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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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Down-regulation of the cwp genes in cells expressing the luciferase gene with a nonsense mutation.(A) Diagrams of the pPT5 and pPT5m plasmids. The pac gene (open box) is under the control of the 5′- and 3′-flanking regions of the gdh gene (striated box). The luciferase reporter gene (luc+, open box) is under the control of the 5′-flanking region of the α2-tubulin (open box) and the 3′-flanking region of the ran gene (dotted box), respectively. The arrows show the directions of gene transcription. Plasmids pPT5 and pPT5m encode a wild-type luciferase gene (luc+) with 550 amino acids and a luciferase mutant (luc+m) in which Leu 411 was mutated to a termination codon (marked by a star) and Asp 500 was mutated to Asn, respectively. (B) Nonsense mutation leads to a decrease of luciferase activity. After stable transfection with the pPT5 and pPT5m constructs, luciferase activity was measured in vegetative cells. The activity of pPT5 transfectants relative to pPT5m transfectants is presented. Values are shown as mean±standard error. (C) Down-regulation of the luciferase gene with a nonsense mutation. Total RNA blots made from vegetative transfectants were hybridized with specific gene probes as indicated (upper panels). Ribosomal RNA loading controls are in the bottom panel. Representative results are shown. Two different luciferase transcripts, a 1.7 kb full-length transcript (shown by an arrow) and a 1.2 kb transcript (shown by a line), were detected in the pPT5 and pPT5m transfectants. The numbers show the relative activity, which reflects expression relative to that in controls.
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pone-0003609-g001: Down-regulation of the cwp genes in cells expressing the luciferase gene with a nonsense mutation.(A) Diagrams of the pPT5 and pPT5m plasmids. The pac gene (open box) is under the control of the 5′- and 3′-flanking regions of the gdh gene (striated box). The luciferase reporter gene (luc+, open box) is under the control of the 5′-flanking region of the α2-tubulin (open box) and the 3′-flanking region of the ran gene (dotted box), respectively. The arrows show the directions of gene transcription. Plasmids pPT5 and pPT5m encode a wild-type luciferase gene (luc+) with 550 amino acids and a luciferase mutant (luc+m) in which Leu 411 was mutated to a termination codon (marked by a star) and Asp 500 was mutated to Asn, respectively. (B) Nonsense mutation leads to a decrease of luciferase activity. After stable transfection with the pPT5 and pPT5m constructs, luciferase activity was measured in vegetative cells. The activity of pPT5 transfectants relative to pPT5m transfectants is presented. Values are shown as mean±standard error. (C) Down-regulation of the luciferase gene with a nonsense mutation. Total RNA blots made from vegetative transfectants were hybridized with specific gene probes as indicated (upper panels). Ribosomal RNA loading controls are in the bottom panel. Representative results are shown. Two different luciferase transcripts, a 1.7 kb full-length transcript (shown by an arrow) and a 1.2 kb transcript (shown by a line), were detected in the pPT5 and pPT5m transfectants. The numbers show the relative activity, which reflects expression relative to that in controls.

Mentions: NMD is related to the presence of a premature stop codon or not [11]–[13]. We have tested NMD effect using a luciferase gene with or without a premature stop codon under the control of the encystation-induced cwp1 promoter [34]. We found that NMD could be present in G. lamblia because the results showed that the mRNA produced from the luciferase gene with a stop codon under the control of the cwp1 promoter was decayed compared with wild type luciferase mRNA [34]. We further used a constitutive promoter to test the NMD. We prepared puromycin-based constructs pPT5 and pPT5m in which the wild type luciferase gene (luc+) and the luciferase gene with a stop codon (luc+m) are controlled by the α2-tubulin promoter, respectively (Fig. 1A), and stably transfected them into G. lamblia. The level of luc+m activity in the pPT5m cell line was reduced by approximately 5,000-fold, relative to that of the wild-type luc+ in the pPT5 cell line (Fig. 1B), indicating that luc+m could be non-functional. This result is similar to the data we previously reported for the luc+m under the control of the cwp1 promoter [34]. The level of luciferase mRNA was lower by ∼50% (P<0.05) in the pPT5m transfectants compared with that in the pPT5 transfectants (Fig. 1C, lanes 1 and 2), indicating that the presence of a nonsense mutation in luc+m triggered a decrease in mRNA levels (NMD). Therefore, NMD can be monitored by a constitutive promoter system. The results from the α2-tubulin and the cwp1 promoters [34] similarly indicate that NMD could be present in G. lamblia. As a control, the ran mRNA levels did not change in the pPT5m transfectants compared with the pPT5 transfectants (Fig. 1C, lanes 1 and 2).


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)

Down-regulation of the cwp genes in cells expressing the luciferase gene with a nonsense mutation.(A) Diagrams of the pPT5 and pPT5m plasmids. The pac gene (open box) is under the control of the 5′- and 3′-flanking regions of the gdh gene (striated box). The luciferase reporter gene (luc+, open box) is under the control of the 5′-flanking region of the α2-tubulin (open box) and the 3′-flanking region of the ran gene (dotted box), respectively. The arrows show the directions of gene transcription. Plasmids pPT5 and pPT5m encode a wild-type luciferase gene (luc+) with 550 amino acids and a luciferase mutant (luc+m) in which Leu 411 was mutated to a termination codon (marked by a star) and Asp 500 was mutated to Asn, respectively. (B) Nonsense mutation leads to a decrease of luciferase activity. After stable transfection with the pPT5 and pPT5m constructs, luciferase activity was measured in vegetative cells. The activity of pPT5 transfectants relative to pPT5m transfectants is presented. Values are shown as mean±standard error. (C) Down-regulation of the luciferase gene with a nonsense mutation. Total RNA blots made from vegetative transfectants were hybridized with specific gene probes as indicated (upper panels). Ribosomal RNA loading controls are in the bottom panel. Representative results are shown. Two different luciferase transcripts, a 1.7 kb full-length transcript (shown by an arrow) and a 1.2 kb transcript (shown by a line), were detected in the pPT5 and pPT5m transfectants. 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-g001: Down-regulation of the cwp genes in cells expressing the luciferase gene with a nonsense mutation.(A) Diagrams of the pPT5 and pPT5m plasmids. The pac gene (open box) is under the control of the 5′- and 3′-flanking regions of the gdh gene (striated box). The luciferase reporter gene (luc+, open box) is under the control of the 5′-flanking region of the α2-tubulin (open box) and the 3′-flanking region of the ran gene (dotted box), respectively. The arrows show the directions of gene transcription. Plasmids pPT5 and pPT5m encode a wild-type luciferase gene (luc+) with 550 amino acids and a luciferase mutant (luc+m) in which Leu 411 was mutated to a termination codon (marked by a star) and Asp 500 was mutated to Asn, respectively. (B) Nonsense mutation leads to a decrease of luciferase activity. After stable transfection with the pPT5 and pPT5m constructs, luciferase activity was measured in vegetative cells. The activity of pPT5 transfectants relative to pPT5m transfectants is presented. Values are shown as mean±standard error. (C) Down-regulation of the luciferase gene with a nonsense mutation. Total RNA blots made from vegetative transfectants were hybridized with specific gene probes as indicated (upper panels). Ribosomal RNA loading controls are in the bottom panel. Representative results are shown. Two different luciferase transcripts, a 1.7 kb full-length transcript (shown by an arrow) and a 1.2 kb transcript (shown by a line), were detected in the pPT5 and pPT5m transfectants. The numbers show the relative activity, which reflects expression relative to that in controls.
Mentions: NMD is related to the presence of a premature stop codon or not [11]–[13]. We have tested NMD effect using a luciferase gene with or without a premature stop codon under the control of the encystation-induced cwp1 promoter [34]. We found that NMD could be present in G. lamblia because the results showed that the mRNA produced from the luciferase gene with a stop codon under the control of the cwp1 promoter was decayed compared with wild type luciferase mRNA [34]. We further used a constitutive promoter to test the NMD. We prepared puromycin-based constructs pPT5 and pPT5m in which the wild type luciferase gene (luc+) and the luciferase gene with a stop codon (luc+m) are controlled by the α2-tubulin promoter, respectively (Fig. 1A), and stably transfected them into G. lamblia. The level of luc+m activity in the pPT5m cell line was reduced by approximately 5,000-fold, relative to that of the wild-type luc+ in the pPT5 cell line (Fig. 1B), indicating that luc+m could be non-functional. This result is similar to the data we previously reported for the luc+m under the control of the cwp1 promoter [34]. The level of luciferase mRNA was lower by ∼50% (P<0.05) in the pPT5m transfectants compared with that in the pPT5 transfectants (Fig. 1C, lanes 1 and 2), indicating that the presence of a nonsense mutation in luc+m triggered a decrease in mRNA levels (NMD). Therefore, NMD can be monitored by a constitutive promoter system. The results from the α2-tubulin and the cwp1 promoters [34] similarly indicate that NMD could be present in G. lamblia. As a control, the ran mRNA levels did not change in the pPT5m transfectants compared with the pPT5 transfectants (Fig. 1C, lanes 1 and 2).

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