Molecular Characterisation Of RIO3Protein Kinase Encoding Gene Of Strongyloides Stercoralis

Protein kinases are a group of enzymes regulating a large variety of cellular processes including cell cycle progression, transcription, DNA replication, and metabolic functions. Based on their structures, protein kianses are divided into two groups:the eukaryotic protein kinases (ePK) and atypical protein kinases (aPK). aPK has13families including RIO protein kinases. RIO protein kinase family includes three members RIO1, RIO2and RIO3. They are a recently described family which is highly conserved yet very divergent from the kinase families with known structure. RIO kinases are present in organism from archaea to human, suggesting a fundamental role in the cell. Studies on yeast RIO kinase have shown that RIO kinases play an important role in ribosome biogenesis. In free-living nematode Caenorhabditis elegans, genome-wide large scale RNA interference revealed that knock-down of RIO1and RIO2affect larval development, leading to phenotypes of larval arrest (Lva), sick larvae, slow growth, larval lethal and embryonic lethal, suggesting these genes are crucial to the development of nematode. In spite of the importance of these molecules, there is little published information on the characterisation and function of them for parasitic nematodes. In the present study, a full-length cDNA (Ss-rio-3) encoding a RIO3protein kinase (Ss-RIO-3) was isolated from parasitic nematode Strongyloides stercoralis. The full length cDNA sequence of Ss-rio-3was1765bp in size containing a5’-UTR of179bp, a3’-UTR of68bp and1521bp of protein coding region encoding a protein of506amino acids. BLASTx analysis revealed that the predicted protein sequence (Ss-RIO-S) had22.5%-37.4%identities/similarities to RIO3orthologs of Ascaris sum, Caenorhabditis elegans, C. briggsae, C. remanei, Loa loa and Haemonchus contortus. Comparison of Ss-RIO-3with other RIO3orthologs revealed the amino acid sequence identity of Ss-RIO-3was most pronounced in the ATP-binding motif, active site and metal binding loop. Phylogenetic trees constructed based on predicted amino acid sequences of Ss-RIO-3and RIO3orthologs from19other species including6nematode species for which full-length cDNA sequences are available in GenBank showed that S.s-RIO-3is grouped with strong bootstrap support within a nematode cluster excluding non-nematode species. Analysis by RT-PCR detected transcription of Ss-rio-3in different stages of S. stercoralis including postparasitic LI larvae, free-living mixed male and female, post-free-living LI, infective L3and parasitic adult female. The full-length genomic region of Ss-rio-3was obtained by long-PCR and cloning. Comparison with cDNA sequence revealed no intron in this gene. The promoter region of Ss-rio-3was also isolated by genome walking method revealing a length of600bp and containing four TATA box (TATAWAW), four GATA motif (WGATAR) and two inverse GATA(TTATC). In addition, the truncated protein of Ss-RIO-3was expressed in E. coli, but the kinase enzyme assay showed that this expressed protein didn’t have kinase activity. In order to localize the protein in S. stercoralis in vivo, an expression vector contaiting Ss-rio-3promoter region and green fluorescent protein (GFP) was constructed. This vector was injected into the gonad of S. stercoralis and the transgenic lines were established. The results revealed no GFP expression in S. stercoralis in vivo.This study provides the first insight into RIO3protein kinase of S. stercoralis and a foundation for further functional genomic studies.