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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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Mapping of the region in the cwp1 gene needed for the UPF1 dependent decay.(A) Diagrams of the pPC1 and plasmids for CWP1 deletion mapping. The pac or cwp1 gene (open box) expression cassette is the same as in Fig. 2 and Fig. 6, respectively. The predicted signal peptide is in gray. The LRRs are indicated as open boxes labeled “L”. (B). Analysis of au5 tagged cwp1 transcripts in vegetative cells. The pPC1D1-6+pRANneo and pPC1D1-6+pNUPF1HA transfectants were cultured in encystation medium for 24 h and then subjected to Northern blot analysis. Total RNA blots were hybridized with the au5 specific probe (au5hyb), and ran gene probe (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-g007: Mapping of the region in the cwp1 gene needed for the UPF1 dependent decay.(A) Diagrams of the pPC1 and plasmids for CWP1 deletion mapping. The pac or cwp1 gene (open box) expression cassette is the same as in Fig. 2 and Fig. 6, respectively. The predicted signal peptide is in gray. The LRRs are indicated as open boxes labeled “L”. (B). Analysis of au5 tagged cwp1 transcripts in vegetative cells. The pPC1D1-6+pRANneo and pPC1D1-6+pNUPF1HA transfectants were cultured in encystation medium for 24 h and then subjected to Northern blot analysis. Total RNA blots were hybridized with the au5 specific probe (au5hyb), and ran gene probe (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 further determined the region within the cwp1 mRNA responsible for the UPF1 dependent decay by constructing a series of deletions (Fig. 7A). Deletion of the sequence encoding the first, second or third to fourth LRRs (nucleotides 193–264, construct D2; nucleotides 265–345, construct D3; nucleotides 346–474, construct D4, Fig. 7A) still resulted in a significant decrease of cwp1-au5 mRNA in the pNUPF1HA co-transfectants relative to the control cell line (Fig. 7B). However, deletion of the sequence encoding the region N terminal to the LRRs (nucleotides 4–192, construct D1, Fig. 7A), the fifth LRR (nucleotides 475–552, construct D5, Fig. 7A) or the region C terminal to the LRRs (nucleotides 553–723, construct D6, Fig. 7A) resulted in an increase of the levels of the cwp1-au5 mRNA to ∼1.4 or 1.7-fold (P<0.05) in the pNUPF1HA co-transfectants relative to the control cell line (Fig. 7B). The results indicate that 5′ (nucleotides 4–192) or 3′ (nucleotides 475–723) sequence of the cwp1 gene may contain the sequence responsible for the UPF1 dependent decay.


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)

Mapping of the region in the cwp1 gene needed for the UPF1 dependent decay.(A) Diagrams of the pPC1 and plasmids for CWP1 deletion mapping. The pac or cwp1 gene (open box) expression cassette is the same as in Fig. 2 and Fig. 6, respectively. The predicted signal peptide is in gray. The LRRs are indicated as open boxes labeled “L”. (B). Analysis of au5 tagged cwp1 transcripts in vegetative cells. The pPC1D1-6+pRANneo and pPC1D1-6+pNUPF1HA transfectants were cultured in encystation medium for 24 h and then subjected to Northern blot analysis. Total RNA blots were hybridized with the au5 specific probe (au5hyb), and ran gene probe (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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0003609-g007: Mapping of the region in the cwp1 gene needed for the UPF1 dependent decay.(A) Diagrams of the pPC1 and plasmids for CWP1 deletion mapping. The pac or cwp1 gene (open box) expression cassette is the same as in Fig. 2 and Fig. 6, respectively. The predicted signal peptide is in gray. The LRRs are indicated as open boxes labeled “L”. (B). Analysis of au5 tagged cwp1 transcripts in vegetative cells. The pPC1D1-6+pRANneo and pPC1D1-6+pNUPF1HA transfectants were cultured in encystation medium for 24 h and then subjected to Northern blot analysis. Total RNA blots were hybridized with the au5 specific probe (au5hyb), and ran gene probe (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 further determined the region within the cwp1 mRNA responsible for the UPF1 dependent decay by constructing a series of deletions (Fig. 7A). Deletion of the sequence encoding the first, second or third to fourth LRRs (nucleotides 193–264, construct D2; nucleotides 265–345, construct D3; nucleotides 346–474, construct D4, Fig. 7A) still resulted in a significant decrease of cwp1-au5 mRNA in the pNUPF1HA co-transfectants relative to the control cell line (Fig. 7B). However, deletion of the sequence encoding the region N terminal to the LRRs (nucleotides 4–192, construct D1, Fig. 7A), the fifth LRR (nucleotides 475–552, construct D5, Fig. 7A) or the region C terminal to the LRRs (nucleotides 553–723, construct D6, Fig. 7A) resulted in an increase of the levels of the cwp1-au5 mRNA to ∼1.4 or 1.7-fold (P<0.05) in the pNUPF1HA co-transfectants relative to the control cell line (Fig. 7B). The results indicate that 5′ (nucleotides 4–192) or 3′ (nucleotides 475–723) sequence of the cwp1 gene may contain the sequence responsible for the UPF1 dependent decay.

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