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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 cwp gene expression.(A) Overexpression of UPF1 decreased the cwp1-3 and myb2 mRNA levels. Total RNA was harvested from vegetative 5′Δ5N-Pac and pPUPF1HA transfectants. Northern blots were hybridized with specific probes as indicated (upper panels). (B)(C) Overexpression of UPF1 decreased the cwp1 and cwp2 mRNA stability. Total RNA was harvested from either 5′Δ5N-Pac (B) or pPUPF1HA (C) transfectants during vegetative growth. The cells were treated without (0 min) or with 45 µg/ml actinomycin D for 13 min to arrest mRNA synthesis. Northern blot were hybridized with specific gene probes as indicated (upper panels). Ribosomal RNA loading controls are in the bottom panels. Representative results are shown. The numbers show the relative activity, which reflects expression relative to that in controls. The cwp1 and cwp2 signals from Fig. 4B and C were a long exposure to show the difference in the AcD treated and untreated samples.
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pone-0003609-g004: Effect of UPF1 overexpression on cwp gene expression.(A) Overexpression of UPF1 decreased the cwp1-3 and myb2 mRNA levels. Total RNA was harvested from vegetative 5′Δ5N-Pac and pPUPF1HA transfectants. Northern blots were hybridized with specific probes as indicated (upper panels). (B)(C) Overexpression of UPF1 decreased the cwp1 and cwp2 mRNA stability. Total RNA was harvested from either 5′Δ5N-Pac (B) or pPUPF1HA (C) transfectants during vegetative growth. The cells were treated without (0 min) or with 45 µg/ml actinomycin D for 13 min to arrest mRNA synthesis. Northern blot were hybridized with specific gene probes as indicated (upper panels). Ribosomal RNA loading controls are in the bottom panels. Representative results are shown. The numbers show the relative activity, which reflects expression relative to that in controls. The cwp1 and cwp2 signals from Fig. 4B and C were a long exposure to show the difference in the AcD treated and untreated samples.

Mentions: We further investigated whether UPF1 overexpression can influence the expression of cwp and other endogenous genes. We found that the levels of native cwp1, cwp2, cwp3, or myb2 mRNA in the vegetative pPUPF1HA cell line decreased by ∼85% or ∼50% (P<0.05) relative to the control cell line which only carries the pac gene (Fig. 4A). The ran mRNA levels did not change significantly in the pPUPF1HA cell line (Fig. 4A). The upf1 mRNA levels increased significantly (P<0.05) in the pPUPF1HA cell line (Fig. 4A).


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 cwp gene expression.(A) Overexpression of UPF1 decreased the cwp1-3 and myb2 mRNA levels. Total RNA was harvested from vegetative 5′Δ5N-Pac and pPUPF1HA transfectants. Northern blots were hybridized with specific probes as indicated (upper panels). (B)(C) Overexpression of UPF1 decreased the cwp1 and cwp2 mRNA stability. Total RNA was harvested from either 5′Δ5N-Pac (B) or pPUPF1HA (C) transfectants during vegetative growth. The cells were treated without (0 min) or with 45 µg/ml actinomycin D for 13 min to arrest mRNA synthesis. Northern blot were hybridized with specific gene probes as indicated (upper panels). Ribosomal RNA loading controls are in the bottom panels. Representative results are shown. The numbers show the relative activity, which reflects expression relative to that in controls. The cwp1 and cwp2 signals from Fig. 4B and C were a long exposure to show the difference in the AcD treated and untreated samples.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2572189&req=5

pone-0003609-g004: Effect of UPF1 overexpression on cwp gene expression.(A) Overexpression of UPF1 decreased the cwp1-3 and myb2 mRNA levels. Total RNA was harvested from vegetative 5′Δ5N-Pac and pPUPF1HA transfectants. Northern blots were hybridized with specific probes as indicated (upper panels). (B)(C) Overexpression of UPF1 decreased the cwp1 and cwp2 mRNA stability. Total RNA was harvested from either 5′Δ5N-Pac (B) or pPUPF1HA (C) transfectants during vegetative growth. The cells were treated without (0 min) or with 45 µg/ml actinomycin D for 13 min to arrest mRNA synthesis. Northern blot were hybridized with specific gene probes as indicated (upper panels). Ribosomal RNA loading controls are in the bottom panels. Representative results are shown. The numbers show the relative activity, which reflects expression relative to that in controls. The cwp1 and cwp2 signals from Fig. 4B and C were a long exposure to show the difference in the AcD treated and untreated samples.
Mentions: We further investigated whether UPF1 overexpression can influence the expression of cwp and other endogenous genes. We found that the levels of native cwp1, cwp2, cwp3, or myb2 mRNA in the vegetative pPUPF1HA cell line decreased by ∼85% or ∼50% (P<0.05) relative to the control cell line which only carries the pac gene (Fig. 4A). The ran mRNA levels did not change significantly in the pPUPF1HA cell line (Fig. 4A). The upf1 mRNA levels increased significantly (P<0.05) in the pPUPF1HA cell line (Fig. 4A).

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