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UPF1, a conserved nonsense-mediated mRNA decay factor, regulates cyst wall protein transcripts in Giardia lamblia.

Chen YH, Su LH, Huang YC, Wang YT, Kao YY, Sun CH - PLoS ONE (2008)

Bottom Line: Previously, we have found that the giardial homologue of a conserved NMD factor, UPF1, may be functionally conserved and involved in NMD and in preventing nonsense suppression.Interestingly, we also found that UPF1 may be involved in regulation of eight other endogenous genes, including up-regulation of the translation elongation factor gene, whose product increases translation which is required for NMD.Our results indicate that NMD factor could contribute to the regulation of not only nonsense containing mRNAs, but also mRNAs of the key encystation-induced genes and other endogenous genes in the early-diverging eukaryote, G. lamblia.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China.

ABSTRACT
The Giardia lamblia cyst wall is required for survival outside the host and infection. Three cyst wall protein (cwp) genes identified to date are highly up-regulated during encystation. However, little is known of the molecular mechanisms governing their gene regulation. Messenger RNAs containing premature stop codons are rapidly degraded by a nonsense-mediated mRNA decay (NMD) system to avoid production of non-functional proteins. In addition to RNA surveillance, NMD also regulates thousands of naturally occurring transcripts through a variety of mechanisms. It is interesting to know the NMD pathway in the primitive eukaryotes. Previously, we have found that the giardial homologue of a conserved NMD factor, UPF1, may be functionally conserved and involved in NMD and in preventing nonsense suppression. In this study, we tested the hypothesis that NMD factors can regulate some naturally occurring transcripts in G. lamblia. We found that overexpression of UPF1 resulted in a significant decrease of the levels of CWP1 and cyst formation and of the endogenous cwp1-3, and myb2 mRNA levels and stability. This indicates that NMD could contribute to the regulation of the cwp1-3 and myb2 transcripts, which are key to G. lamblia differentiation into cyst. Interestingly, we also found that UPF1 may be involved in regulation of eight other endogenous genes, including up-regulation of the translation elongation factor gene, whose product increases translation which is required for NMD. Our results indicate that NMD factor could contribute to the regulation of not only nonsense containing mRNAs, but also mRNAs of the key encystation-induced genes and other endogenous genes in the early-diverging eukaryote, G. lamblia.

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Effect of UPF1 overexpression on endogenous gene expression.Total RNA blots made from the 5′Δ5N-Pac and pPUPF1HA transfectants were hybridized with specific gene probes as described. Equal RNA loading was confirmed by ethidium bromide staining of ribosomal RNA (data not shown). Representative results are shown. The numbers show the relative activity, which reflects expression relative to that in controls.
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pone-0003609-g005: Effect of UPF1 overexpression on endogenous gene expression.Total RNA blots made from the 5′Δ5N-Pac and pPUPF1HA transfectants were hybridized with specific gene probes as described. Equal RNA loading was confirmed by ethidium bromide staining of ribosomal RNA (data not shown). Representative results are shown. The numbers show the relative activity, which reflects expression relative to that in controls.

Mentions: In the previous studies, we have found that the expression of the cwp1, cwp2, myb2 genes was upregulated in stable cell line with drug selection and that the expression of other eight genes was also upregulated [35]. They encodes enzymes involved in anaerobic glycolysis, phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (G3PD), enzymes for arginine hydrolysis, ornithine carbamoyltransferase (OCT) and carbamate kinase (CK), enzymes involved in protein folding, cyclophilin (CY), co-chaperone-like protein p21, and Bip, and open reading frame (ORF) 16424 with unknown function [1], [35], [36]. We wished to understand the importance of UPF1 for expression of these genes. To achieve this goal, we compared expression of these genes in the UPF1 overexpressed cell line and the control cell line. Of these eight genes, three were upregulated by UPF1 overexpression, including cy, p21, and bip (P<0.05) (Fig. 5). Interestingly, one gene was downregulated by UPF1 overexpression (g3pd, P<0.05) (Fig. 5). The transcript levels of the other four genes, pgk, oct, ck, and orf 16424, were not changed significantly by UPF1 overexpression (Fig. 5). We also found that two other genes with no change in mRNA levels in the stable cell lines, glutamate dehydrogenase (gdh), and thioredoxin peroxidase (tp), were downregulated by UPF1 overexpression (P<0.05) (Fig. 5). In addition, we found two newly identified genes, translation elongation factor (tef, γ subunit of translation elongation factor 1B) [37] and arginine deiminase (ad) [1], were upregulated by UPF1 overexpression (P<0.05) (Fig. 5).


UPF1, a conserved nonsense-mediated mRNA decay factor, regulates cyst wall protein transcripts in Giardia lamblia.

Chen YH, Su LH, Huang YC, Wang YT, Kao YY, Sun CH - PLoS ONE (2008)

Effect of UPF1 overexpression on endogenous gene expression.Total RNA blots made from the 5′Δ5N-Pac and pPUPF1HA transfectants were hybridized with specific gene probes as described. Equal RNA loading was confirmed by ethidium bromide staining of ribosomal RNA (data not shown). Representative results are shown. The numbers show the relative activity, which reflects expression relative to that in controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2572189&req=5

pone-0003609-g005: Effect of UPF1 overexpression on endogenous gene expression.Total RNA blots made from the 5′Δ5N-Pac and pPUPF1HA transfectants were hybridized with specific gene probes as described. Equal RNA loading was confirmed by ethidium bromide staining of ribosomal RNA (data not shown). Representative results are shown. The numbers show the relative activity, which reflects expression relative to that in controls.
Mentions: In the previous studies, we have found that the expression of the cwp1, cwp2, myb2 genes was upregulated in stable cell line with drug selection and that the expression of other eight genes was also upregulated [35]. They encodes enzymes involved in anaerobic glycolysis, phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (G3PD), enzymes for arginine hydrolysis, ornithine carbamoyltransferase (OCT) and carbamate kinase (CK), enzymes involved in protein folding, cyclophilin (CY), co-chaperone-like protein p21, and Bip, and open reading frame (ORF) 16424 with unknown function [1], [35], [36]. We wished to understand the importance of UPF1 for expression of these genes. To achieve this goal, we compared expression of these genes in the UPF1 overexpressed cell line and the control cell line. Of these eight genes, three were upregulated by UPF1 overexpression, including cy, p21, and bip (P<0.05) (Fig. 5). Interestingly, one gene was downregulated by UPF1 overexpression (g3pd, P<0.05) (Fig. 5). The transcript levels of the other four genes, pgk, oct, ck, and orf 16424, were not changed significantly by UPF1 overexpression (Fig. 5). We also found that two other genes with no change in mRNA levels in the stable cell lines, glutamate dehydrogenase (gdh), and thioredoxin peroxidase (tp), were downregulated by UPF1 overexpression (P<0.05) (Fig. 5). In addition, we found two newly identified genes, translation elongation factor (tef, γ subunit of translation elongation factor 1B) [37] and arginine deiminase (ad) [1], were upregulated by UPF1 overexpression (P<0.05) (Fig. 5).

Bottom Line: Previously, we have found that the giardial homologue of a conserved NMD factor, UPF1, may be functionally conserved and involved in NMD and in preventing nonsense suppression.Interestingly, we also found that UPF1 may be involved in regulation of eight other endogenous genes, including up-regulation of the translation elongation factor gene, whose product increases translation which is required for NMD.Our results indicate that NMD factor could contribute to the regulation of not only nonsense containing mRNAs, but also mRNAs of the key encystation-induced genes and other endogenous genes in the early-diverging eukaryote, G. lamblia.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China.

ABSTRACT
The Giardia lamblia cyst wall is required for survival outside the host and infection. Three cyst wall protein (cwp) genes identified to date are highly up-regulated during encystation. However, little is known of the molecular mechanisms governing their gene regulation. Messenger RNAs containing premature stop codons are rapidly degraded by a nonsense-mediated mRNA decay (NMD) system to avoid production of non-functional proteins. In addition to RNA surveillance, NMD also regulates thousands of naturally occurring transcripts through a variety of mechanisms. It is interesting to know the NMD pathway in the primitive eukaryotes. Previously, we have found that the giardial homologue of a conserved NMD factor, UPF1, may be functionally conserved and involved in NMD and in preventing nonsense suppression. In this study, we tested the hypothesis that NMD factors can regulate some naturally occurring transcripts in G. lamblia. We found that overexpression of UPF1 resulted in a significant decrease of the levels of CWP1 and cyst formation and of the endogenous cwp1-3, and myb2 mRNA levels and stability. This indicates that NMD could contribute to the regulation of the cwp1-3 and myb2 transcripts, which are key to G. lamblia differentiation into cyst. Interestingly, we also found that UPF1 may be involved in regulation of eight other endogenous genes, including up-regulation of the translation elongation factor gene, whose product increases translation which is required for NMD. Our results indicate that NMD factor could contribute to the regulation of not only nonsense containing mRNAs, but also mRNAs of the key encystation-induced genes and other endogenous genes in the early-diverging eukaryote, G. lamblia.

Show MeSH
Related in: MedlinePlus