ABSTRACT
SMYB1 is a Schistosoma mansoni protein highly similar to members of the Y-box binding protein family. Similar to other homologues, SMYB1 is able to bind double- and single-stranded DNA, as well as RNA molecules. The characterization of proteins involved in the regulation of gene expression in S. mansoni is of great importance for the understanding of molecular events that control morphological and physiological changes in this parasite. Here we demonstrate that SMYB1 is located in the cytoplasm of cells from different life-cycle stages of S. mansoni, suggesting that this protein is probably acting in mRNA metabolism in the cytoplasm and corroborating previous findings from our group that showed its ability to bind RNA. Protein-protein interactions are important events in all biological processes, since most proteins execute their functions through large supramolecular structures. Yeast two-hybrid screenings using SMYB1 as bait identified a partner in S. mansoni similar to the SmD3 protein of Drosophila melanogaster (SmRNP), which is important in the assembly of small nuclear ribonucleoprotein complexes. Also, pull-down assays were conducted using immobilized GST-SMYB1 proteins and confirmed the SMYB1-SmRNP interaction. The interaction of SMYB1 with a protein involved in mRNA processing suggests that it may act in processes such as turnover, transport and stabilization of RNA molecules.
Subject(s)
Helminth Proteins/metabolism , RNA, Helminth/metabolism , RNA, Messenger/metabolism , Schistosoma mansoni/metabolism , Animals , Antibodies, Helminth/blood , Antibodies, Helminth/immunology , Biological Transport , Cytoplasm/metabolism , Female , Gene Library , Helminth Proteins/genetics , Immunohistochemistry , Male , RNA, Helminth/genetics , RNA, Messenger/genetics , Rabbits , Schistosoma mansoni/genetics , Two-Hybrid System TechniquesABSTRACT
The SCF (Skp1-Cul1-F-box) complex is one of the several E3 ligase enzymes and it catalyzes protein ubiquitination and degradation by the 26S proteasome. Rbx1 is a member of the SCF complex in humans and HRT1 is its yeast orthologue. A cDNA encoding a Schistosoma mansoni Rbx1 homolog was cloned and functionally characterized. Heterologous functional complementation in yeast showed that the worm SmRbx gene was able to complement the HRT1yeast null mutation. Gene deletion constructs for N- and C-termini truncated proteins were used to transform hrt1(-) yeast mutant strains, allowing us to observe that regions reported to be involved in the interaction with cullin1 (Cul1) were essential for SmRbx function. Yeast two-hybrid assays using SmRbx and yeast Cul1 confirmed that SmRbx, but not the mutant SmRbxDelta24N, lacking the N-terminus of the protein, was capable of interacting with Cul1. These results suggest that SmRbx protein is involved in the SCF complex formation.
Subject(s)
Helminth Proteins/genetics , Schistosoma mansoni/genetics , Ubiquitin/metabolism , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cullin Proteins/genetics , Cullin Proteins/metabolism , DNA, Complementary/chemistry , DNA, Helminth/chemistry , Expressed Sequence Tags , Female , Genetic Complementation Test , Genetic Vectors , Helminth Proteins/chemistry , Humans , Male , Molecular Sequence Data , Mutation , Reverse Transcriptase Polymerase Chain Reaction , SKP Cullin F-Box Protein Ligases , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Schistosoma mansoni/metabolismABSTRACT
PUR-alpha is a highly conserved protein in eukaryotes belonging to the family of single-stranded DNA-binding proteins. Because PUR-alpha is a multifunctional protein that participates in several regulatory events at the level of gene transcription, it became relevant to investigate the structural features of Schistosoma mansoni PUR-alpha (SmPUR-alpha) that could be correlated to its mode of action. Using deletion constructs on a dot blot assay we mapped the domains of GST-SmPUR-alpha fusion protein involved in the interactions with DNA and RNA. Individually, the N-terminal amino acid residues 1-26 and the C-terminal residues 196-276 of GST-SmPUR-alpha which did not contain nucleic acid-binding domains, did not bind ssDNA or RNA. In contrast, domains encompassing the N-terminal and Class I and C-terminal plus Class I exhibited the highest binding affinity. Seemingly, the latter (GST-SmPUR-alpha 174-276) played a major role in nucleic acid interaction as judged by affinity alone. Other combinations of the deletion constructs displayed either intermediary or no binding affinity to the DNA or RNA probes. Gel shift competition assay showed that GST-SmPUR-alpha bound to ssDNA with higher affinity than to RNA. Because SmPUR-alpha contains two putative phosphorylation sites the protein was tested as a substrate to casein kinase II. GST-SmPUR-alpha could be phosphorylated in vitro by casein kinase II at both, the N- and C-terminus of the protein. The multifunctional nature of SmPUR-alpha was demonstrated by experiments measuring the physical interaction between SmPUR-alpha and the transcription factor SMYB1. This was determined in vivo (yeast two hybrid) and in vitro (GST-pull down). Furthermore, we showed that SmPUR-alpha and SMYB1 acted synergistically to bind preferentially to pyrimidine-rich sequences.
Subject(s)
DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Helminth Proteins/genetics , Helminth Proteins/metabolism , Protein Interaction Mapping , Schistosoma mansoni/physiology , Transcription Factors/metabolism , Animals , Binding Sites , Blotting, Western , Casein Kinase II/metabolism , DNA, Single-Stranded/metabolism , Electrophoretic Mobility Shift Assay , Genes, Helminth , Phosphorylation , Protein Binding , RNA , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Two-Hybrid System TechniquesABSTRACT
The zinc finger motifs (Cys2His2) are found in several proteins playing a role in the regulation of transcripton. SmZF1, a Schistosoma mansoni gene encoding a zinc finger protein was initially isolated from an adult worm cDNA library, as a partial cDNA. The full sequence of the gene was obtained by subcloning and sequencing cDNA and genomic fragments. The collated gene sequence is 2181 nt and the complete cDNA sequence is 705 bp containing the full open reading frame of the gene. Analysis of the genome sequence revealed the presence of three introns interrupting the coding region. The open reading frame theoretically encodes a protein of 164 amino acids, with a calculated molecular mass of 18,667Da. The predicted protein contains three zinc finger motifs, usually present in transcription regulatory proteins. PCR amplification with specific primers for the gene allowed for the detection of the target in egg, cercariae, schistosomulum and adult worm cDNA libraries indicating the expression of the mRNA in these life cycle stages of S. mansoni. This pattern of expression suggests the gene plays a role in vital functions of different life cycle stages of the parasite. Future research will be directed to elucidate the functional role of SmZF1