Limits...
The archaeal DnaG protein needs Csl4 for binding to the exosome and enhances its interaction with adenine-rich RNAs.

Hou L, Klug G, Evguenieva-Hackenberg E - RNA Biol (2013)

Bottom Line: We found that the archaeal DnaG binds to the Csl4-exosome but not to the Rrp4-exosome of Sulfolobus solfataricus.DnaG is the second poly(A)-binding protein besides Rrp4 in the heteromeric, RNA-binding cap of the S. solfataricus exosome.This apparently reflects the need for effective and selective recruitment of adenine-rich RNAs to the exosome in the RNA metabolism of S. solfataricus.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Molecular Biology; Heinrich-Buff-Ring; Giessen, Germany.

ABSTRACT
The archaeal RNA-degrading exosome contains a catalytically active hexameric core, an RNA-binding cap formed by Rrp4 and Csl4 and the protein annotated as DnaG (bacterial type primase) with so-far-unknown functions in RNA metabolism. We found that the archaeal DnaG binds to the Csl4-exosome but not to the Rrp4-exosome of Sulfolobus solfataricus. In vitro assays revealed that DnaG is a poly(A)-binding protein enhancing the degradation of adenine-rich transcripts by the Csl4-exosome. DnaG is the second poly(A)-binding protein besides Rrp4 in the heteromeric, RNA-binding cap of the S. solfataricus exosome. This apparently reflects the need for effective and selective recruitment of adenine-rich RNAs to the exosome in the RNA metabolism of S. solfataricus.

Show MeSH
Figure 1. DnaG needs Csl4 for the interaction with the exosome. Silver-stained 12% SDS-gels showing results of experiments analyzing the interaction between DnaG and the exosome. In, input, the mixture of proteins used; FT, flow-through; W1, W5, the first and the last washing fractions; E, the elution fraction. (A) Rrp4-exosome or Csl4-exosome was mixed with DnaG and the interacting proteins were co-immunoprecipitated with Rrp41-specific antibodies. His-tagged proteins were used. (B) The Rrp41-Rrp42 hexamer (hexameric ring) alone or mixed with DnaG was subjected to co-immunoprecipitation (CoIP) with DnaG-specific antibodies. His-tagged proteins were used. The protein fractions were analyzed by SDS-PAGE and silver staining (upper panel) and by western blot hybridization with Rrp41-specific antibodies. (C) Strep-tagged Csl4 and His-tagged DnaG were mixed and a pull-down assay with Strep-Tactin Sepharose beads was performed. M, protein marker, the migration behavior of the proteins is given in kDa on the left side. An E. coli protein binding to Ni-NTA, which was routinely present in the DnaG-fractions used for the reconstitution experiment, is marked with an asterisk in (A and C). (D) His-tagged Rrp4 and His-tagged DnaG were mixed and CoIP with Rrp4-specific antibodies was performed. The detected proteins are marked on the right side. (E) Reconstituted and purified Csl4-exosome and DnaG-Csl4-exosome. (F) Reconstituted and purified Rrp4-Csl4-exosome and DnaG-Rrp4-Csl4-exosome. For the experiments shown in (E and F), Strep-Csl4 and His-tagged Rrp4, Rrp41, Rrp42 and DnaG proteins were used. Purification was performed with Strep-Tactin followed by Ni-NTA chromatography.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3672285&req=5

Figure 1: Figure 1. DnaG needs Csl4 for the interaction with the exosome. Silver-stained 12% SDS-gels showing results of experiments analyzing the interaction between DnaG and the exosome. In, input, the mixture of proteins used; FT, flow-through; W1, W5, the first and the last washing fractions; E, the elution fraction. (A) Rrp4-exosome or Csl4-exosome was mixed with DnaG and the interacting proteins were co-immunoprecipitated with Rrp41-specific antibodies. His-tagged proteins were used. (B) The Rrp41-Rrp42 hexamer (hexameric ring) alone or mixed with DnaG was subjected to co-immunoprecipitation (CoIP) with DnaG-specific antibodies. His-tagged proteins were used. The protein fractions were analyzed by SDS-PAGE and silver staining (upper panel) and by western blot hybridization with Rrp41-specific antibodies. (C) Strep-tagged Csl4 and His-tagged DnaG were mixed and a pull-down assay with Strep-Tactin Sepharose beads was performed. M, protein marker, the migration behavior of the proteins is given in kDa on the left side. An E. coli protein binding to Ni-NTA, which was routinely present in the DnaG-fractions used for the reconstitution experiment, is marked with an asterisk in (A and C). (D) His-tagged Rrp4 and His-tagged DnaG were mixed and CoIP with Rrp4-specific antibodies was performed. The detected proteins are marked on the right side. (E) Reconstituted and purified Csl4-exosome and DnaG-Csl4-exosome. (F) Reconstituted and purified Rrp4-Csl4-exosome and DnaG-Rrp4-Csl4-exosome. For the experiments shown in (E and F), Strep-Csl4 and His-tagged Rrp4, Rrp41, Rrp42 and DnaG proteins were used. Purification was performed with Strep-Tactin followed by Ni-NTA chromatography.

Mentions: To study the mechanisms of interaction between DnaG and the exosome, we reconstituted complexes with homomeric RNA-binding caps containing either Rrp4 or Csl4 and tested them for interaction with DnaG by co-immunoprecipitation with Rrp41-specific antibodies. His-tagged versions of all proteins were used for these tests. Figure 1A shows that the Rrp4-exosome does not interact with DnaG, and demonstrates a clear binding of DnaG to the Csl4-exosome.


The archaeal DnaG protein needs Csl4 for binding to the exosome and enhances its interaction with adenine-rich RNAs.

Hou L, Klug G, Evguenieva-Hackenberg E - RNA Biol (2013)

Figure 1. DnaG needs Csl4 for the interaction with the exosome. Silver-stained 12% SDS-gels showing results of experiments analyzing the interaction between DnaG and the exosome. In, input, the mixture of proteins used; FT, flow-through; W1, W5, the first and the last washing fractions; E, the elution fraction. (A) Rrp4-exosome or Csl4-exosome was mixed with DnaG and the interacting proteins were co-immunoprecipitated with Rrp41-specific antibodies. His-tagged proteins were used. (B) The Rrp41-Rrp42 hexamer (hexameric ring) alone or mixed with DnaG was subjected to co-immunoprecipitation (CoIP) with DnaG-specific antibodies. His-tagged proteins were used. The protein fractions were analyzed by SDS-PAGE and silver staining (upper panel) and by western blot hybridization with Rrp41-specific antibodies. (C) Strep-tagged Csl4 and His-tagged DnaG were mixed and a pull-down assay with Strep-Tactin Sepharose beads was performed. M, protein marker, the migration behavior of the proteins is given in kDa on the left side. An E. coli protein binding to Ni-NTA, which was routinely present in the DnaG-fractions used for the reconstitution experiment, is marked with an asterisk in (A and C). (D) His-tagged Rrp4 and His-tagged DnaG were mixed and CoIP with Rrp4-specific antibodies was performed. The detected proteins are marked on the right side. (E) Reconstituted and purified Csl4-exosome and DnaG-Csl4-exosome. (F) Reconstituted and purified Rrp4-Csl4-exosome and DnaG-Rrp4-Csl4-exosome. For the experiments shown in (E and F), Strep-Csl4 and His-tagged Rrp4, Rrp41, Rrp42 and DnaG proteins were used. Purification was performed with Strep-Tactin followed by Ni-NTA chromatography.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672285&req=5

Figure 1: Figure 1. DnaG needs Csl4 for the interaction with the exosome. Silver-stained 12% SDS-gels showing results of experiments analyzing the interaction between DnaG and the exosome. In, input, the mixture of proteins used; FT, flow-through; W1, W5, the first and the last washing fractions; E, the elution fraction. (A) Rrp4-exosome or Csl4-exosome was mixed with DnaG and the interacting proteins were co-immunoprecipitated with Rrp41-specific antibodies. His-tagged proteins were used. (B) The Rrp41-Rrp42 hexamer (hexameric ring) alone or mixed with DnaG was subjected to co-immunoprecipitation (CoIP) with DnaG-specific antibodies. His-tagged proteins were used. The protein fractions were analyzed by SDS-PAGE and silver staining (upper panel) and by western blot hybridization with Rrp41-specific antibodies. (C) Strep-tagged Csl4 and His-tagged DnaG were mixed and a pull-down assay with Strep-Tactin Sepharose beads was performed. M, protein marker, the migration behavior of the proteins is given in kDa on the left side. An E. coli protein binding to Ni-NTA, which was routinely present in the DnaG-fractions used for the reconstitution experiment, is marked with an asterisk in (A and C). (D) His-tagged Rrp4 and His-tagged DnaG were mixed and CoIP with Rrp4-specific antibodies was performed. The detected proteins are marked on the right side. (E) Reconstituted and purified Csl4-exosome and DnaG-Csl4-exosome. (F) Reconstituted and purified Rrp4-Csl4-exosome and DnaG-Rrp4-Csl4-exosome. For the experiments shown in (E and F), Strep-Csl4 and His-tagged Rrp4, Rrp41, Rrp42 and DnaG proteins were used. Purification was performed with Strep-Tactin followed by Ni-NTA chromatography.
Mentions: To study the mechanisms of interaction between DnaG and the exosome, we reconstituted complexes with homomeric RNA-binding caps containing either Rrp4 or Csl4 and tested them for interaction with DnaG by co-immunoprecipitation with Rrp41-specific antibodies. His-tagged versions of all proteins were used for these tests. Figure 1A shows that the Rrp4-exosome does not interact with DnaG, and demonstrates a clear binding of DnaG to the Csl4-exosome.

Bottom Line: We found that the archaeal DnaG binds to the Csl4-exosome but not to the Rrp4-exosome of Sulfolobus solfataricus.DnaG is the second poly(A)-binding protein besides Rrp4 in the heteromeric, RNA-binding cap of the S. solfataricus exosome.This apparently reflects the need for effective and selective recruitment of adenine-rich RNAs to the exosome in the RNA metabolism of S. solfataricus.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Molecular Biology; Heinrich-Buff-Ring; Giessen, Germany.

ABSTRACT
The archaeal RNA-degrading exosome contains a catalytically active hexameric core, an RNA-binding cap formed by Rrp4 and Csl4 and the protein annotated as DnaG (bacterial type primase) with so-far-unknown functions in RNA metabolism. We found that the archaeal DnaG binds to the Csl4-exosome but not to the Rrp4-exosome of Sulfolobus solfataricus. In vitro assays revealed that DnaG is a poly(A)-binding protein enhancing the degradation of adenine-rich transcripts by the Csl4-exosome. DnaG is the second poly(A)-binding protein besides Rrp4 in the heteromeric, RNA-binding cap of the S. solfataricus exosome. This apparently reflects the need for effective and selective recruitment of adenine-rich RNAs to the exosome in the RNA metabolism of S. solfataricus.

Show MeSH