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 5. DnaG enhances the interaction between poly(A)-RNA and the Rrp4-Csl4-exosome. (A) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing 25 fmol radioactively labeled poly(A) 30-mer and 45 pmol unlabeled A-rich transcript (59 nt). 0.3 pmol of DnaG, the Rrp4-Csl4-exosome or the DnaG-Rrp4-Csl4-exosome were present in each reaction mixture as indicated above the panel. The Strep-Csl4-containing exosomes shown in Figure 1F were used. The incubation time (in min) at 60°C is indicated. The 30-meric poly(A) substrate and the accumulating degradation product of 25 nt (see ref. 21) are marked on the right side. Control, negative control without protein. (B) Graphical representation of the results shown in (A) and from two additional independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Figure 5. DnaG enhances the interaction between poly(A)-RNA and the Rrp4-Csl4-exosome. (A) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing 25 fmol radioactively labeled poly(A) 30-mer and 45 pmol unlabeled A-rich transcript (59 nt). 0.3 pmol of DnaG, the Rrp4-Csl4-exosome or the DnaG-Rrp4-Csl4-exosome were present in each reaction mixture as indicated above the panel. The Strep-Csl4-containing exosomes shown in Figure 1F were used. The incubation time (in min) at 60°C is indicated. The 30-meric poly(A) substrate and the accumulating degradation product of 25 nt (see ref. 21) are marked on the right side. Control, negative control without protein. (B) Graphical representation of the results shown in (A) and from two additional independent experiments.

Mentions: In vivo Csl4 and Rrp4 are forming together the RNA-binding cap of the soluble exosome.16 Thus, the question arose whether DnaG can influence the interaction of the Rrp4-Csl4-exosome with poly(A)-RNA, since this exosome already contains the poly(A)-binding protein Rrp4.21 This was tested in degradation assays with the Rrp4-Csl4-exosome and the DnaG-Rrp4-Csl4-exosome (Fig. 5). The used protein complexes are shown in Figure 1F. A substrate mixture of 25 fmol labeled, 30-meric poly(A)-RNA and 45 pmol unlabeled, adenine-rich transcript of 59 nt was used. Figure 5A shows that the poly(A)-RNA was converted into a 25 nt degradation intermediate routinely observed in degradation assays with the Rrp4-exosome (compare with Fig. 2C). This intermediate accumulates when longer substrates are present in the reaction mixture.21 The degradation of the 30-meric poly(A)-RNA was faster in the presence of DnaG (Fig. 5A and B). We conclude that DnaG enhances the efficiency of interaction between poly(A)-RNA and the exosome containing Rrp4 and Csl4.


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 5. DnaG enhances the interaction between poly(A)-RNA and the Rrp4-Csl4-exosome. (A) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing 25 fmol radioactively labeled poly(A) 30-mer and 45 pmol unlabeled A-rich transcript (59 nt). 0.3 pmol of DnaG, the Rrp4-Csl4-exosome or the DnaG-Rrp4-Csl4-exosome were present in each reaction mixture as indicated above the panel. The Strep-Csl4-containing exosomes shown in Figure 1F were used. The incubation time (in min) at 60°C is indicated. The 30-meric poly(A) substrate and the accumulating degradation product of 25 nt (see ref. 21) are marked on the right side. Control, negative control without protein. (B) Graphical representation of the results shown in (A) and from two additional independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Figure 5. DnaG enhances the interaction between poly(A)-RNA and the Rrp4-Csl4-exosome. (A) A phosphorimage of a 16% polyacrylamide gel with degradation assays containing 25 fmol radioactively labeled poly(A) 30-mer and 45 pmol unlabeled A-rich transcript (59 nt). 0.3 pmol of DnaG, the Rrp4-Csl4-exosome or the DnaG-Rrp4-Csl4-exosome were present in each reaction mixture as indicated above the panel. The Strep-Csl4-containing exosomes shown in Figure 1F were used. The incubation time (in min) at 60°C is indicated. The 30-meric poly(A) substrate and the accumulating degradation product of 25 nt (see ref. 21) are marked on the right side. Control, negative control without protein. (B) Graphical representation of the results shown in (A) and from two additional independent experiments.
Mentions: In vivo Csl4 and Rrp4 are forming together the RNA-binding cap of the soluble exosome.16 Thus, the question arose whether DnaG can influence the interaction of the Rrp4-Csl4-exosome with poly(A)-RNA, since this exosome already contains the poly(A)-binding protein Rrp4.21 This was tested in degradation assays with the Rrp4-Csl4-exosome and the DnaG-Rrp4-Csl4-exosome (Fig. 5). The used protein complexes are shown in Figure 1F. A substrate mixture of 25 fmol labeled, 30-meric poly(A)-RNA and 45 pmol unlabeled, adenine-rich transcript of 59 nt was used. Figure 5A shows that the poly(A)-RNA was converted into a 25 nt degradation intermediate routinely observed in degradation assays with the Rrp4-exosome (compare with Fig. 2C). This intermediate accumulates when longer substrates are present in the reaction mixture.21 The degradation of the 30-meric poly(A)-RNA was faster in the presence of DnaG (Fig. 5A and B). We conclude that DnaG enhances the efficiency of interaction between poly(A)-RNA and the exosome containing Rrp4 and Csl4.

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