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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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Sucrose gradients of cell extracts. (A) Yeast extract of the BY4742 strain was separated on a 7–47% sucrose gradient as described in the text, and ∼0.5-ml fractions were collected as indicated. The absorption profile at 254 nm is shown. Cells were incubated with 0.1 μg/ml of cycloheximide for 10 min prior to harvesting. Most of the Ded1 sedimented in complexes that were larger than those for the majority of the other detected proteins (indicated as *). (B) Western blot analysis of the fractions separated on a 10% SDS PAGE. Every other fraction was loaded so that the entire profile could be shown on a single gel. Ext, 10% of the original extract was loaded on the gel as a reference. (C) Ponceau red stained membrane of the electrophoretically transferred proteins from the SDS PAGE. MW, molecular weight markers shown in kDa.
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Figure 6: Sucrose gradients of cell extracts. (A) Yeast extract of the BY4742 strain was separated on a 7–47% sucrose gradient as described in the text, and ∼0.5-ml fractions were collected as indicated. The absorption profile at 254 nm is shown. Cells were incubated with 0.1 μg/ml of cycloheximide for 10 min prior to harvesting. Most of the Ded1 sedimented in complexes that were larger than those for the majority of the other detected proteins (indicated as *). (B) Western blot analysis of the fractions separated on a 10% SDS PAGE. Every other fraction was loaded so that the entire profile could be shown on a single gel. Ext, 10% of the original extract was loaded on the gel as a reference. (C) Ponceau red stained membrane of the electrophoretically transferred proteins from the SDS PAGE. MW, molecular weight markers shown in kDa.

Mentions: Ded1 has been proposed to associate with the 43S ribosome late during its assembly on the eIF4F translation-initiation complex, but prior to ribosome scanning to the AUG start codon, and to promote subsequent formation of the 48S ribosome [see (76) and references therein]. Ded1 is thought to enhance scanning of mRNAs and particularly those with long, structured, 5′ UTRs. Our results suggested that Ded1 associated with cap complexes on mRNAs very early and perhaps even before transcription was terminated. To help clarify this apparent contradiction, we undertook polysome analyses of yeast cells growing in early exponential phase (OD595 ∼0.8). Extracts were loaded on sucrose gradients and the complexes separated by centrifugation. Fractions were collected across the gradients, the recovered proteins separated by SDS polyacrylamide electrophoresis and then the gel was subjected to western blot analysis with IgGs against various proteins (Figure 6; Supplemental Figure S6A).


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)

Sucrose gradients of cell extracts. (A) Yeast extract of the BY4742 strain was separated on a 7–47% sucrose gradient as described in the text, and ∼0.5-ml fractions were collected as indicated. The absorption profile at 254 nm is shown. Cells were incubated with 0.1 μg/ml of cycloheximide for 10 min prior to harvesting. Most of the Ded1 sedimented in complexes that were larger than those for the majority of the other detected proteins (indicated as *). (B) Western blot analysis of the fractions separated on a 10% SDS PAGE. Every other fraction was loaded so that the entire profile could be shown on a single gel. Ext, 10% of the original extract was loaded on the gel as a reference. (C) Ponceau red stained membrane of the electrophoretically transferred proteins from the SDS PAGE. MW, molecular weight markers shown in kDa.
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Figure 6: Sucrose gradients of cell extracts. (A) Yeast extract of the BY4742 strain was separated on a 7–47% sucrose gradient as described in the text, and ∼0.5-ml fractions were collected as indicated. The absorption profile at 254 nm is shown. Cells were incubated with 0.1 μg/ml of cycloheximide for 10 min prior to harvesting. Most of the Ded1 sedimented in complexes that were larger than those for the majority of the other detected proteins (indicated as *). (B) Western blot analysis of the fractions separated on a 10% SDS PAGE. Every other fraction was loaded so that the entire profile could be shown on a single gel. Ext, 10% of the original extract was loaded on the gel as a reference. (C) Ponceau red stained membrane of the electrophoretically transferred proteins from the SDS PAGE. MW, molecular weight markers shown in kDa.
Mentions: Ded1 has been proposed to associate with the 43S ribosome late during its assembly on the eIF4F translation-initiation complex, but prior to ribosome scanning to the AUG start codon, and to promote subsequent formation of the 48S ribosome [see (76) and references therein]. Ded1 is thought to enhance scanning of mRNAs and particularly those with long, structured, 5′ UTRs. Our results suggested that Ded1 associated with cap complexes on mRNAs very early and perhaps even before transcription was terminated. To help clarify this apparent contradiction, we undertook polysome analyses of yeast cells growing in early exponential phase (OD595 ∼0.8). Extracts were loaded on sucrose gradients and the complexes separated by centrifugation. Fractions were collected across the gradients, the recovered proteins separated by SDS polyacrylamide electrophoresis and then the gel was subjected to western blot analysis with IgGs against various proteins (Figure 6; Supplemental Figure S6A).

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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Related in: MedlinePlus