in However, in late (48 h) liver stage schizonts, PyPuf3::GFP the rodent-infectious P. yoelii parasite, as analyses throughout expression was not detectable (Fig. 3D, Fig. S3B). These data the entire parasite life cycle are practical in this species. PyPuf3 indicate that the parasite can rapidly modulate the abundance of was successfully tagged with GFP at its C-terminus as confirmed PyPuf3 during parasite maturation, and in addition to its putative by genotyping PCR (Fig. S2D). Similar to P. falciparum, role in the nucleolus, that it may also play additional roles in the immunofluorescence assay (IFA) micrographs showed that cytoplasm in the mosquito stages of parasite development. Journal of Cell Science

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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).

PfPuf3 is associated with ribosomal proteins a highly stringent false discovery rate (FDR) at 1% by using the In order to identify proteins that associate with PfPuf3 that might Significance Analysis of INTeractome (SAINT) algorithm indicate its role in the nucleolus, we used a tandem affinity (Table 1). Among them, 26 proteins are putative 60S ribosomal purification (TAP) procedure combined with mass spectrometry proteins. The remaining five include the nuclear import receptor (MS) to analyze PfPuf3 in asexual blood-stage parasites. The karyopherin β, the putative nucleolar GTP-binding protein 1 presence of PfPuf3–PTP in the transgenic parasite lysates (Nog1), the putative ribosome biogenesis protein MRT4, and and TAP fractions was verified by western blotting using rabbit histones H3 and H4. All of these are associated with ribosome anti-protein C antibodies, and was not detected in wild-type biogenesis in model organisms (Chook and Süel, 2011; parasites (Fig. S4). Liquid chromatography coupled to tandem Harnpicharnchai et al., 2001; Jakel and Gorlich, 1998; Jensen MS (LC/MS/MS) analysis of the proteins after TAP detected a et al., 2003; Kallstrom et al., 2003; Rout et al., 1997). These data total of 124 proteins, including PfPuf3, in three biological suggest that PfPuf3 might be specifically involved in the replicates. A total of 31 proteins were identified after establishing biogenesis of the 60S ribosome. Journal of Cell Science

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Table 1. Proteins identified from the TAP purification of PfPuf3-PTP followed by MS Gene ID Gene annotation Predicted functions SpecSum FDR PFF1030w (PF3D7_0621300) PfPuf3 Ribosomal biogenesis 494 PFE0350c (PF3D7_0507100) 60S ribosomal protein L4 Ribosomal structure 58 0 PF14_0230 (PF3D7_1424100) 60S ribosomal protein L5 Ribosomal structure 50 0 PF14_0296 (PF3D7_1431700) 60S ribosomal protein L14 Ribosomal structure 32 0 PF10_0043 (PF3D7_1004000) 60S ribosomal protein L13 Ribosomal structure 46 0 PF08_0075 (PF3D7_0814000) 60S ribosomal protein L13 Ribosomal structure 38 0 PFC0300c (PF3D7_0307200) 60S ribosomal protein L7 Ribosomal structure 49 0 PF10_0272 (PF3D7_1027800) 60S ribosomal protein L3 Ribosomal structure 46 0 PF11_0106 (PF3D7_1109900) 60S ribosomal protein L36 Ribosomal structure 18 0 PF14_0579 (PF3D7_1460700) 60S ribosomal protein L27 Ribosomal structure 26 0.0002 PF07_0043 (PF3D7_0710600) 60S ribosomal protein L34-A Ribosomal structure 13 0.0002 PF13_0213 (PF3D7_1338200) 60S ribosomal protein L6-2 Ribosomal structure 35 0.0004 PF11_0260 (PF3D7_1124900) 60S ribosomal protein L35 Ribosomal structure 13 0.0004 PFF0700c (PF3D7_0614500) 60S ribosomal protein L19 Ribosomal structure 12 0.0004 PF13_0224 (PF3D7_1341200) 60S ribosomal protein L18 Ribosomal structure 34 0.0004 PF11_0313 (PF3D7_1130200) 60S ribosomal protein P0 Ribosomal structure 32 0.0005 PFE0850c (PF3D7_0517000) 60S ribosomal protein L12 Ribosomal structure 28 0.0006 PFF0625w (PF3D7_0612900) Nucleolar GTP-binding protein 1(Nog1) 60S assembly pathway; Maturation and 43 0.0008 export of 60S and 40S ribosomal subunits PF07_0079 (PF3D7_0719600) 60S ribosomal protein L11a Ribosomal structure 23 0.0009 PFE1195w (PF3D7_0524000) Karyopherin β Import and export through the nuclear pore 182 0.0014 PFD0770c (PF3D7_0415900) 60S ribosomal protein L15 Ribosomal structure 30 0.0014 PF10_0187 (PF3D7_1019400) 60S ribosomal protein L30e Ribosomal structure 13 0.0014 PF11_0061 (PF3D7_1105000) Histone H4 Nucleosome assembly and regulation 9 0.0016 PF13_0132 (PF3D7_1323400) 60S ribosomal protein L23a Ribosomal structure 9 0.0016 PF13_0129 (PF3D7_1323100) 60S ribosomal protein L6 Ribosomal structure 23 0.0017 PFF0510w (PF3D7_0610400) Histone H3 Nucleosome assembly and regulation 9 0.0019 MAL13P1.341 (PF3D7_1367600) Ribosome biogenesis protein MRT4 Ribosome biogenesis 24 0.0021 PF11_0438 (PF3D7_1142600) 60S ribosomal protein L35Ae Ribosomal structure 19 0.0024 PF13_0171 (PF3D7_1331800) 60S ribosomal protein L23 Ribosomal structure 6 0.0028 PF08_0039 (PF3D7_0821700) 60S ribosomal protein L22 Ribosomal structure 11 0.0032 PF14_0240 (PF3D7_1426000) 60S ribosomal protein L21e Ribosomal structure 25 0.0037 PFE0845c (PF3D7_0516900) 60S ribosomal protein L8 Ribosomal structure 26 0.0077

PfPuf3 is associated with ITS2 sequences of rRNA DISCUSSION To identify potential target RNAs of PfPuf3, an RNA More than 100 Puf protein members have been identified in various immunoprecipitation (RIP) with anti-protein C antibodies eukaryotes from unicellular species to human. They fall into three followed by RNA-seq analysis was conducted for the transgenic distinct subfamilies. Here, we have identified a novel Puf member, PfPuf3–PTP and control 3D7 parasites in the asexual blood stage. Puf3, that is found in each of the sequenced Plasmodium genomes Illumina sequencing showed that in both immunoprecipitated and and shares limited sequence identity with the classical Puf protein input RNAs from the PfPuf3–PTP parasite, the overwhelming Pum. A phylogenetic analysis showed that Puf3 diverges from Puf1 majority of the sequence reads were from rRNAs, mostly 28S, 18S and Puf2, and is grouped with the HsPuf-A/ScPuf6 subfamily and 5.8S rRNAs from the A-type rDNA clusters located in (Fig. 1A). We provide data that indicates that Puf3 is likely involved chromosomes 5 and 7. Specifically, 28S rRNAs from in large ribosomal subunit biogenesis, and perhaps in the maturation chromosomes 5 and 7 had 1.47- and 1.49-fold enrichment, of the 28S rRNA. respectively, whereas reads from the 18S and 5.8S rRNAs were A previous study showed that HsPuf-A/ScPuf6 subfamily 0.47- and 0.25-fold lower in the immunoprecipitated RNA than the proteins are involved in large ribosomal subunit biogenesis (Li input RNA (Fig. 4A). In comparison, ITS1 and ITS2 sequences et al., 2009; Qiu et al., 2014; Yang et al., 2016). Consistent with this, from these two rDNA clusters were at least 100 times less we also found that PfPuf3 is a nucleolar protein and colocalized abundant than the mature rRNA sequences (Fig. 4B). Despite this, with a nucleolar marker PfNop1 in the asexual stages (Figueiredo ITS2 sequences from chromosomes 5 and 7 had 14.7- and 18.4- et al., 2005; Mancio-Silva et al., 2008, 2010). In addition, PfPuf3 fold enrichment, respectively, in the immunoprecipitated RNA was co-purified with 26 putative 60S ribosomal proteins and five compared to the input RNA, whereas there was essentially no assembly factors including the nuclear import receptor karyopherin enrichment for the ITS1 sequences (Fig. 4B). A side-by-side β, the putative nucleolar GTP-binding protein 1 (Nog1), a putative comparison of sequence enrichment in the RIP versus input ribosome biogenesis protein MRT4, and H3 and H4. These proteins between transgenic PfPuf3–PTP and wild-type 3D7 control all have important functions in the biogenesis of the large ribosomal parasites clearly demonstrated significant enrichment of the subunit in yeast (Chook and Süel, 2011; Jensen et al., 2003; Keener ITS2 sequences from both rDNA clusters (Fig. 4C). This is et al., 1997; Rodríguez-Mateos et al., 2009a,b; Woolford and further illustrated when sequence reads from the two A-type rDNA Baserga, 2013). Although none of these factors have been well clusters were combined (Fig. 4D). Taken together, this analysis studied in P. falciparum, the association of PfPuf3 and other 60S suggests that PfPuf3 is primarily associated with the ITS2 ribosomal proteins suggests that they may have conserved functions sequences. in ribosome assembly. It is noteworthy that whereas these 32 Journal of Cell Science

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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. proteins associated with the 60S ribosomes were identified under expression. While the reason for the PfPuf3 decline is not known, stringent conditions, additional 40S ribosomal proteins were also we speculate that at the earlier stage of nuclear division, the nucleoli detected when the stringency was lowered. These results suggest might be less well organized in the newly formed nuclei, and that PfPuf3 might be involved in 60S ribosomal subunit biogenesis. presumably rRNA transcription and ribosome assembly decrease to Further analysis of PfPuf3 and putative ribosome assembly factors a minimum due to a lesser requirement for the activities and in the Puf3 complex may yield mechanistic insights into how these functions of PfPuf3 and ribosome assembly factors. In schizonts, factors cooperate in the 28S rRNA maturation and large ribosome rRNA synthesis and ribosome assembly occur in individual subunit assembly. merozoites, coincidentally with increased PfPuf3 expression and It is noteworthy that PfPuf3 expression peaks in early localization of PfPuf3 in individual nucleoli. Furthermore, the three trophozoites, which is compatible with active protein synthesis putative ribosome assembly factors (karyopherin β, Nog1 and during trophozoite growth (Fig. 2A). In late trophozoite stage when MRT4) all show a parallel mRNA expression profile to that of nuclear division begins, PfPuf3 showed minimal levels of PfPuf3, suggesting that they might be integral components of the Journal of Cell Science

7 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212597. doi:10.1242/jcs.212597 ribosome assembly complex during different stages of the IDC. modeling when sequence similarities between the targets and templates are Expression and localization studies of PyPuf3 in asexual blood 15–25% (Chen et al., 2009). This program employs a S2A2 matrix and a stages show a similar pattern. position-specific sequence profile (PSSM) generated by PSI-BLAST, which together ‘blend’ the amino acid and structural propensities. For homology- Interestingly, PyPuf3 expression in mosquito and liver stage ‘ ’ parasites is different. Detectable expression is limited to banded foci based modeling, instead of using the default Automatic template selection, we specified the 2.15 Å resolution crystal structure of HsPuf-A as the in oocysts, and foci in oocyst sporozoites, and large foci in early modeling template based on the preliminary model search result. liver stage parasites (Fig. 3C,D). In addition to DAPI-adjacent puncta, cytosolic puncta are also observed for PyPuf3 in oocyst Generation of transgenic P. falciparum lines sporozoites, which is reminiscent of what is seen for S. cerevisiae To disrupt PfPuf3, a PfPuf3 fragment [nucleotides (nts) 90–1005] was Puf6, which undertakes the function of classical Puf proteins in the amplified from P. falciparum genomic DNA using primers F1 and R1 (all translational repression of mRNA (Gu et al., 2004). This shifting primers used in this study are listed in Table S1). Then the PCR product was pattern of PyPuf3 expression and localization during mosquito stage cloned into pBluescript, to fuse with GFP, and pDT 3′ UTR. This cassette was development, as compared to putative nucleolar localization in subcloned into pHD22Y at the BamHI and NotI sites to produce pHD22Y/ asexual blood stage, oocyst sporozoite and mid-liver stage parasites Puf3KO-GFP (Fidock and Wellems, 1997). To tag PfPuf3 with GFP and PTP suggests that PyPuf3 may play additional roles in sporozoites. at the C-terminus (Schimanski et al., 2005; Takebe et al., 2007; Xu et al., – Further investigations should address what these potential functions 2010), a PfPuf3 nt 1450 2361 was amplified using primers F2 and R2, and cloned into pBluescript SK, to fuse with GFP and PTP, and pDT 3′ UTR. This of Puf3 are. Classical Puf proteins adopt a crescent shape and cassette was digested with BamHI and NotI and subcloned into pHD22Y to recognize single-stranded RNA in a sequence-specific manner. generate pHD22Y/Puf3-GFP/PTP. Colocalization studies were performed Similarly, the S. cerevisiae Nop9 family proteins bind specifically with the PfPuf3::GFP parasites containing an additional pCC4 plasmid for to the ITS2 D-A2 fragment of 20S pre-rRNA to protect ITS1 from expression of the nucleolar marker PfNop1 with a C-terminal tdTomato tag. premature cleavage in the nucleolus (Zhang et al., 2016). Distinct To create this plasmid, the full-length PfNop1 and tdtomato coding sequences from these two subfamilies, the HsPuf-A/ScPuf6 family proteins were amplified with two pairs of primers (Nop1F and Nop1R, tdTomF and can interact with single- or double-stranded RNA through non- tdTomR). The Nop1 PCR product was cloned into pBluescript to fuse with specific sequence contacts with the phosphate backbone (Qiu et al., tdTomato and pDT 3′ UTR. The PfNop1 expression cassette was cloned into 2014). Homology modeling of Puf3 shows that excellent the modified vector at SpeIandNotI sites to obtain the construct PfNop1- superimposition of Puf3 and HsPuf-A is possible, suggesting that tdTom-pCC4. For transfection, P. falciparum 3D7 and NF54 parasites were maintained and synchronized through sorbitol treatment (Lambros and Puf3 may also nonspecifically interact with nucleic acids. Our RIP Vanderberg, 1979). Parasite transfection was performed by using the RBC and subsequent RNA-seq analysis with PTP-tagged PfPuf3 clearly loading method (Rug and Maier, 2013). To enrich parasites that have showed association of PfPuf3 with the 28S rRNA and the ITS2, integrated the constructs into the genome, parasites were subjected to two strongly suggesting that PfPuf3 is involved in the processing of the rounds of on–off drug selection (Crabb and Cowman, 1996). Drug-resistant large ribosome subunit. While significant enrichment of the ITS2 parasites were screened by integration-specific PCR and Southern blotting to RNA over ITS1 RNA suggests a sequence-specific interaction, it is detect plasmid integration, while western blotting was used to confirm the unknown whether the interaction between PfPuf3 and the ITS2 generation of the variant protein. Single clones of parasites with stable region of the pre-rRNA is direct, or whether it indirectly associates integration of the constructs were obtained by limiting dilution (Rosario, 1981). via a specific ITS2-binding protein. P. yoelii Puf3 Whereas Puf-A appears to play critical roles in both human and Generation of transgenic with Py tagging and zebrafish development (Fan et al., 2013; Kuo et al., 2009), the knockout To study the function of the PfPuf3 ortholog PyPuf3 in P. yoelii, two clonal S. cerevisiae Puf6 is nonessential under standard culture conditions. transgenic parasite lines were created by double homologous recombination. Deletion of S. cerevisiae Puf6 results in a slow-growth phenotype at First, the PyPuf3 coding sequence was replaced with a drug resistance 20°C and it is linked to 60S ribosome biogenesis defects (Li et al., cassette (HsDHFR) and a GFPmut2 expression cassette by using targeting 2009). Our evidence strongly indicates a similar involvement of sequences that were PCR amplified from genomic DNA with the Phusion Plasmodium Puf3 in the biogenesis of the large ribosome subunit, polymerase (NEB) supplemented with 5 mM MgCl2 via primers yKO5F, and this function is likely essential in Plasmodium, as the Puf3 gene yKO5R, yKO3F and yKO3R (Table S1). The PCR products were gel could not be deleted in either P. falciparum or P. yoelii. Future purified, fused by means of sequence overlap extension PCR, and cloned studies could use conditional knockdown systems to investigate into the pDEF-GFP vector (pSL0444). Second, the PyPuf3 protein was which steps of the 60S ribosome assembly are affected by Puf3, tagged with GFP at its C-terminus using the same strategy as above, but whether this interaction is direct or indirect, and whether the binding using primers yKO3F, yKO3R, yCtOF and yCtOR for the amplification of targeting sequences (Table S1). PCR products were fused and inserted into a involves a specific sequence motif in the ITS2. pDEF-GFP-Ct plasmid (pSL0442). Linearized plasmids were introduced into P. yoelii (17XNL) parasites using an Amaxa Nucleofector 2b. MATERIALS AND METHODS Transfected parasites were intravenously injected into Swiss Webster mice Identification of PfPuf3, and phylogeny and homology-based (Envigo) and selected with pyrimethamine supplied in the drinking water as modeling previously described (Munoz et al., 2017). The presence of transgenic PfPuf3 (PF3D7_0621300, PFF1030w) was identified by searching the parasites (PyPuf3−,PyPuf3::gfp) was verified by genotyping PCR and by PlasmoDB database (http://www.plasmodb.org) with the Pum domain using the presence of GFP. For the P. yoelii work, a total of 22 female Swiss a hidden Markov model. A total of 19 GenBank entries with complete Puf Webster mice were used. The animal use protocol was approved by the domains, representing three Puf subfamilies, were retrieved for phylogenetic Pennsylvania State University Institutional Animal Care and Use analysis. The Puf domains of individual Puf proteins were trimmed and used Committee (#42628). to generate the data matrix to infer phylogenetic relationships among these Puf family members. Multiple alignment was performed using the PfPuf3 expression during IDC CLUSTALW program and phylogenetic analysis was performed using To study PfPuf3 protein expression during the IDC, synchronization was MEGA 7.0. Preliminary prediction of the protein model was carried out by performed by two rounds of sorbitol treatment at the ring state. submitting the full-length PfPuf3 sequence to the online (PS)2-v2 server Synchronized parasites were lysed by sonication (three pulses of 10 s

(http://140.113.239.111/~ps2v2/index.php), which is good for homologous each). Protein concentration was measured by using the Pierce BCA Protein Journal of Cell Science

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Assay Kit (Thermo Fisher Scientific), and equal amounts of parasite lysate temperature, 3 mM MgCl2, 0.5 mM DTT, 0.1% (v/v) Tween 20] containing (50 μg) at each developmental stage were separated by 10% SDS-PAGE and a protease inhibitor cocktail (one pill in 10 ml PA150 buffer, Roche). Then transferred to nitrocellulose membranes. Western blotting was performed by the parasites were manually lysed by 60–100 strokes in a Dounce using a standard procedure with rabbit anti-Protein C (1:1000, GenScript, homogenizer with a tight pestle and centrifuged at 16,000 g for 20 min at Cat# A00637) and rabbit anti-PfAldolase (1:3000, kindly provided by Dr 4°C. The supernatant was incubated with 100 μl (settled volume) of IgG– Tobias Spielmann, Parasitology Section, Bernhard Nocht Institute for agarose beads (GE Healthcare) at 4°C for 2 h. The beads were washed three Tropical Medicine, Germany) as the primary antibodies and horseradish times with pre-cooled PA150 and equilibrated twice with the TEV buffer peroxidase-conjugated goat anti-rabbit-IgG (1:3000) as the secondary [150 mM KCl, 20 mM Tris-HCl, pH 7.7 at room temperature, 3 mM MgCl2, antibodies. The results were visualized with the ECL detection system using 0.5 mM EDTA, 1 mM DTT, 0.1% (v/v) Tween 20]. Then the beads were X-ray film (Invitrogen, Cat# WP20005). incubated overnight with 2 ml of TEV buffer containing 100–150 U of TEV protease with gentle rotation at 4°C. The supernatant was obtained by Cell fractionation centrifugation at 500 g for 5 min at 4°C. Subsequently, 200 μl of anti-protein To estimate the distribution of PfPuf3 in the cytoplasmic and nuclear C affinity matrix (Roche) equilibrated with 1 ml of PC150 [150 mM KCl, compartments of the parasite, ∼100 μl of parasite pellet of the PfPuf3–PTP 20 mM Tris-HCl, pH 7.7 at room temperature, 3 mM MgCl2, 1 mM CaCl2, line was resuspended in 500 μl of a hypotonic buffer A [10 mM Hepes, pH 0.1% (v/v) Tween 20] was added into the supernatant with 7.5 μl1M 7.9 at room temperature, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5 mM CaCl2. After incubation for 2 h at 4°C, the beads were washed four times EDTA and 1% (v/v) protease-inhibitor cocktail (Roche)] and incubated on ice with PC150 and eluted with a buffer containing 10 mM EGTA and 5 mM for 10–15 min. The parasites were mechanically lysed by at least 40 strokes in EDTA. Western blotting was used to check for the presence of PfPuf3 in the a Dounce homogenizer with a loose pestle and then centrifuged at 700 g for purified complex; PfAldolase served as a control. The eluted protein 20 min at 4°C. The supernatant was centrifuged at 10,000 g for 10 min to complex was analyzed by nano-LC/MS/MS with a Waters NanoAcquity obtain the cytoplasmic extract. The pellet was resuspended in five volumes of HPLC system interfaced to a Q Exactive™ Hybrid Quadrupole-Orbitrap buffer B [20 mM HEPES, pH 7.9 at room temperature, 20% (v/v) glycerol, mass spectrometer (Thermo Scientific). Peptides were loaded on a trapping 200 mM KCl, 0.5 mM DTT, 0.5 mM EDTA, 0.5% (v/v) NP40 and protease column and eluted over a 75 μm analytical column at 350 nl/min. MS and inhibitor cocktail] and dounced for 40 times with a tight pestle. The MS/MS were performed at 70,000 full width and half maximum (FWHM) homogenate was centrifuged at 10,000 g for 10 min to obtain the nuclear and 17,500 FWHM resolution, respectively. The 15 most abundant ions extract. Protein extracts were resolved by 10% SDS-PAGE and detected by were selected for MS/MS and Mascot Generic Files (MGF) from RAW files immunoblotting using the anti-Protein C antibodies (1:1000, GenScript, Cat# were extracted by use of Proteome Discoverer v1.4 (Thermo Scientific). A00637) and anti-histone H3 antibodies (1:1000, Sigma, Cat# H0164) as a Parasite proteins were identified by searching the Uniprot P. falciparum control for nuclear proteins. For a cytoplasmic protein control, a rabbit protein database (v01/2014, 5369 entries) concatenated with the SWISS- antiserum against aldolase was used, which specifically reacts with PROT human database (20,160 entries). The combined database (25,529 PfAldolase in the parasite (Knapp et al., 1990). entries) was reversed and appended back to the forward database (51,058 entries total). All peak list files (MGFs) were analyzed by using Mascot Fluorescence microscopy (Matrix Science; London, UK; version 2.5.1). Search parameters included A P. falciparum 3D7 line with PfPuf3::GFP and tdTomato-tagged PfNop1 trypsin digestion (C-terminal K and R cleavage) full cleavage with 2 missed were used for protein localization studies. Culture medium was removed sites, and a fixed modification of +57 on C (carbamidomethyl), and variable gently and infected red blood cells were resuspended in 1× PBS with Hoechst modifications −17 on n (Gln->pyro-Glu), +1 on NQ (deamidated), +16 on 33342 (1:8000) (Chazotte, 2011) and incubated at room temperature for M (oxidation), +42 on n (acetyl), fragment ion mass tolerance of 0.02 Da, 5 min. Cells were centrifuged at 500 g for 3 min, and washed with 1× PBS. peptide mass tolerance of 10 ppm. Mascot DAT files were parsed into the Then cells were placed onto a slide and covered with a coverslip. Images were Scaffold software for validation, filtering and to create a nonredundant list captured by using a Nikon Eclipse E600 epifluorescence microscope. per sample. Data were filtered with a 1% FDR for both protein and peptide PyPuf3 expression in P. yoelii was examined by using the transgenic analyses, and proteins reported had at least two unique peptides detected. PyPuf3::GFP parasite line. PyPuf3::GFP expression was observed in blood Unique proteins were identified after controlling the FDR at 1% using the stages, oocyst and salivary gland sporozoites, and liver stages by performing SAINT algorithm (Table S2) (Choi et al., 2012). Data from these proteomic an indirect immunofluorescence assay (IFA), while its expression in day 7 experiments have been deposited to the ProteomeXchange Consortium and day 10 oocysts was observed by live-cell fluorescence microscopy. All (http://proteomecentral.proteomexchange.org) via the PRIDE database samples for IFA were prepared as previously described (Lindner et al., (dataset identifier PXD007978). 2014). Parasites were stained with the following primary antibodies: rabbit anti-GFP (1:1000, Invitrogen, Cat# A11122), rabbit anti-PyACP (1:1000), RIP and RNA-Seq mouse anti-GFP (1:1000, DSHB, Clone 4C9), rabbit anti-merozoite surface The transgenic parasite line PfPuf3-PTP was employed for RIP and RNA- protein (MSP) (1:1000, BEI Resources, MRA23) and mouse anti-P. yoelii seq analysis. RNA was isolated from the eluate after the TEV protease circumsporozoite protein (PyCSP) (1:1000, Clone 2F6) (Lindner et al., cleavage step. RNA libraries were made using the KAPA stranded RNA-seq 2013b) antibodies. Secondary antibodies used for all stages were Alexa library preparation kit instruction (Roche). Briefly, RNA was fragmented by Fluor-conjugated (Alexa Fluor 488 or Alexa Fluor 594) and specific to heating at 94°C for 8 min. Then cDNA was generated from the RNA rabbit or mouse IgG (1:1000, Invitrogen, Cat# A11001, A11005, A11008, template with random primers by means of a reverse transcriptase reaction. A11012). 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain nuclei The first-strand cDNA was converted to double-stranded DNA, which was and samples were mounted with the VectaShield antifade reagent (VWR), subsequently end-repaired to create blunt ends. Addition of a single covered with a coverslip, and sealed with nail polish prior to visualization. nucleotide to the 3′ end of the double-stranded DNA fragments was Fluorescence and DIC images were taken by using a Zeiss fluorescence and performed. Afterwards, 3′ dTMP adapters were ligated to the 3′ dAMP phase contrast microscope (Zeiss Axioscope A1 with 8-bit AxioCam ICc1 library fragments. PCR amplification was performed to increase the amount camera) with a 40× or 100× oil objective and processed with Zen imaging of the library, and the RNA library was analyzed for size distribution and software. quality. Finally, the libraries were sequenced on an Illumina HiSeq 2500 to produce 150 base pair single end reads. Illumina adapter sequence removal Isolation of a PfPuf3 protein complex and MS analysis and quality trimming of reads were performed with Trimmomatic (Bolger TAP of PfPuf3 was performed by using the PfPuf3-PTP parasite line or et al., 2014). A sliding window approach was used to trim the reads once the wild-type (3D7) parasites as control (Schimanski et al., 2005; Takebe et al., average quality within a 4 base pair window dropped below the threshold of 2007; Xu et al., 2010). Parasites were isolated as described above. The 18. Only reads that had a minimum length of 50 base pairs were retained. parasite pellet was lysed with five times the volume of the parasite pellet of Reads were then mapped to the P. falciparum reference genome pre-cooled PA150 buffer [150 mM KCl, 20 mM Tris-HCl, pH 7.7 at room (Pf3D7_01_v3) with Tophat2 (Kim et al., 2013). Reads that mapped to Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212597. doi:10.1242/jcs.212597 each gene were counted by using featureCounts (Liao et al., 2014). Read Fan, C.-C., Lee, L.-Y., Yu, M.-Y., Tzen, C.-Y., Chou, C. and Chang, M.-S. (2013). counts were normalized to their library size by using a DESeq normalization Upregulated hPuf-A promotes breast cancer tumorigenesis. Tumour Biol. 34, method (Anders and Huber, 2010). Normalized read counts were used for 2557-2564. Fidock, D. A. and Wellems, T. E. (1997). Transformation with human dihydrofolate further analysis. Final RNA-seq datasets for both wild-type and PfPuf3-PTP reductase renders malaria parasites insensitive to WR99210 but does not affect have been deposited in the GEO database under the accession number the intrinsic activity of proguanil. Proc. Natl. Acad. Sci. USA 94, 10931-10936. GSE105126, and are also provided as Table S3. Figueiredo, L. M., Rocha, E. P., Mancio-Silva, L., Prevost, C., Hernandez- Verdun, D. and Scherf, A. (2005). The unusually large Plasmodium telomerase Competing interests reverse-transcriptase localizes in a discrete compartment associated with the The authors declare no competing or financial interests. nucleolus. Nucleic Acids Res. 33, 1111-1122. Garcıa-Rodŕ ıguez,́ L. J., Gay, A. C. and Pon, L. A. (2007). Puf3p, a Pumilio family RNA binding protein, localizes to mitochondria and regulates mitochondrial Author contributions biogenesis and motility in budding yeast. J. Cell Biol. 176, 197-207. Conceptualization: L.C.; Methodology: X. Liang; Software: G.D., I.A.; Formal Goldstrohm, A. C., Hook, B. A., Seay, D. J. and Wickens, M. (2006). PUF proteins analysis: X. Liang, K.H., G.D., A.S., J.M., S.L., L.C.; Investigation: X. Liang, K.H., bind Pop2p to regulate messenger RNAs. Nat. Struct. Mol. Biol. 13, 533-539. F.S., A.S., X. Li, I.A., J.M.; Data curation: X. Liang, A.S.; Writing - original draft: Gomes-Santos, C. S. S., Braks, J., Prudêncio, M., Carret, C., Gomes, A. R., X. Liang, K.H., J.M.; Writing - review & editing: S.L., L.C.; Supervision: S.L., L.C.; Pain, A., Feltwell, T., Khan, S., Waters, A. and Janse, C. (2011). Transition of Project administration: L.C.; Funding acquisition: S.L., L.C. Plasmodium sporozoites into liver stage-like forms is regulated by the RNA binding protein Pumilio. PLoS Pathog. 7, e1002046. Funding Gray, N. K. and Wickens, M. (1998). Control of translation initiation in animals. This research was supported by the National Institutes of Health (grants Annu. Rev. Cell Dev. Biol. 14, 399-458. R01AI104946 to L.C., R01AI123341 and K22AI101039 to S.L.). Deposited in PMC Gu, W., Deng, Y., Zenklusen, D. and Singer, R. H. (2004). A new yeast PUF family for release after 12 months. protein, Puf6p, represses ASH1 mRNA translation and is required for its localization. Genes Dev. 18, 1452-1465. 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