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A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18.

Chi B, Wang K, Du Y, Gui B, Chang X, Wang L, Fan J, Chen S, Wu X, Li G, Cheng H - Nucleic Acids Res. (2014)

Bottom Line: We found that PRE drastically enhances the cytoplasmic accumulation of cDNA transcripts independent of any viral protein.Together, our data indicate that SEP1 enhances mRNA export by recruiting TREX via ZC3H18.The new mRNA export factors that we identified might be involved in cap- and splicing-dependent TREX recruitment to cellular mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Molecular Andrology, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

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PRE promotes the cytoplasmic accumulation of cDNA transcripts. (A) Schematic of the β-globin reporter constructs. The CMV promoter, BGH polyA sites and the location of the FISH probe (vector probe) that detects a region of pcDNA3 vector (indicated by a short line) are shown. (B) CMV-DNA constructs were transfected into HeLa cells. Twenty-four hours after transfection, FISH was preformed to detect the indicated mRNAs. DAPI staining was used to indicate the nuclei. C/N ratios were determined for 30 cells per construct in each experiment. The graph shows the average C/N ratios from three independent experiments, and error bars indicate the standard deviations. Statistical analysis was performed using Student's t test. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Schematic of the cG constructs with truncated forms of PRE. The remaining parts of PRE are shown with nucleotide numbers relative to the HBV genomic sequence. (D) Constructs shown in (C) were transfected into HeLa cells followed by FISH analysis at 24 h after transfection. Numbers below the images represent the C/N ratios (mean value ± SD). Quantification was carried out as in (B).(.
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Figure 1: PRE promotes the cytoplasmic accumulation of cDNA transcripts. (A) Schematic of the β-globin reporter constructs. The CMV promoter, BGH polyA sites and the location of the FISH probe (vector probe) that detects a region of pcDNA3 vector (indicated by a short line) are shown. (B) CMV-DNA constructs were transfected into HeLa cells. Twenty-four hours after transfection, FISH was preformed to detect the indicated mRNAs. DAPI staining was used to indicate the nuclei. C/N ratios were determined for 30 cells per construct in each experiment. The graph shows the average C/N ratios from three independent experiments, and error bars indicate the standard deviations. Statistical analysis was performed using Student's t test. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Schematic of the cG constructs with truncated forms of PRE. The remaining parts of PRE are shown with nucleotide numbers relative to the HBV genomic sequence. (D) Constructs shown in (C) were transfected into HeLa cells followed by FISH analysis at 24 h after transfection. Numbers below the images represent the C/N ratios (mean value ± SD). Quantification was carried out as in (B).(.

Mentions: Previous studies suggested that PRE enhances the expression of intronless reporter mRNAs by promoting nuclear export (18). To determine the role of PRE in promoting nuclear export of intronless mRNAs, we constructed β-globin reporter plasmids with PRE inserted in the sense (cG-PRE) or anti-sense direction (cG-rPRE) at the 3′ end of the open reading frame (Figure 1A). These constructs, as well as wild-type β-globin (wG) and its cDNA counterpart (cG), were transfected into HeLa cells, and nucleocytoplasmic distribution of these mRNAs was determined using FISH (Figure 1B). As expected, 24 h after transfection, the wG mRNA mainly accumulated in the cytoplasm, whereas the cG mRNA was mostly retained in the nucleus (Figure 1B, wG and cG). Significantly, the cG-PRE mRNA was largely detected in the cytoplasm. In contrast, the cG-rPRE mRNA was mainly nuclear (Figure 1B). These results indicate that PRE enhances the cytoplasmic accumulation of the cG mRNA. To test whether PRE also enhances cytoplasmic accumulation of other intronless mRNAs, we next used Smad reporter constructs. When PRE was inserted at the 3′ end, the Smad cDNA transcript (cS) that was otherwise retained in the nucleus was accumulated in the cytoplasm to the similar extent to the spliced Smad mRNA (wS) (Supplementary Figure S1). Thus, our data indicate that the role of PRE in enhancing nuclear export of cDNA transcripts is general.


A Sub-Element in PRE enhances nuclear export of intronless mRNAs by recruiting the TREX complex via ZC3H18.

Chi B, Wang K, Du Y, Gui B, Chang X, Wang L, Fan J, Chen S, Wu X, Li G, Cheng H - Nucleic Acids Res. (2014)

PRE promotes the cytoplasmic accumulation of cDNA transcripts. (A) Schematic of the β-globin reporter constructs. The CMV promoter, BGH polyA sites and the location of the FISH probe (vector probe) that detects a region of pcDNA3 vector (indicated by a short line) are shown. (B) CMV-DNA constructs were transfected into HeLa cells. Twenty-four hours after transfection, FISH was preformed to detect the indicated mRNAs. DAPI staining was used to indicate the nuclei. C/N ratios were determined for 30 cells per construct in each experiment. The graph shows the average C/N ratios from three independent experiments, and error bars indicate the standard deviations. Statistical analysis was performed using Student's t test. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Schematic of the cG constructs with truncated forms of PRE. The remaining parts of PRE are shown with nucleotide numbers relative to the HBV genomic sequence. (D) Constructs shown in (C) were transfected into HeLa cells followed by FISH analysis at 24 h after transfection. Numbers below the images represent the C/N ratios (mean value ± SD). Quantification was carried out as in (B).(.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: PRE promotes the cytoplasmic accumulation of cDNA transcripts. (A) Schematic of the β-globin reporter constructs. The CMV promoter, BGH polyA sites and the location of the FISH probe (vector probe) that detects a region of pcDNA3 vector (indicated by a short line) are shown. (B) CMV-DNA constructs were transfected into HeLa cells. Twenty-four hours after transfection, FISH was preformed to detect the indicated mRNAs. DAPI staining was used to indicate the nuclei. C/N ratios were determined for 30 cells per construct in each experiment. The graph shows the average C/N ratios from three independent experiments, and error bars indicate the standard deviations. Statistical analysis was performed using Student's t test. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Schematic of the cG constructs with truncated forms of PRE. The remaining parts of PRE are shown with nucleotide numbers relative to the HBV genomic sequence. (D) Constructs shown in (C) were transfected into HeLa cells followed by FISH analysis at 24 h after transfection. Numbers below the images represent the C/N ratios (mean value ± SD). Quantification was carried out as in (B).(.
Mentions: Previous studies suggested that PRE enhances the expression of intronless reporter mRNAs by promoting nuclear export (18). To determine the role of PRE in promoting nuclear export of intronless mRNAs, we constructed β-globin reporter plasmids with PRE inserted in the sense (cG-PRE) or anti-sense direction (cG-rPRE) at the 3′ end of the open reading frame (Figure 1A). These constructs, as well as wild-type β-globin (wG) and its cDNA counterpart (cG), were transfected into HeLa cells, and nucleocytoplasmic distribution of these mRNAs was determined using FISH (Figure 1B). As expected, 24 h after transfection, the wG mRNA mainly accumulated in the cytoplasm, whereas the cG mRNA was mostly retained in the nucleus (Figure 1B, wG and cG). Significantly, the cG-PRE mRNA was largely detected in the cytoplasm. In contrast, the cG-rPRE mRNA was mainly nuclear (Figure 1B). These results indicate that PRE enhances the cytoplasmic accumulation of the cG mRNA. To test whether PRE also enhances cytoplasmic accumulation of other intronless mRNAs, we next used Smad reporter constructs. When PRE was inserted at the 3′ end, the Smad cDNA transcript (cS) that was otherwise retained in the nucleus was accumulated in the cytoplasm to the similar extent to the spliced Smad mRNA (wS) (Supplementary Figure S1). Thus, our data indicate that the role of PRE in enhancing nuclear export of cDNA transcripts is general.

Bottom Line: We found that PRE drastically enhances the cytoplasmic accumulation of cDNA transcripts independent of any viral protein.Together, our data indicate that SEP1 enhances mRNA export by recruiting TREX via ZC3H18.The new mRNA export factors that we identified might be involved in cap- and splicing-dependent TREX recruitment to cellular mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Molecular Andrology, State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.

Show MeSH
Related in: MedlinePlus