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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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Overexpression of UPF1 reduced the levels of CWP1 and cyst formation.(A) Diagrams of the pPUPF1HA plasmid. The pac gene (open box) expression cassette is the same as in Fig. 2. The upf1 gene is under the control of its own 5′-flanking region (open box) and the 3′-flanking region of the ran gene (dotted box). The filled black box indicates the coding sequence of the HA epitope tag. (B) Overexpression of UPF1 reduced the levels of cyst formation. The 5′Δ5N-Pac and pPUPF1HA stable transfectants were cultured in growth medium to late log/early stationary phase (Veg). Cyst count was performed on these late log/early stationary phase cultures (1.5×106 cells/ml). In another study, the 5′Δ5N-Pac and pPUPF1HA stable transfectants were cultured in encystation medium for 24 h and then subjected to cyst count (Enc). The sum of total cysts is expressed as relative expression level over control. Values are shown as mean±standard error. (C) Overexpression of UPF1 reduced the CWP1 level. The 5′Δ5N-Pac and pPUPF1HA stable transfectants were cultured in encystation medium for 24 h and then subjected to SDS-PAGE and Western blot. The blot was probed by anti-CWP1 antibody. Equal amounts of proteins loaded were confirmed by detection of giardial RAN protein. Representative results are shown.
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pone-0003609-g003: Overexpression of UPF1 reduced the levels of CWP1 and cyst formation.(A) Diagrams of the pPUPF1HA plasmid. The pac gene (open box) expression cassette is the same as in Fig. 2. The upf1 gene is under the control of its own 5′-flanking region (open box) and the 3′-flanking region of the ran gene (dotted box). The filled black box indicates the coding sequence of the HA epitope tag. (B) Overexpression of UPF1 reduced the levels of cyst formation. The 5′Δ5N-Pac and pPUPF1HA stable transfectants were cultured in growth medium to late log/early stationary phase (Veg). Cyst count was performed on these late log/early stationary phase cultures (1.5×106 cells/ml). In another study, the 5′Δ5N-Pac and pPUPF1HA stable transfectants were cultured in encystation medium for 24 h and then subjected to cyst count (Enc). The sum of total cysts is expressed as relative expression level over control. Values are shown as mean±standard error. (C) Overexpression of UPF1 reduced the CWP1 level. The 5′Δ5N-Pac and pPUPF1HA stable transfectants were cultured in encystation medium for 24 h and then subjected to SDS-PAGE and Western blot. The blot was probed by anti-CWP1 antibody. Equal amounts of proteins loaded were confirmed by detection of giardial RAN protein. Representative results are shown.

Mentions: Because of the reverse correlation between the levels of the cwp1/2 and upf1 mRNAs as described above, we further investigated the effect of giardial UPF1 on cyst formation. We stably transfected the UPF1 expression plasmid pPUPF1HA into G. lamblia (Fig. 3A). The UPF1-HA protein was expressed in the stable transfectants as detected in immunofluorescence assays [34]. No significant change in growth rate was observed in the pPUPF1HA cell line (data not shown). We found that the cyst number in the vegetative or encysting pPUPF1HA cell line decreased by ∼30% or ∼50% (P<0.05) relative to the control cell line which only carries the pac gene (5′Δ5N-Pac) (Fig. 3B). We further asked whether the levels of cyst wall protein 1 decreased with the decrease of the cyst formation. As shown by Western blot analysis, UPF1 overexpression also resulted in a reduction of the levels of CWP1 protein (Fig. 3C). As a control, similar levels of intensity of the giardial RAN protein (∼30 kDa) were detected by anti-human RAN antibody (Fig. 3C). The results suggest that UPF1 may function in reducing the level of CWP1 and cyst formation.


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)

Overexpression of UPF1 reduced the levels of CWP1 and cyst formation.(A) Diagrams of the pPUPF1HA plasmid. The pac gene (open box) expression cassette is the same as in Fig. 2. The upf1 gene is under the control of its own 5′-flanking region (open box) and the 3′-flanking region of the ran gene (dotted box). The filled black box indicates the coding sequence of the HA epitope tag. (B) Overexpression of UPF1 reduced the levels of cyst formation. The 5′Δ5N-Pac and pPUPF1HA stable transfectants were cultured in growth medium to late log/early stationary phase (Veg). Cyst count was performed on these late log/early stationary phase cultures (1.5×106 cells/ml). In another study, the 5′Δ5N-Pac and pPUPF1HA stable transfectants were cultured in encystation medium for 24 h and then subjected to cyst count (Enc). The sum of total cysts is expressed as relative expression level over control. Values are shown as mean±standard error. (C) Overexpression of UPF1 reduced the CWP1 level. The 5′Δ5N-Pac and pPUPF1HA stable transfectants were cultured in encystation medium for 24 h and then subjected to SDS-PAGE and Western blot. The blot was probed by anti-CWP1 antibody. Equal amounts of proteins loaded were confirmed by detection of giardial RAN protein. Representative results are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2572189&req=5

pone-0003609-g003: Overexpression of UPF1 reduced the levels of CWP1 and cyst formation.(A) Diagrams of the pPUPF1HA plasmid. The pac gene (open box) expression cassette is the same as in Fig. 2. The upf1 gene is under the control of its own 5′-flanking region (open box) and the 3′-flanking region of the ran gene (dotted box). The filled black box indicates the coding sequence of the HA epitope tag. (B) Overexpression of UPF1 reduced the levels of cyst formation. The 5′Δ5N-Pac and pPUPF1HA stable transfectants were cultured in growth medium to late log/early stationary phase (Veg). Cyst count was performed on these late log/early stationary phase cultures (1.5×106 cells/ml). In another study, the 5′Δ5N-Pac and pPUPF1HA stable transfectants were cultured in encystation medium for 24 h and then subjected to cyst count (Enc). The sum of total cysts is expressed as relative expression level over control. Values are shown as mean±standard error. (C) Overexpression of UPF1 reduced the CWP1 level. The 5′Δ5N-Pac and pPUPF1HA stable transfectants were cultured in encystation medium for 24 h and then subjected to SDS-PAGE and Western blot. The blot was probed by anti-CWP1 antibody. Equal amounts of proteins loaded were confirmed by detection of giardial RAN protein. Representative results are shown.
Mentions: Because of the reverse correlation between the levels of the cwp1/2 and upf1 mRNAs as described above, we further investigated the effect of giardial UPF1 on cyst formation. We stably transfected the UPF1 expression plasmid pPUPF1HA into G. lamblia (Fig. 3A). The UPF1-HA protein was expressed in the stable transfectants as detected in immunofluorescence assays [34]. No significant change in growth rate was observed in the pPUPF1HA cell line (data not shown). We found that the cyst number in the vegetative or encysting pPUPF1HA cell line decreased by ∼30% or ∼50% (P<0.05) relative to the control cell line which only carries the pac gene (5′Δ5N-Pac) (Fig. 3B). We further asked whether the levels of cyst wall protein 1 decreased with the decrease of the cyst formation. As shown by Western blot analysis, UPF1 overexpression also resulted in a reduction of the levels of CWP1 protein (Fig. 3C). As a control, similar levels of intensity of the giardial RAN protein (∼30 kDa) were detected by anti-human RAN antibody (Fig. 3C). The results suggest that UPF1 may function in reducing the level of CWP1 and cyst formation.

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