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. 2002 Nov 1;30(21):4607-17.
doi: 10.1093/nar/gkf600.

The malaria parasite Plasmodium falciparum encodes members of the Puf RNA-binding protein family with conserved RNA binding activity

Liwang Cui  1 , Qi Fan, Jinfang Li
Affiliations

The malaria parasite Plasmodium falciparum encodes members of the Puf RNA-binding protein family with conserved RNA binding activity

Liwang Cui et al. Nucleic Acids Res. .

Abstract

A novel class of RNA-binding proteins, Puf, regulates translation and RNA stability by binding to specific sequences in the 3'-untranslated region of target mRNAs. Members of this protein family share a conserved Puf domain consisting of eight 36 amino acid imperfect repeats. Here we report two Puf family member genes, PfPuf1 and PfPuf2, from the human malaria parasite Plasmodium falciparum. Both genes are spliced with four and three introns clustered within or near the Puf domains, respectively. Northern and RT-PCR analysis indicated that both genes were differentially expressed in gametocytes during erythrocytic development of the parasite. Except for similarities in the Puf domain and expression profile, the deduced PfPuf1 and PfPuf2 proteins differ considerably in size and structure. PfPuf1 has 1894 amino acids and a central Puf domain, whereas PfPuf2 is much smaller with a C-terminal Puf domain. The presence of at least two Puf members in other Plasmodium species suggests that these proteins play evolutionarily similar roles during parasite development. Both in vivo studies using the yeast three-hybrid system and in vitro binding assays using the recombinant Puf domain of PfPuf1 expressed in bacteria demonstrated intrinsic binding activity of the PfPuf1 Puf domain to the NRE sequences in the hunchback RNA, the target sequence for Drosophila Pumilio protein. Altogether, these results suggest that PfPufs might function during sexual differentiation and development in Plasmodium through a conserved mechanism of translational regulation of their target mRNAs.

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Figures

Figure 1
Figure 1
Genomic organization of PfPuf genes. (A) Genomic Southern of PfPuf1. In each lane, 3 µg of P.falciparum DNA was digested with HindIII, Sau3AI and XbaI and electrophoresed in a 0.7% agarose gel. The blot was probed with the 32P-labeled 244 bp PfPuf1 cDNA fragment. DNA markers are in kb. The location of the probe for genomic Southern and northern analyses is indicated in (B). (B) A schematic representation of the genomic structure of PfPuf loci. Exons are indicated as boxes and introns and intergenic regions as solid lines. The conserved RNA-binding domain is shown as checkered boxes. Other repetitive sequences in PfPuf1 (Fig. 2A) are indicated as hatched boxes. The orientations of the genes are indicated by arrows. Genes flanking the Puf genes are shown as solid arrows.
Figure 2
Figure 2
RNA-binding domains of the deduced PfPuf proteins and other short repeats of PfPuf1. PfPuf1 and PfPuf2 sequences were deposited in GenBank with accession nos AY098937 and AY099486. (A) Short repetitive sequences of PfPuf1. Four types of short repeats are aligned and their locations in the protein and consensus sequences indicated above the sequences. (B) Alignment of Puf domains (eight imperfect repeats) of five Plasmodium Pufs and Drosophila Pum. Plasmodium Puf genes are PfPuf1 (Pf1), PfPuf2 (Pf2), P.yoelii Puf1 (Py1), P.yoelii Puf2 (Py2) and P.knowlesi Puf2 (Pk2). Matching amino acids (at least 4 of 6) are shaded and gaps (–) are introduced to optimize alignment. Asterisks indicate amino acids that are likely to confer RNA binding specificity. Three boxes, labeled H1, H2 and H3, respectively, indicate regions that were determined to form three α-helices in Pum and human Pumilio1.
Figure 2
Figure 2
RNA-binding domains of the deduced PfPuf proteins and other short repeats of PfPuf1. PfPuf1 and PfPuf2 sequences were deposited in GenBank with accession nos AY098937 and AY099486. (A) Short repetitive sequences of PfPuf1. Four types of short repeats are aligned and their locations in the protein and consensus sequences indicated above the sequences. (B) Alignment of Puf domains (eight imperfect repeats) of five Plasmodium Pufs and Drosophila Pum. Plasmodium Puf genes are PfPuf1 (Pf1), PfPuf2 (Pf2), P.yoelii Puf1 (Py1), P.yoelii Puf2 (Py2) and P.knowlesi Puf2 (Pk2). Matching amino acids (at least 4 of 6) are shaded and gaps (–) are introduced to optimize alignment. Asterisks indicate amino acids that are likely to confer RNA binding specificity. Three boxes, labeled H1, H2 and H3, respectively, indicate regions that were determined to form three α-helices in Pum and human Pumilio1.
Figure 3
Figure 3
A phylogenetic tree showing the relationship between the amino acid sequences of Puf members. GenBank entries with significant homology to the Puf domain of PfPuf1 were retrieved for detailed analysis. Among these, 34 members with complete Puf domains and five Plasmodium Pufs were used for phylogenetic analysis. Only the Puf domains were used for alignment. Each entry is identified by its GenBank accession no.
Figure 4
Figure 4
Expression of PfPuf genes in blood stage parasites. (A) Northern analysis of PfPuf1 expression. Aliquots of 10 µg of total RNA from P.falciparum asexual stages (A), purified stage IV–V gametocytes (S) and gametocytes 10 min after stimulation with 100 µM xanthurenic acid (Ex) were electrophoresed in 1% agarose/formaldehyde gels and transferred to nylon membranes for hybridization to 32P-labeled 244 bp PfPuf1 cDNA fragment. The upper panel shows the autoradiograph of the hybridization and the lower panel shows the rRNAs in the ethidium bromide stained gel as loading controls. RNA sizes (in kb) are indicated. The faint bands of ∼4 kb might be due to cross-hybridization to the PfPuf2 mRNA. (B) RT–PCR analysis of PfPuf expression during erythrocytic development of the parasite. RT–PCR was performed on total RNA isolated from synchronized asexual parasites as rings (R), trophozoites (T), schizonts (S) and gametocytes at stage I (I), stage II (II) and mixed stages IV and V (V). G indicates PCR amplification from P.falciparum genomic DNA. The actin I gene was used as an internal control, which showed a relatively constitutive expression in erythrocytic stages.
Figure 5
Figure 5
Expression and purification of the PfPuf1 RNA-binding domain in a bacterial expression system. (A) SDS–PAGE analysis of protein samples. Lane 1, lysate of induced bacteria; lane 2, lysate passed through a Ni–NTA agarose column; lane 3, 15 mM imidazole wash; lane 4, 30 mM imidazole wash; lane 5, 50 mM imidazole wash; lane 6, 80 mM imidazole wash; lane 7, 200 mM imidazole elution. Proteins were electrophoresed in a 4–12% NuPAGE gradient gel and visualized by Coomassie blue staining. (B) An immunoblot of PfPuf1 RNA-binding domain expression. The samples were separated in a 4–12% SDS–PAGE gel and transferred to nitrocellulose membrane for immunoblotting with monoclonal anti-His tag antibody. Lane 1, lysate of uninduced bacteria; lane 2, lysate of induced bacteria; lane 3, 30 mM imidazole wash; lane 4, 80 mM imidazole wash; lane 5, 200 mM imidazole elution. The 40 kDa PfPuf polypeptide eluted in 200 mM imidazole is indicated by an arrow. Two bands corresponding to the dimers and trimers of the 40 kDa protein are indicated with arrowheads. The MultiMark multi-colored standard (Invitrogen) is indicated in kDa (M).
Figure 6
Figure 6
RNA binding analysis of recombinant PfPuf1 RNA-binding domain. (A) A gel retardation assay using ∼30 fmol of 32P-labeled NRE RNA and various concentrations (0–240 nM) of recombinant PfPuf1. The Kd was estimated to be <20 nM. (B) A gel retardation assay using ∼30 fmol of 32P-labeled NRE RNA and 60 nM recombinant PfPuf1 in the presence of a 10–100× excess of cold competitor RNAs. NRE and three mutants (Box A, Box B and both Boxes A and B) were included as competitors.
Figure 7
Figure 7
In vivo interactions of the PfPuf1 Puf domain with the Drosophila NRE sequence in yeast. Yeast strain L40uraMS2 was transformed with the plasmid pYESTrp2-PfPuf1 to express the hybrid protein of the B42 transactivation domain and the PfPuf1 Puf domain. One of the bait plasmids expressing MS2, MS2–NRE, MS2–IRE or MS2–antisense NRE (MS2– asNRE) was co-transformed with the hybrid protein plasmid. The MS2–NRE and MS2–asNRE were co-transformed with pYESTrp2-IRP as additional negative controls. The yeast expressing the hybrid protein B42–IRP and bait RNA MS2–IRE was included as the positive control. Both filter and liquid assays for β-galactosidase are shown. For liquid assay, the number represents the mean ± SD using at least three yeast colonies.
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