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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 decreased mRNA levels of vector-based cwp1 gene.(A) Diagrams of the pPC1 and pNUPF1HA plasmids. The neo or pac gene (open box) expression cassette is the same as in Fig. 2. In pPC1, the cwp1 gene (open boxes) is flanked by its own 5′-flanking region (gray box) and 3′-flanking region of the ran gene (dotted box) and the filled black box indicates the coding sequence of the AU5 epitope tag. In pNUPF1HA, the upf1 gene is under the control of its own promoter (open box) and the 3′-flanking of the ran gene (dotted box) and the filled black box indicates the coding sequence of the HA epitope tag. (B) Northern blot analysis of au5 tagged cwp1 transcripts in vegetative cells (upper panel). Total RNA blots made from pPC1+pRANneo or pPC1+pNUPF1HA transfectants were hybridized with the au5 specific probe (au5hyb), and specific gene probes as indicated (upper panels). Ribosomal RNA loading controls are in the bottom panel. Representative results are shown. The numbers show the relative activity, which reflects expression relative to that in controls.
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pone-0003609-g006: Overexpression of UPF1 decreased mRNA levels of vector-based cwp1 gene.(A) Diagrams of the pPC1 and pNUPF1HA plasmids. The neo or pac gene (open box) expression cassette is the same as in Fig. 2. In pPC1, the cwp1 gene (open boxes) is flanked by its own 5′-flanking region (gray box) and 3′-flanking region of the ran gene (dotted box) and the filled black box indicates the coding sequence of the AU5 epitope tag. In pNUPF1HA, the upf1 gene is under the control of its own promoter (open box) and the 3′-flanking of the ran gene (dotted box) and the filled black box indicates the coding sequence of the HA epitope tag. (B) Northern blot analysis of au5 tagged cwp1 transcripts in vegetative cells (upper panel). Total RNA blots made from pPC1+pRANneo or pPC1+pNUPF1HA transfectants were hybridized with the au5 specific probe (au5hyb), and specific gene probes as indicated (upper panels). Ribosomal RNA loading controls are in the bottom panel. Representative results are shown. The numbers show the relative activity, which reflects expression relative to that in controls.

Mentions: We next tested whether overexpression of UPF1 influenced vector-expressed cwp1 gene. We stably transfected the UPF1 expression plasmid pNUPF1HA (Fig. 6A) together with construct pPC1 in which the cwp1 gene is controlled by its own promoter and contains an AU5 epitope tag at their C terminus (Fig. 6A). The CWP1-AU5 protein was expressed in the stable transfectants as detected in immunofluorescence assays and Western blot analysis (data not shown). Northern blot analysis showed that the levels of the cwp1-au5 mRNA decreased by ∼80% (P<0.05) in the pPC1+pNUPF1HA co-transfectants relative to the pPC1+pRANneo control cell line during vegetative growth (Fig. 6B). The levels of the cwp1 (including endogenous cwp1 and vector-derived cwp1-au5) and cwp2 mRNA also decreased by ∼60–70% (P<0.05) in the UPF1 overexpressed cell line, pPC1+pNUPF1HA (Fig. 6B). The results indicate that overexpression of UPF1 not only can decrease the expression of the endogenous cwp genes but also can decrease the expression of the vector-expressed cwp1 gene.


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 decreased mRNA levels of vector-based cwp1 gene.(A) Diagrams of the pPC1 and pNUPF1HA plasmids. The neo or pac gene (open box) expression cassette is the same as in Fig. 2. In pPC1, the cwp1 gene (open boxes) is flanked by its own 5′-flanking region (gray box) and 3′-flanking region of the ran gene (dotted box) and the filled black box indicates the coding sequence of the AU5 epitope tag. In pNUPF1HA, the upf1 gene is under the control of its own promoter (open box) and the 3′-flanking of the ran gene (dotted box) and the filled black box indicates the coding sequence of the HA epitope tag. (B) Northern blot analysis of au5 tagged cwp1 transcripts in vegetative cells (upper panel). Total RNA blots made from pPC1+pRANneo or pPC1+pNUPF1HA transfectants were hybridized with the au5 specific probe (au5hyb), and specific gene probes as indicated (upper panels). Ribosomal RNA loading controls are in the bottom panel. Representative results are shown. The numbers show the relative activity, which reflects expression relative to that in controls.
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Related In: Results  -  Collection

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pone-0003609-g006: Overexpression of UPF1 decreased mRNA levels of vector-based cwp1 gene.(A) Diagrams of the pPC1 and pNUPF1HA plasmids. The neo or pac gene (open box) expression cassette is the same as in Fig. 2. In pPC1, the cwp1 gene (open boxes) is flanked by its own 5′-flanking region (gray box) and 3′-flanking region of the ran gene (dotted box) and the filled black box indicates the coding sequence of the AU5 epitope tag. In pNUPF1HA, the upf1 gene is under the control of its own promoter (open box) and the 3′-flanking of the ran gene (dotted box) and the filled black box indicates the coding sequence of the HA epitope tag. (B) Northern blot analysis of au5 tagged cwp1 transcripts in vegetative cells (upper panel). Total RNA blots made from pPC1+pRANneo or pPC1+pNUPF1HA transfectants were hybridized with the au5 specific probe (au5hyb), and specific gene probes as indicated (upper panels). Ribosomal RNA loading controls are in the bottom panel. Representative results are shown. The numbers show the relative activity, which reflects expression relative to that in controls.
Mentions: We next tested whether overexpression of UPF1 influenced vector-expressed cwp1 gene. We stably transfected the UPF1 expression plasmid pNUPF1HA (Fig. 6A) together with construct pPC1 in which the cwp1 gene is controlled by its own promoter and contains an AU5 epitope tag at their C terminus (Fig. 6A). The CWP1-AU5 protein was expressed in the stable transfectants as detected in immunofluorescence assays and Western blot analysis (data not shown). Northern blot analysis showed that the levels of the cwp1-au5 mRNA decreased by ∼80% (P<0.05) in the pPC1+pNUPF1HA co-transfectants relative to the pPC1+pRANneo control cell line during vegetative growth (Fig. 6B). The levels of the cwp1 (including endogenous cwp1 and vector-derived cwp1-au5) and cwp2 mRNA also decreased by ∼60–70% (P<0.05) in the UPF1 overexpressed cell line, pPC1+pNUPF1HA (Fig. 6B). The results indicate that overexpression of UPF1 not only can decrease the expression of the endogenous cwp genes but also can decrease the expression of the vector-expressed cwp1 gene.

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