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Toward understanding the functional role of Ss-RIOK-1, a RIO protein kinase-encoding gene of Strongyloides stercoralis.

Yuan W, Lok JB, Stoltzfus JD, Gasser RB, Fang F, Lei WQ, Fang R, Zhou YQ, Zhao JL, Hu M - PLoS Negl Trop Dis (2014)

Bottom Line: Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation.Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis.The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.

ABSTRACT

Background: Some studies of Saccharomyces cerevisiae and mammals have shown that RIO protein kinases (RIOKs) are involved in ribosome biogenesis, cell cycle progression and development. However, there is a paucity of information on their functions in parasitic nematodes. We aimed to investigate the function of RIOK-1 encoding gene from Strongyloides stercoralis, a nematode parasitizing humans and dogs.

Methodology/principal findings: The RIOK-1 protein-encoding gene Ss-riok-1 was characterized from S. stercoralis. The full-length cDNA, gDNA and putative promoter region of Ss-riok-1 were isolated and sequenced. The cDNA comprises 1,828 bp, including a 377 bp 5'-UTR, a 17 bp 3'-UTR and a 1,434 bp ORF encoding a protein of 477 amino acids containing a RIOK-1 signature motif. The genomic sequence of the Ss-riok-1 coding region is 1,636 bp in length and has three exons and two introns. The putative promoter region comprises 4,280 bp and contains conserved promoter elements, including four CAAT boxes, 12 GATA boxes, eight E-boxes (CANNTG) and 38 TATA boxes. The Ss-riok-1 gene is transcribed throughout all developmental stages with the highest transcript abundance in the infective third-stage larva (iL3). Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation. Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis.

Conclusions/significance: The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes.

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Related in: MedlinePlus

The anatomical expression pattern of Ss-riok-1 in the early post-free-living first stage larvae.(A, B) and in infective third-stage larvae (C, D). (A, B) strong GFP expression is found at the sphincter connecting pharynx and intestine (S). (C, D) GFP expressed in the pharynx muscle (PM) and body wall muscle (BM). Scale bars = 100 µm.
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pntd-0003062-g006: The anatomical expression pattern of Ss-riok-1 in the early post-free-living first stage larvae.(A, B) and in infective third-stage larvae (C, D). (A, B) strong GFP expression is found at the sphincter connecting pharynx and intestine (S). (C, D) GFP expressed in the pharynx muscle (PM) and body wall muscle (BM). Scale bars = 100 µm.

Mentions: To determine the anatomic expression pattern of Ss-riok-1, larval progeny of FL Female of S. stercoralis transformed with the construct pRP1 were screened for GFP expression. Some of the immature eggs had GFP expression, even when they were still in the vulva of the female adults (data not shown). After 24 h, newly hatched transgenic PFL L1s exhibited GFP expression throughout the body, with strongest expression at the boundary between pharynx and intestine (Fig. 6A and B). After 72 h, strong GFP expression under the Ss-riok-1 promoter was seen in the nervous system, including some head neurons, body neurons and tail neurons as well as in pharynx and hypodermis of transgenic PFL L1s and PFL L2s (Fig. 7A and B). Processes of head neurons go through the body of these larvae, connecting to neurons in the body and tail. The nervous system of S. stercoralis has not been mapped in its entirety, so that a neural map of the free-living nematode C. elegans[50], [51] was employed as a model to tentatively identify of neurons expressing GFP under the Ss-riok-1 promoter. Using this comparative approach, we concluded that body neurons expressing the Ss-riok-1-based reporter are likely sensory neurons and ventral nerve cord motor neurons (Fig. 7C, D, E and F). Furthermore, as PFL L1s developed towards iL3s in the next 4–5 days in culture at 22°C, Ss-riok-1-specific reporter expression was localized to zones in the body wall muscle of the parasite (Fig. 6 C and D).


Toward understanding the functional role of Ss-RIOK-1, a RIO protein kinase-encoding gene of Strongyloides stercoralis.

Yuan W, Lok JB, Stoltzfus JD, Gasser RB, Fang F, Lei WQ, Fang R, Zhou YQ, Zhao JL, Hu M - PLoS Negl Trop Dis (2014)

The anatomical expression pattern of Ss-riok-1 in the early post-free-living first stage larvae.(A, B) and in infective third-stage larvae (C, D). (A, B) strong GFP expression is found at the sphincter connecting pharynx and intestine (S). (C, D) GFP expressed in the pharynx muscle (PM) and body wall muscle (BM). Scale bars = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125297&req=5

pntd-0003062-g006: The anatomical expression pattern of Ss-riok-1 in the early post-free-living first stage larvae.(A, B) and in infective third-stage larvae (C, D). (A, B) strong GFP expression is found at the sphincter connecting pharynx and intestine (S). (C, D) GFP expressed in the pharynx muscle (PM) and body wall muscle (BM). Scale bars = 100 µm.
Mentions: To determine the anatomic expression pattern of Ss-riok-1, larval progeny of FL Female of S. stercoralis transformed with the construct pRP1 were screened for GFP expression. Some of the immature eggs had GFP expression, even when they were still in the vulva of the female adults (data not shown). After 24 h, newly hatched transgenic PFL L1s exhibited GFP expression throughout the body, with strongest expression at the boundary between pharynx and intestine (Fig. 6A and B). After 72 h, strong GFP expression under the Ss-riok-1 promoter was seen in the nervous system, including some head neurons, body neurons and tail neurons as well as in pharynx and hypodermis of transgenic PFL L1s and PFL L2s (Fig. 7A and B). Processes of head neurons go through the body of these larvae, connecting to neurons in the body and tail. The nervous system of S. stercoralis has not been mapped in its entirety, so that a neural map of the free-living nematode C. elegans[50], [51] was employed as a model to tentatively identify of neurons expressing GFP under the Ss-riok-1 promoter. Using this comparative approach, we concluded that body neurons expressing the Ss-riok-1-based reporter are likely sensory neurons and ventral nerve cord motor neurons (Fig. 7C, D, E and F). Furthermore, as PFL L1s developed towards iL3s in the next 4–5 days in culture at 22°C, Ss-riok-1-specific reporter expression was localized to zones in the body wall muscle of the parasite (Fig. 6 C and D).

Bottom Line: Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation.Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis.The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.

ABSTRACT

Background: Some studies of Saccharomyces cerevisiae and mammals have shown that RIO protein kinases (RIOKs) are involved in ribosome biogenesis, cell cycle progression and development. However, there is a paucity of information on their functions in parasitic nematodes. We aimed to investigate the function of RIOK-1 encoding gene from Strongyloides stercoralis, a nematode parasitizing humans and dogs.

Methodology/principal findings: The RIOK-1 protein-encoding gene Ss-riok-1 was characterized from S. stercoralis. The full-length cDNA, gDNA and putative promoter region of Ss-riok-1 were isolated and sequenced. The cDNA comprises 1,828 bp, including a 377 bp 5'-UTR, a 17 bp 3'-UTR and a 1,434 bp ORF encoding a protein of 477 amino acids containing a RIOK-1 signature motif. The genomic sequence of the Ss-riok-1 coding region is 1,636 bp in length and has three exons and two introns. The putative promoter region comprises 4,280 bp and contains conserved promoter elements, including four CAAT boxes, 12 GATA boxes, eight E-boxes (CANNTG) and 38 TATA boxes. The Ss-riok-1 gene is transcribed throughout all developmental stages with the highest transcript abundance in the infective third-stage larva (iL3). Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation. Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis.

Conclusions/significance: The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes.

Show MeSH
Related in: MedlinePlus