Characterization of PfPuf2, member of the Puf family RNA-binding proteins from the malaria parasite Plasmodium falciparum.
Puf proteins are a family of evolutionarily conserved translational regulators in eukaryotes. The malaria parasite has two Puf proteins (PfPuf1 and PfPuf2) that share 25% homology in the RNA binding domain. Here we confirmed the preferential expression of PfPuf2 in gametocyte stages using Northern analysis. The transcriptional initiation site of this gene, mapped using RNA ligase-mediated rapid amplification of cDNA end and primer extension, is located approximately 300 bp upstream from the translational start codon. The 3' end of PfPuf2 is located approximately 250 bp downstream from the stop codon. The total length of the RNA is approximately 2.1 kb, consistent with the mRNA size determined by Northern analysis. Recombinant PfPuf2 proteins expressed in bacteria were purified and used to produce polyclonal antibodies. Western blot further established the preferential synthesis of PfPuf2 in gametocyte stages. Using the Nanos-responsive elements (NRE) in the Hunchback mRNA of Drosophila melanogaster as an artificial target sequence, we tested the binding of PfPuf2 Puf domain to this sequence using the yeast three-hybrid system. The results showed that PfPuf2 Puf domain bound specifically to NRE, suggesting that PfPuf2 may be involved in translational regulation of target genes using a conserved mechanism of the Puf family proteins.
Fan
Qi
Q
Department of Entomology, Pennsylvania State University, University Park, Pennsylvania, USA.
Li
Jinfang
J
Kariuki
Michael
M
Cui
Liwang
L
D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D018076:DNA, Complementary; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins