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The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus.

Senissar M, Le Saux A, Belgareh-Touzé N, Adam C, Banroques J, Tanner NK - Nucleic Acids Res. (2014)

Bottom Line: In addition, we show that Ded1 is genetically linked to these factors.Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes.We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Expression Génétique Microbienne, CNRS FRE3630 (UPR9073), in association with Université Paris Diderot, Sorbonne Paris Cité, Paris 75005, France Université Paris-Sud, Ecole Doctorale 426 GGC, Orsay, France.

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Ded1 shuttled between the cytoplasm and nucleus. (A) and (B) Fluorescence microscopy of cells that were fixed with formaldehyde when stained with DAPI to reveal the nuclei and mitochondria (seen as speckles). Ded1-GFP was primarily located in the cytoplasm of wildtype (G50) cells at all the temperatures tested. (C) In contrast, Ded1-GFP shows a strong nuclear location in the mex67-5/xpo1-1 mutant when incubated at nonpermissive temperatures for 30 min in living cells stained with DAPI. (D) Fluorescence microscopy (GFP) and Nomarski interference contrast microscopy of living yeast cells. Ded1-GFP was mostly located in the cytoplasm of mex67-5/xpo1-1 cells at the permissive temperatures (24°C), but a small amount was visible in the nuclei as well (arrows). The vacuoles/lysosomes are indicated with arrowheads. (E) In contrast, Cbp80-GFP was almost exclusively located in the nuclei. (F) Ded1-GFP that was deleted for the 30 amino-terminal residues containing the NES sequence (Δ30N) and the eIF4E binding site accumulated in the nuclei of yeast even in wildtype cells.
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Figure 1: Ded1 shuttled between the cytoplasm and nucleus. (A) and (B) Fluorescence microscopy of cells that were fixed with formaldehyde when stained with DAPI to reveal the nuclei and mitochondria (seen as speckles). Ded1-GFP was primarily located in the cytoplasm of wildtype (G50) cells at all the temperatures tested. (C) In contrast, Ded1-GFP shows a strong nuclear location in the mex67-5/xpo1-1 mutant when incubated at nonpermissive temperatures for 30 min in living cells stained with DAPI. (D) Fluorescence microscopy (GFP) and Nomarski interference contrast microscopy of living yeast cells. Ded1-GFP was mostly located in the cytoplasm of mex67-5/xpo1-1 cells at the permissive temperatures (24°C), but a small amount was visible in the nuclei as well (arrows). The vacuoles/lysosomes are indicated with arrowheads. (E) In contrast, Cbp80-GFP was almost exclusively located in the nuclei. (F) Ded1-GFP that was deleted for the 30 amino-terminal residues containing the NES sequence (Δ30N) and the eIF4E binding site accumulated in the nuclei of yeast even in wildtype cells.

Mentions: We next asked where the Ded1-GFP fusion protein was located within the cell. Fluorescence microscopy of living cells revealed that the protein was primarily located within the cytoplasm, but the nuclei showed a faint signal relative to the vacuoles/lysosomes. To verify this, we fixed the cells and then stained them with DAPI to identify the nuclei (Figure 1A and B). These experiments confirmed that Ded1 was largely excluded from the nuclei, but that the faint signals corresponded to the nuclei rather than to the vacuoles. Both plasmid-expressed and integrated Ded1-GFP showed the same distributions, which indicated that the expression levels did not alter the results. However, the intensity of the Ded1-GFP signal varied between cells in both the plasmid-borne and integrated cells. This implied that the expression levels of Ded1 were variable between cells (Figure 1A and B and Supplemental Figure S3). Thus, our results were consistent with the possibility that a small amount of Ded1 was located in the nucleus.


The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus.

Senissar M, Le Saux A, Belgareh-Touzé N, Adam C, Banroques J, Tanner NK - Nucleic Acids Res. (2014)

Ded1 shuttled between the cytoplasm and nucleus. (A) and (B) Fluorescence microscopy of cells that were fixed with formaldehyde when stained with DAPI to reveal the nuclei and mitochondria (seen as speckles). Ded1-GFP was primarily located in the cytoplasm of wildtype (G50) cells at all the temperatures tested. (C) In contrast, Ded1-GFP shows a strong nuclear location in the mex67-5/xpo1-1 mutant when incubated at nonpermissive temperatures for 30 min in living cells stained with DAPI. (D) Fluorescence microscopy (GFP) and Nomarski interference contrast microscopy of living yeast cells. Ded1-GFP was mostly located in the cytoplasm of mex67-5/xpo1-1 cells at the permissive temperatures (24°C), but a small amount was visible in the nuclei as well (arrows). The vacuoles/lysosomes are indicated with arrowheads. (E) In contrast, Cbp80-GFP was almost exclusively located in the nuclei. (F) Ded1-GFP that was deleted for the 30 amino-terminal residues containing the NES sequence (Δ30N) and the eIF4E binding site accumulated in the nuclei of yeast even in wildtype cells.
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Related In: Results  -  Collection

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Figure 1: Ded1 shuttled between the cytoplasm and nucleus. (A) and (B) Fluorescence microscopy of cells that were fixed with formaldehyde when stained with DAPI to reveal the nuclei and mitochondria (seen as speckles). Ded1-GFP was primarily located in the cytoplasm of wildtype (G50) cells at all the temperatures tested. (C) In contrast, Ded1-GFP shows a strong nuclear location in the mex67-5/xpo1-1 mutant when incubated at nonpermissive temperatures for 30 min in living cells stained with DAPI. (D) Fluorescence microscopy (GFP) and Nomarski interference contrast microscopy of living yeast cells. Ded1-GFP was mostly located in the cytoplasm of mex67-5/xpo1-1 cells at the permissive temperatures (24°C), but a small amount was visible in the nuclei as well (arrows). The vacuoles/lysosomes are indicated with arrowheads. (E) In contrast, Cbp80-GFP was almost exclusively located in the nuclei. (F) Ded1-GFP that was deleted for the 30 amino-terminal residues containing the NES sequence (Δ30N) and the eIF4E binding site accumulated in the nuclei of yeast even in wildtype cells.
Mentions: We next asked where the Ded1-GFP fusion protein was located within the cell. Fluorescence microscopy of living cells revealed that the protein was primarily located within the cytoplasm, but the nuclei showed a faint signal relative to the vacuoles/lysosomes. To verify this, we fixed the cells and then stained them with DAPI to identify the nuclei (Figure 1A and B). These experiments confirmed that Ded1 was largely excluded from the nuclei, but that the faint signals corresponded to the nuclei rather than to the vacuoles. Both plasmid-expressed and integrated Ded1-GFP showed the same distributions, which indicated that the expression levels did not alter the results. However, the intensity of the Ded1-GFP signal varied between cells in both the plasmid-borne and integrated cells. This implied that the expression levels of Ded1 were variable between cells (Figure 1A and B and Supplemental Figure S3). Thus, our results were consistent with the possibility that a small amount of Ded1 was located in the nucleus.

Bottom Line: In addition, we show that Ded1 is genetically linked to these factors.Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes.We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs.

View Article: PubMed Central - PubMed

Affiliation: Expression Génétique Microbienne, CNRS FRE3630 (UPR9073), in association with Université Paris Diderot, Sorbonne Paris Cité, Paris 75005, France Université Paris-Sud, Ecole Doctorale 426 GGC, Orsay, France.

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