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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 2. DnaG influences the degradation properties of the Csl4-exosome but not of the Rrp4-exosome, and confers poly(A) specificity to the Csl4-exosome. (A) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing 8 pmol radioactively labeled poly(A) 30-mer and 0.3 pmol of the Csl4-exosome or the Csl4-exosome supplemented with DnaG. The time of incubation at 60°C is also indicated (in min). The poly(A) substrate and the degradation products (degr. products) are marked on the right side. The size of the degradation products was previously estimated.26 Control, negative control without protein. (B) Graphical representation of the results shown in (A) and from two additional independent experiments. (C) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing 8 pmol radioactively labeled poly(A) 30-mer and 0.3 pmol of the Rrp4-exosome or the Rrp4-exosome supplemented with DnaG. For further descriptions, see (A). (D) Graphical representation of the results shown in (C) and from an additional independent experiment. (E‒G) Graphs showing the relative amount of the remaining substrate (in %) against the time (in min) in degradation assays (data from three independent experiments). In each reaction, 0.3 pmol protein complex was used. The protein complexes, labeled RNAs and non-labeled competitors, and their amounts per reaction mixture are indicated. (H) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing the proteins indicated above the panel. 0.3 pmol of either Rrp41, Rrp42, Rrp4, Csl4, DnaG, the hexameric ring or the DnaG-Csl4-exosome were incubated for 10 min at 60°C with 8 fmol (lanes 1‒7) or 1 pmol (lanes 8‒12) radioactively labeled poly(A) 30-mer. Control, negative control without protein. Only His-tagged proteins were used for the experiments in this figure.
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Figure 2: Figure 2. DnaG influences the degradation properties of the Csl4-exosome but not of the Rrp4-exosome, and confers poly(A) specificity to the Csl4-exosome. (A) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing 8 pmol radioactively labeled poly(A) 30-mer and 0.3 pmol of the Csl4-exosome or the Csl4-exosome supplemented with DnaG. The time of incubation at 60°C is also indicated (in min). The poly(A) substrate and the degradation products (degr. products) are marked on the right side. The size of the degradation products was previously estimated.26 Control, negative control without protein. (B) Graphical representation of the results shown in (A) and from two additional independent experiments. (C) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing 8 pmol radioactively labeled poly(A) 30-mer and 0.3 pmol of the Rrp4-exosome or the Rrp4-exosome supplemented with DnaG. For further descriptions, see (A). (D) Graphical representation of the results shown in (C) and from an additional independent experiment. (E‒G) Graphs showing the relative amount of the remaining substrate (in %) against the time (in min) in degradation assays (data from three independent experiments). In each reaction, 0.3 pmol protein complex was used. The protein complexes, labeled RNAs and non-labeled competitors, and their amounts per reaction mixture are indicated. (H) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing the proteins indicated above the panel. 0.3 pmol of either Rrp41, Rrp42, Rrp4, Csl4, DnaG, the hexameric ring or the DnaG-Csl4-exosome were incubated for 10 min at 60°C with 8 fmol (lanes 1‒7) or 1 pmol (lanes 8‒12) radioactively labeled poly(A) 30-mer. Control, negative control without protein. Only His-tagged proteins were used for the experiments in this figure.

Mentions: To study the function of DnaG in RNA metabolism, we compared the RNA-degrading properties of the Csl4-exosome in the presence and in the absence of DnaG using a 30-meric poly(A)-RNA as substrate. The reaction mixtures were incubated at 60°C in the presence of inorganic phosphate (Pi) and resolved on 16% polyacrylamide gels (Fig. 2A). The exosome degraded the substrate to final products with a length of 4–5 nt.26 Intermediates with lengths between 15 and 6 nt were most probably products of a distributive degradation in contrast to the processive degradation of longer molecules.20,26 The percentage of the remaining 30-meric poly(A) RNA per lane was determined. The data show that DnaG stimulates the degradation of the substrate by the Csl4-exosome (Fig. 2B). As a negative control, the experiments were performed with the Rrp4-exosome, which does not interact with DnaG. In agreement with previous results, a 25 nt intermediate degradation product was detected in the assays with the Rrp4-exosome (Fig. 2C).21 Quantification of the remaining 30-meric poly(A)-RNA revealed that DnaG did not influence its degradation by the Rrp4-exosome (Fig. 2D). Thus, the positive effect of DnaG on the degradation of the 30-meric poly(A)-RNA by the Csl4-exosome is specific.


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 2. DnaG influences the degradation properties of the Csl4-exosome but not of the Rrp4-exosome, and confers poly(A) specificity to the Csl4-exosome. (A) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing 8 pmol radioactively labeled poly(A) 30-mer and 0.3 pmol of the Csl4-exosome or the Csl4-exosome supplemented with DnaG. The time of incubation at 60°C is also indicated (in min). The poly(A) substrate and the degradation products (degr. products) are marked on the right side. The size of the degradation products was previously estimated.26 Control, negative control without protein. (B) Graphical representation of the results shown in (A) and from two additional independent experiments. (C) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing 8 pmol radioactively labeled poly(A) 30-mer and 0.3 pmol of the Rrp4-exosome or the Rrp4-exosome supplemented with DnaG. For further descriptions, see (A). (D) Graphical representation of the results shown in (C) and from an additional independent experiment. (E‒G) Graphs showing the relative amount of the remaining substrate (in %) against the time (in min) in degradation assays (data from three independent experiments). In each reaction, 0.3 pmol protein complex was used. The protein complexes, labeled RNAs and non-labeled competitors, and their amounts per reaction mixture are indicated. (H) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing the proteins indicated above the panel. 0.3 pmol of either Rrp41, Rrp42, Rrp4, Csl4, DnaG, the hexameric ring or the DnaG-Csl4-exosome were incubated for 10 min at 60°C with 8 fmol (lanes 1‒7) or 1 pmol (lanes 8‒12) radioactively labeled poly(A) 30-mer. Control, negative control without protein. Only His-tagged proteins were used for the experiments in this figure.
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Figure 2: Figure 2. DnaG influences the degradation properties of the Csl4-exosome but not of the Rrp4-exosome, and confers poly(A) specificity to the Csl4-exosome. (A) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing 8 pmol radioactively labeled poly(A) 30-mer and 0.3 pmol of the Csl4-exosome or the Csl4-exosome supplemented with DnaG. The time of incubation at 60°C is also indicated (in min). The poly(A) substrate and the degradation products (degr. products) are marked on the right side. The size of the degradation products was previously estimated.26 Control, negative control without protein. (B) Graphical representation of the results shown in (A) and from two additional independent experiments. (C) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing 8 pmol radioactively labeled poly(A) 30-mer and 0.3 pmol of the Rrp4-exosome or the Rrp4-exosome supplemented with DnaG. For further descriptions, see (A). (D) Graphical representation of the results shown in (C) and from an additional independent experiment. (E‒G) Graphs showing the relative amount of the remaining substrate (in %) against the time (in min) in degradation assays (data from three independent experiments). In each reaction, 0.3 pmol protein complex was used. The protein complexes, labeled RNAs and non-labeled competitors, and their amounts per reaction mixture are indicated. (H) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing the proteins indicated above the panel. 0.3 pmol of either Rrp41, Rrp42, Rrp4, Csl4, DnaG, the hexameric ring or the DnaG-Csl4-exosome were incubated for 10 min at 60°C with 8 fmol (lanes 1‒7) or 1 pmol (lanes 8‒12) radioactively labeled poly(A) 30-mer. Control, negative control without protein. Only His-tagged proteins were used for the experiments in this figure.
Mentions: To study the function of DnaG in RNA metabolism, we compared the RNA-degrading properties of the Csl4-exosome in the presence and in the absence of DnaG using a 30-meric poly(A)-RNA as substrate. The reaction mixtures were incubated at 60°C in the presence of inorganic phosphate (Pi) and resolved on 16% polyacrylamide gels (Fig. 2A). The exosome degraded the substrate to final products with a length of 4–5 nt.26 Intermediates with lengths between 15 and 6 nt were most probably products of a distributive degradation in contrast to the processive degradation of longer molecules.20,26 The percentage of the remaining 30-meric poly(A) RNA per lane was determined. The data show that DnaG stimulates the degradation of the substrate by the Csl4-exosome (Fig. 2B). As a negative control, the experiments were performed with the Rrp4-exosome, which does not interact with DnaG. In agreement with previous results, a 25 nt intermediate degradation product was detected in the assays with the Rrp4-exosome (Fig. 2C).21 Quantification of the remaining 30-meric poly(A)-RNA revealed that DnaG did not influence its degradation by the Rrp4-exosome (Fig. 2D). Thus, the positive effect of DnaG on the degradation of the 30-meric poly(A)-RNA by the Csl4-exosome is specific.

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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