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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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Reverse correlation between the expression levels of the cwp1/2 and upf1 mRNA in stable cell lines.Diagrams of the pRANneo and 5′Δ5N-Pac plasmids for stable transfection are shown in inset. The neo or pac gene (open box) is under the control of the 5′- and 3′-flanking regions of the ran (dotted box) or gdh gene (striated box). Total RNA blots made from vegetative untransfected cells (UT), pRANneo or 5′Δ5N-Pac transfectants were hybridized with specific probes as indicated. Hybridization signals were imaged and quantified as indicated in Materials and Methods. The results are expressed as relative expression level over untransfected control. Values are shown as mean±standard error.
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pone-0003609-g002: Reverse correlation between the expression levels of the cwp1/2 and upf1 mRNA in stable cell lines.Diagrams of the pRANneo and 5′Δ5N-Pac plasmids for stable transfection are shown in inset. The neo or pac gene (open box) is under the control of the 5′- and 3′-flanking regions of the ran (dotted box) or gdh gene (striated box). Total RNA blots made from vegetative untransfected cells (UT), pRANneo or 5′Δ5N-Pac transfectants were hybridized with specific probes as indicated. Hybridization signals were imaged and quantified as indicated in Materials and Methods. The results are expressed as relative expression level over untransfected control. Values are shown as mean±standard error.

Mentions: Previously we found that the levels of the upf1 mRNA decreased with increasing levels of the cwp1 and cwp2 mRNA during encystation [34]. We also demonstrated that stable transfection systems can increase the levels of the cwp1, cwp2 and cwp3 transcripts during vegetative growth [35]. We further asked whether stable transfection influenced the upf1 gene expression. We found that the level of the upf1 mRNA decreased by ∼50–70% (P<0.05) in cells stably transfected with the pRANneo or 5′Δ5N-Pac, which contain the neomycin or puromycin selective marker, relative to untransfected cells (Fig. 2). As reported previously, the levels of the cwp1/2 mRNA increased significantly and the levels of the ran and ribosomal mRNA did not change significantly in the transfected cell lines (Fig. 2) [35].


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)

Reverse correlation between the expression levels of the cwp1/2 and upf1 mRNA in stable cell lines.Diagrams of the pRANneo and 5′Δ5N-Pac plasmids for stable transfection are shown in inset. The neo or pac gene (open box) is under the control of the 5′- and 3′-flanking regions of the ran (dotted box) or gdh gene (striated box). Total RNA blots made from vegetative untransfected cells (UT), pRANneo or 5′Δ5N-Pac transfectants were hybridized with specific probes as indicated. Hybridization signals were imaged and quantified as indicated in Materials and Methods. The results are expressed as relative expression level over untransfected control. Values are shown as mean±standard error.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003609-g002: Reverse correlation between the expression levels of the cwp1/2 and upf1 mRNA in stable cell lines.Diagrams of the pRANneo and 5′Δ5N-Pac plasmids for stable transfection are shown in inset. The neo or pac gene (open box) is under the control of the 5′- and 3′-flanking regions of the ran (dotted box) or gdh gene (striated box). Total RNA blots made from vegetative untransfected cells (UT), pRANneo or 5′Δ5N-Pac transfectants were hybridized with specific probes as indicated. Hybridization signals were imaged and quantified as indicated in Materials and Methods. The results are expressed as relative expression level over untransfected control. Values are shown as mean±standard error.
Mentions: Previously we found that the levels of the upf1 mRNA decreased with increasing levels of the cwp1 and cwp2 mRNA during encystation [34]. We also demonstrated that stable transfection systems can increase the levels of the cwp1, cwp2 and cwp3 transcripts during vegetative growth [35]. We further asked whether stable transfection influenced the upf1 gene expression. We found that the level of the upf1 mRNA decreased by ∼50–70% (P<0.05) in cells stably transfected with the pRANneo or 5′Δ5N-Pac, which contain the neomycin or puromycin selective marker, relative to untransfected cells (Fig. 2). As reported previously, the levels of the cwp1/2 mRNA increased significantly and the levels of the ran and ribosomal mRNA did not change significantly in the transfected cell lines (Fig. 2) [35].

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