Limits...
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.

Show MeSH

Related in: MedlinePlus

Ded1 was retained by cytoplasmic and nuclear cap-binding complexes. The indicated proteins were incubated with 7-methyl-GTP Sepharose beads, washed and then eluted with 1 mM free 7-methyl-GTP. The eluted proteins were separated by electrophoresis on 10% SDS polyacrylamide gels (SDS PAGE) and stained with Coomassie blue. (A) Proteins associated with the predominately cytoplasmic eIF4E protein bound to the beads. The eIF4G9 protein consisted of amino acids 160–492 of eIF4G1. Pab1 (Pab) gave a weak signal, but its presence was verified by western blot analysis. (B) Proteins associated with the predominately nuclear Cbp20 protein bound to the beads. Nab2 had a very weak affinity for the beads in the absence of the other proteins. The markers reflect the sizes of the Prestain Protein Ladder (Euromedex).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150762&req=5

Figure 4: Ded1 was retained by cytoplasmic and nuclear cap-binding complexes. The indicated proteins were incubated with 7-methyl-GTP Sepharose beads, washed and then eluted with 1 mM free 7-methyl-GTP. The eluted proteins were separated by electrophoresis on 10% SDS polyacrylamide gels (SDS PAGE) and stained with Coomassie blue. (A) Proteins associated with the predominately cytoplasmic eIF4E protein bound to the beads. The eIF4G9 protein consisted of amino acids 160–492 of eIF4G1. Pab1 (Pab) gave a weak signal, but its presence was verified by western blot analysis. (B) Proteins associated with the predominately nuclear Cbp20 protein bound to the beads. Nab2 had a very weak affinity for the beads in the absence of the other proteins. The markers reflect the sizes of the Prestain Protein Ladder (Euromedex).

Mentions: The preceding experiments indicated that Ded1 was in complexes containing the identified proteins, but they did not demonstrate a direct or functional interaction between the proteins. However, it seemed likely that Ded1 was part of the nuclear and cytoplasmic cap-binding complexes. To test this, we purified the different recombinant proteins from E. coli (Supplemental Figure S5) and tested their ability to form complexes on 7-methyl-GTP Sepharose beads. Both Cbp20 and eIF4E have high affinity for 7-methylguanosine. We bound these proteins to the beads, added various combinations of the purified proteins and then eluted the proteins retained (Figure 4). To facilitate the analyses, we used a shorten version of eIF4G1 (eIF4G9; amino acids 160–492) that was known to retain its capacity to interact with Pab1 and eIF4E (43). Of the proteins tested, only the polyadenosine-binding protein Nab2 showed a weak, nonspecific affinity for the beads.


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 was retained by cytoplasmic and nuclear cap-binding complexes. The indicated proteins were incubated with 7-methyl-GTP Sepharose beads, washed and then eluted with 1 mM free 7-methyl-GTP. The eluted proteins were separated by electrophoresis on 10% SDS polyacrylamide gels (SDS PAGE) and stained with Coomassie blue. (A) Proteins associated with the predominately cytoplasmic eIF4E protein bound to the beads. The eIF4G9 protein consisted of amino acids 160–492 of eIF4G1. Pab1 (Pab) gave a weak signal, but its presence was verified by western blot analysis. (B) Proteins associated with the predominately nuclear Cbp20 protein bound to the beads. Nab2 had a very weak affinity for the beads in the absence of the other proteins. The markers reflect the sizes of the Prestain Protein Ladder (Euromedex).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150762&req=5

Figure 4: Ded1 was retained by cytoplasmic and nuclear cap-binding complexes. The indicated proteins were incubated with 7-methyl-GTP Sepharose beads, washed and then eluted with 1 mM free 7-methyl-GTP. The eluted proteins were separated by electrophoresis on 10% SDS polyacrylamide gels (SDS PAGE) and stained with Coomassie blue. (A) Proteins associated with the predominately cytoplasmic eIF4E protein bound to the beads. The eIF4G9 protein consisted of amino acids 160–492 of eIF4G1. Pab1 (Pab) gave a weak signal, but its presence was verified by western blot analysis. (B) Proteins associated with the predominately nuclear Cbp20 protein bound to the beads. Nab2 had a very weak affinity for the beads in the absence of the other proteins. The markers reflect the sizes of the Prestain Protein Ladder (Euromedex).
Mentions: The preceding experiments indicated that Ded1 was in complexes containing the identified proteins, but they did not demonstrate a direct or functional interaction between the proteins. However, it seemed likely that Ded1 was part of the nuclear and cytoplasmic cap-binding complexes. To test this, we purified the different recombinant proteins from E. coli (Supplemental Figure S5) and tested their ability to form complexes on 7-methyl-GTP Sepharose beads. Both Cbp20 and eIF4E have high affinity for 7-methylguanosine. We bound these proteins to the beads, added various combinations of the purified proteins and then eluted the proteins retained (Figure 4). To facilitate the analyses, we used a shorten version of eIF4G1 (eIF4G9; amino acids 160–492) that was known to retain its capacity to interact with Pab1 and eIF4E (43). Of the proteins tested, only the polyadenosine-binding protein Nab2 showed a weak, nonspecific affinity for the beads.

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.

Show MeSH
Related in: MedlinePlus