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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.

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Figure 3. Poly(A)-RNA binding by DnaG, the Csl4-exosome and the DnaG-Csl4-exosome. Shown are phosphorimages of EMSA assays in native 5% polyacrylamide gels. Twenty-five fmol labeled poly(A)-RNA 30-mer was used in each reaction. Only His-tagged proteins were used for the experiments in (A and B). Strep-tagged Csl4 was used for the reconstitution of the complexes (see Fig. 1E) used in (C and D). (A) Binding assays with the Csl4-exosome or with DnaG in different amounts as indicated above the panel. His-tagged proteins were used. (B) Binding of DnaG (amount indicated above each lane) to radiolabeled poly(A) 30-mer in the absence or presence of unlabeled competitor RNAs. Lanes 1, 2 and 11, no competitor was used. Lanes 3‒10, competitors in the following amounts were used: Lanes 3 and 4, 11 pmol MCS-RNA; lanes 5 and 6; 22 pmol MCS-RNA; lanes 7 and 8, 11 pmol poly(A); lanes 9 and 10, 22 pmol poly(A). (C) Binding assays with 2.5 pmol of DnaG, the Csl4-exosome or the DnaG-Csl4-exosome, as indicated above the panel. The amounts of the unlabeled MCS-RNA-derived 30-mer used as competitor are also indicated above the corresponding lanes. (D) Binding assays with 0.6 pmol of DnaG, the Csl4-exosome or the DnaG-Csl4-exosome, as indicated above the panel. The percentages of unbound poly(A) substrate in each lane is indicated below the panels (results from three independent experiments). The proportion of unbound substrate in the control lane was set to 100%. Control, negative control without protein and without competitor. The migration in the gel of the unbound poly(A) 30-mer and of the protein/RNA complexes (complexes) is indicated on the right side.
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Figure 3: Figure 3. Poly(A)-RNA binding by DnaG, the Csl4-exosome and the DnaG-Csl4-exosome. Shown are phosphorimages of EMSA assays in native 5% polyacrylamide gels. Twenty-five fmol labeled poly(A)-RNA 30-mer was used in each reaction. Only His-tagged proteins were used for the experiments in (A and B). Strep-tagged Csl4 was used for the reconstitution of the complexes (see Fig. 1E) used in (C and D). (A) Binding assays with the Csl4-exosome or with DnaG in different amounts as indicated above the panel. His-tagged proteins were used. (B) Binding of DnaG (amount indicated above each lane) to radiolabeled poly(A) 30-mer in the absence or presence of unlabeled competitor RNAs. Lanes 1, 2 and 11, no competitor was used. Lanes 3‒10, competitors in the following amounts were used: Lanes 3 and 4, 11 pmol MCS-RNA; lanes 5 and 6; 22 pmol MCS-RNA; lanes 7 and 8, 11 pmol poly(A); lanes 9 and 10, 22 pmol poly(A). (C) Binding assays with 2.5 pmol of DnaG, the Csl4-exosome or the DnaG-Csl4-exosome, as indicated above the panel. The amounts of the unlabeled MCS-RNA-derived 30-mer used as competitor are also indicated above the corresponding lanes. (D) Binding assays with 0.6 pmol of DnaG, the Csl4-exosome or the DnaG-Csl4-exosome, as indicated above the panel. The percentages of unbound poly(A) substrate in each lane is indicated below the panels (results from three independent experiments). The proportion of unbound substrate in the control lane was set to 100%. Control, negative control without protein and without competitor. The migration in the gel of the unbound poly(A) 30-mer and of the protein/RNA complexes (complexes) is indicated on the right side.

Mentions: The results shown in Figure 2 raised the question whether DnaG is a poly(A)-binding subunit of the S. solfataricus exosome. This question was addressed by electrophoretic mobility shift assays (EMSA) aiming to compare the poly(A)-binding properties of the Csl4-exosome and of DnaG. Figure 3A shows that the 30-meric poly(A)-RNA (25 fmol per reaction) was bound much stronger by DnaG than by the Csl4-exosome when comparable molar amounts of the proteins were used. For example, the substrate was shifted as a stable band by 1.2 pmol DnaG, while no shift was observed with the same amount of Csl4-exosome (compare lanes 2 and 9 in Fig. 3A). To analyze the poly(A)-specificity of DnaG, competition experiments were performed (Fig. 3B). Addition of 11 pmol or 22 pmol of unlabeled, 30-meric MCS RNA did not abolish the binding of 25 fmol labeled poly(A)-RNA by 2.5 pmol of DnaG (compare lane 1 to lanes 3 and 5). In contrast, when unlabeled poly(A)-RNA was added to the reaction mixtures, the labeled poly(A)-RNA was detected only in its unbound form (lanes 7 and 9). These results show that DnaG preferentially binds poly(A)-RNA.


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 3. Poly(A)-RNA binding by DnaG, the Csl4-exosome and the DnaG-Csl4-exosome. Shown are phosphorimages of EMSA assays in native 5% polyacrylamide gels. Twenty-five fmol labeled poly(A)-RNA 30-mer was used in each reaction. Only His-tagged proteins were used for the experiments in (A and B). Strep-tagged Csl4 was used for the reconstitution of the complexes (see Fig. 1E) used in (C and D). (A) Binding assays with the Csl4-exosome or with DnaG in different amounts as indicated above the panel. His-tagged proteins were used. (B) Binding of DnaG (amount indicated above each lane) to radiolabeled poly(A) 30-mer in the absence or presence of unlabeled competitor RNAs. Lanes 1, 2 and 11, no competitor was used. Lanes 3‒10, competitors in the following amounts were used: Lanes 3 and 4, 11 pmol MCS-RNA; lanes 5 and 6; 22 pmol MCS-RNA; lanes 7 and 8, 11 pmol poly(A); lanes 9 and 10, 22 pmol poly(A). (C) Binding assays with 2.5 pmol of DnaG, the Csl4-exosome or the DnaG-Csl4-exosome, as indicated above the panel. The amounts of the unlabeled MCS-RNA-derived 30-mer used as competitor are also indicated above the corresponding lanes. (D) Binding assays with 0.6 pmol of DnaG, the Csl4-exosome or the DnaG-Csl4-exosome, as indicated above the panel. The percentages of unbound poly(A) substrate in each lane is indicated below the panels (results from three independent experiments). The proportion of unbound substrate in the control lane was set to 100%. Control, negative control without protein and without competitor. The migration in the gel of the unbound poly(A) 30-mer and of the protein/RNA complexes (complexes) is indicated on the right side.
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Figure 3: Figure 3. Poly(A)-RNA binding by DnaG, the Csl4-exosome and the DnaG-Csl4-exosome. Shown are phosphorimages of EMSA assays in native 5% polyacrylamide gels. Twenty-five fmol labeled poly(A)-RNA 30-mer was used in each reaction. Only His-tagged proteins were used for the experiments in (A and B). Strep-tagged Csl4 was used for the reconstitution of the complexes (see Fig. 1E) used in (C and D). (A) Binding assays with the Csl4-exosome or with DnaG in different amounts as indicated above the panel. His-tagged proteins were used. (B) Binding of DnaG (amount indicated above each lane) to radiolabeled poly(A) 30-mer in the absence or presence of unlabeled competitor RNAs. Lanes 1, 2 and 11, no competitor was used. Lanes 3‒10, competitors in the following amounts were used: Lanes 3 and 4, 11 pmol MCS-RNA; lanes 5 and 6; 22 pmol MCS-RNA; lanes 7 and 8, 11 pmol poly(A); lanes 9 and 10, 22 pmol poly(A). (C) Binding assays with 2.5 pmol of DnaG, the Csl4-exosome or the DnaG-Csl4-exosome, as indicated above the panel. The amounts of the unlabeled MCS-RNA-derived 30-mer used as competitor are also indicated above the corresponding lanes. (D) Binding assays with 0.6 pmol of DnaG, the Csl4-exosome or the DnaG-Csl4-exosome, as indicated above the panel. The percentages of unbound poly(A) substrate in each lane is indicated below the panels (results from three independent experiments). The proportion of unbound substrate in the control lane was set to 100%. Control, negative control without protein and without competitor. The migration in the gel of the unbound poly(A) 30-mer and of the protein/RNA complexes (complexes) is indicated on the right side.
Mentions: The results shown in Figure 2 raised the question whether DnaG is a poly(A)-binding subunit of the S. solfataricus exosome. This question was addressed by electrophoretic mobility shift assays (EMSA) aiming to compare the poly(A)-binding properties of the Csl4-exosome and of DnaG. Figure 3A shows that the 30-meric poly(A)-RNA (25 fmol per reaction) was bound much stronger by DnaG than by the Csl4-exosome when comparable molar amounts of the proteins were used. For example, the substrate was shifted as a stable band by 1.2 pmol DnaG, while no shift was observed with the same amount of Csl4-exosome (compare lanes 2 and 9 in Fig. 3A). To analyze the poly(A)-specificity of DnaG, competition experiments were performed (Fig. 3B). Addition of 11 pmol or 22 pmol of unlabeled, 30-meric MCS RNA did not abolish the binding of 25 fmol labeled poly(A)-RNA by 2.5 pmol of DnaG (compare lane 1 to lanes 3 and 5). In contrast, when unlabeled poly(A)-RNA was added to the reaction mixtures, the labeled poly(A)-RNA was detected only in its unbound form (lanes 7 and 9). These results show that DnaG preferentially binds poly(A)-RNA.

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
Related in: MedlinePlus