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The closely related RNA helicases, UAP56 and URH49, preferentially form distinct mRNA export machineries and coordinately regulate mitotic progression.

Yamazaki T, Fujiwara N, Yukinaga H, Ebisuya M, Shiki T, Kurihara T, Kioka N, Kambe T, Nagao M, Nishida E, Masuda S - Mol. Biol. Cell (2010)

Bottom Line: Consistent with their target mRNAs, depletion of UAP56 causes mitotic delay and sister chromatid cohesion defects, whereas depletion of URH49 causes chromosome arm resolution defects and failure of cytokinesis.In addition, depletion of the other human TREX components or CIP29 causes mitotic defects similar to those observed in UAP56- or URH49-depleted cells, respectively.Taken together, the two closely related RNA helicases have evolved to form distinct mRNA export machineries, which regulate mitosis at different steps.

View Article: PubMed Central - PubMed

Affiliation: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.

ABSTRACT
Nuclear export of mRNA is an essential process for eukaryotic gene expression. The TREX complex couples gene expression from transcription and splicing to mRNA export. Sub2, a core component of the TREX complex in yeast, has diversified in humans to two closely related RNA helicases, UAP56 and URH49. Here, we show that URH49 forms a novel URH49-CIP29 complex, termed the AREX (alternative mRNA export) complex, whereas UAP56 forms the human TREX complex. The mRNAs regulated by these helicases are different at the genome-wide level. The two sets of target mRNAs contain distinct subsets of key mitotic regulators. Consistent with their target mRNAs, depletion of UAP56 causes mitotic delay and sister chromatid cohesion defects, whereas depletion of URH49 causes chromosome arm resolution defects and failure of cytokinesis. In addition, depletion of the other human TREX components or CIP29 causes mitotic defects similar to those observed in UAP56- or URH49-depleted cells, respectively. Taken together, the two closely related RNA helicases have evolved to form distinct mRNA export machineries, which regulate mitosis at different steps.

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hTREXi results in mitotic delay with chromosome misalignment. (A) Knockdown of Aly, hHpr1 or CIP29 was confirmed by immunoblotting. HeLa cells transfected with the indicated siRNAs were cultured for 48 h. β-actin was used as a loading control. IB; immunoblotting. (B) Typical mitotic figures are shown in the indicated siRNA-transfected HeLa cells expressing GFP-CENP-A. GFP-CENP-A and β-tubulin signals indicate the location of the centromere and spindle body, respectively. The arrowhead indicates the misaligned chromosome. Scale bar, 10 μm. (C) The proportion of cells in each mitotic phase is shown in several knockdown cells. Each value is the mean with SD of three independent experiments (more than 70 cells were evaluated in each experiment). The ratios of cells in prometaphase to metaphase (PM/M ratio) are shown as the means with SD of three independent experiments. p values were calculated by comparison with the control: *p < 0.05, **p < 0.01, Student's t test. (D) Representative successive live cell images in knockdown cells. The number at the bottom right indicates the time (min) from nuclear envelope breakdown (NEBD). The arrowhead indicates the misaligned chromosome. Scale bar, 10 μm. (E) The time from NEBD-to-anaphase transition or mitotic cell death (MCD) was measured. At least 30 cells were observed in each experiment. Each value is the mean with SD of three independent experiments. The data from Figure 2D for Control and UAP56, and URH49 knockdown cells are shown to enable comparison. p values were calculated by comparison with the control: *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test. (F) The duration is shown as the mean with SD of three independent experiments. The data from Figure 2E for Control and UAP56 and URH49 knockdown cells are shown to enable comparison. p values were calculated by comparison with the control if not indicated otherwise: *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test.
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Figure 6: hTREXi results in mitotic delay with chromosome misalignment. (A) Knockdown of Aly, hHpr1 or CIP29 was confirmed by immunoblotting. HeLa cells transfected with the indicated siRNAs were cultured for 48 h. β-actin was used as a loading control. IB; immunoblotting. (B) Typical mitotic figures are shown in the indicated siRNA-transfected HeLa cells expressing GFP-CENP-A. GFP-CENP-A and β-tubulin signals indicate the location of the centromere and spindle body, respectively. The arrowhead indicates the misaligned chromosome. Scale bar, 10 μm. (C) The proportion of cells in each mitotic phase is shown in several knockdown cells. Each value is the mean with SD of three independent experiments (more than 70 cells were evaluated in each experiment). The ratios of cells in prometaphase to metaphase (PM/M ratio) are shown as the means with SD of three independent experiments. p values were calculated by comparison with the control: *p < 0.05, **p < 0.01, Student's t test. (D) Representative successive live cell images in knockdown cells. The number at the bottom right indicates the time (min) from nuclear envelope breakdown (NEBD). The arrowhead indicates the misaligned chromosome. Scale bar, 10 μm. (E) The time from NEBD-to-anaphase transition or mitotic cell death (MCD) was measured. At least 30 cells were observed in each experiment. Each value is the mean with SD of three independent experiments. The data from Figure 2D for Control and UAP56, and URH49 knockdown cells are shown to enable comparison. p values were calculated by comparison with the control: *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test. (F) The duration is shown as the mean with SD of three independent experiments. The data from Figure 2E for Control and UAP56 and URH49 knockdown cells are shown to enable comparison. p values were calculated by comparison with the control if not indicated otherwise: *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test.

Mentions: To reveal the in vivo biological functions of the hTREX complex and the AREX complex, the hTHO complex, Aly, and CIP29 were knocked down in HeLa cells. Each siRNA efficiently knocked down its corresponding mRNA and protein (Figure 6A). In hHpr1i cells, the expression of fSAP79 and fSAP24, other components of the hTREX complex, was down-regulated. A similar phenomenon was previously observed in S. cerevisiae lacking the THO components (Strasser et al., 2002). Immunodepletion of hTho2 from HeLa cell NE also codepleted the rest of the THO components (Masuda et al., 2005). Together, this implies that the THO complex is unstable if one of its components is depleted. In these cells, the effect on nuclear export of bulk poly(A)+ RNA was examined. hHpr1i, Alyi, and CIP29i cells accumulated bulk poly(A)+ RNA in the nucleus (Supplemental Figure S5). This suggests that CIP29 is involved in the mRNA export pathway.


The closely related RNA helicases, UAP56 and URH49, preferentially form distinct mRNA export machineries and coordinately regulate mitotic progression.

Yamazaki T, Fujiwara N, Yukinaga H, Ebisuya M, Shiki T, Kurihara T, Kioka N, Kambe T, Nagao M, Nishida E, Masuda S - Mol. Biol. Cell (2010)

hTREXi results in mitotic delay with chromosome misalignment. (A) Knockdown of Aly, hHpr1 or CIP29 was confirmed by immunoblotting. HeLa cells transfected with the indicated siRNAs were cultured for 48 h. β-actin was used as a loading control. IB; immunoblotting. (B) Typical mitotic figures are shown in the indicated siRNA-transfected HeLa cells expressing GFP-CENP-A. GFP-CENP-A and β-tubulin signals indicate the location of the centromere and spindle body, respectively. The arrowhead indicates the misaligned chromosome. Scale bar, 10 μm. (C) The proportion of cells in each mitotic phase is shown in several knockdown cells. Each value is the mean with SD of three independent experiments (more than 70 cells were evaluated in each experiment). The ratios of cells in prometaphase to metaphase (PM/M ratio) are shown as the means with SD of three independent experiments. p values were calculated by comparison with the control: *p < 0.05, **p < 0.01, Student's t test. (D) Representative successive live cell images in knockdown cells. The number at the bottom right indicates the time (min) from nuclear envelope breakdown (NEBD). The arrowhead indicates the misaligned chromosome. Scale bar, 10 μm. (E) The time from NEBD-to-anaphase transition or mitotic cell death (MCD) was measured. At least 30 cells were observed in each experiment. Each value is the mean with SD of three independent experiments. The data from Figure 2D for Control and UAP56, and URH49 knockdown cells are shown to enable comparison. p values were calculated by comparison with the control: *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test. (F) The duration is shown as the mean with SD of three independent experiments. The data from Figure 2E for Control and UAP56 and URH49 knockdown cells are shown to enable comparison. p values were calculated by comparison with the control if not indicated otherwise: *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test.
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Figure 6: hTREXi results in mitotic delay with chromosome misalignment. (A) Knockdown of Aly, hHpr1 or CIP29 was confirmed by immunoblotting. HeLa cells transfected with the indicated siRNAs were cultured for 48 h. β-actin was used as a loading control. IB; immunoblotting. (B) Typical mitotic figures are shown in the indicated siRNA-transfected HeLa cells expressing GFP-CENP-A. GFP-CENP-A and β-tubulin signals indicate the location of the centromere and spindle body, respectively. The arrowhead indicates the misaligned chromosome. Scale bar, 10 μm. (C) The proportion of cells in each mitotic phase is shown in several knockdown cells. Each value is the mean with SD of three independent experiments (more than 70 cells were evaluated in each experiment). The ratios of cells in prometaphase to metaphase (PM/M ratio) are shown as the means with SD of three independent experiments. p values were calculated by comparison with the control: *p < 0.05, **p < 0.01, Student's t test. (D) Representative successive live cell images in knockdown cells. The number at the bottom right indicates the time (min) from nuclear envelope breakdown (NEBD). The arrowhead indicates the misaligned chromosome. Scale bar, 10 μm. (E) The time from NEBD-to-anaphase transition or mitotic cell death (MCD) was measured. At least 30 cells were observed in each experiment. Each value is the mean with SD of three independent experiments. The data from Figure 2D for Control and UAP56, and URH49 knockdown cells are shown to enable comparison. p values were calculated by comparison with the control: *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test. (F) The duration is shown as the mean with SD of three independent experiments. The data from Figure 2E for Control and UAP56 and URH49 knockdown cells are shown to enable comparison. p values were calculated by comparison with the control if not indicated otherwise: *p < 0.05, **p < 0.01, ***p < 0.001, Student's t test.
Mentions: To reveal the in vivo biological functions of the hTREX complex and the AREX complex, the hTHO complex, Aly, and CIP29 were knocked down in HeLa cells. Each siRNA efficiently knocked down its corresponding mRNA and protein (Figure 6A). In hHpr1i cells, the expression of fSAP79 and fSAP24, other components of the hTREX complex, was down-regulated. A similar phenomenon was previously observed in S. cerevisiae lacking the THO components (Strasser et al., 2002). Immunodepletion of hTho2 from HeLa cell NE also codepleted the rest of the THO components (Masuda et al., 2005). Together, this implies that the THO complex is unstable if one of its components is depleted. In these cells, the effect on nuclear export of bulk poly(A)+ RNA was examined. hHpr1i, Alyi, and CIP29i cells accumulated bulk poly(A)+ RNA in the nucleus (Supplemental Figure S5). This suggests that CIP29 is involved in the mRNA export pathway.

Bottom Line: Consistent with their target mRNAs, depletion of UAP56 causes mitotic delay and sister chromatid cohesion defects, whereas depletion of URH49 causes chromosome arm resolution defects and failure of cytokinesis.In addition, depletion of the other human TREX components or CIP29 causes mitotic defects similar to those observed in UAP56- or URH49-depleted cells, respectively.Taken together, the two closely related RNA helicases have evolved to form distinct mRNA export machineries, which regulate mitosis at different steps.

View Article: PubMed Central - PubMed

Affiliation: Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.

ABSTRACT
Nuclear export of mRNA is an essential process for eukaryotic gene expression. The TREX complex couples gene expression from transcription and splicing to mRNA export. Sub2, a core component of the TREX complex in yeast, has diversified in humans to two closely related RNA helicases, UAP56 and URH49. Here, we show that URH49 forms a novel URH49-CIP29 complex, termed the AREX (alternative mRNA export) complex, whereas UAP56 forms the human TREX complex. The mRNAs regulated by these helicases are different at the genome-wide level. The two sets of target mRNAs contain distinct subsets of key mitotic regulators. Consistent with their target mRNAs, depletion of UAP56 causes mitotic delay and sister chromatid cohesion defects, whereas depletion of URH49 causes chromosome arm resolution defects and failure of cytokinesis. In addition, depletion of the other human TREX components or CIP29 causes mitotic defects similar to those observed in UAP56- or URH49-depleted cells, respectively. Taken together, the two closely related RNA helicases have evolved to form distinct mRNA export machineries, which regulate mitosis at different steps.

Show MeSH
Related in: MedlinePlus