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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 4. DnaG enhances the interaction between heteropolymeric, adenine-rich transcripts and the Csl4-exosome. The relative amount of the remaining labeled substrate (in %) is plotted over time (in min). (A) Degradation assays with 0.3 pmol of the DnaG-Csl4-exosome, 0.2 pmol MCS-RNA (97 nt) and 0.2 pmol MCS-RNA carrying a heteropolymeric, adenine-rich tail of 19 nt (MCS-RNA19hetero). The two substrates were present together in reaction mixtures, in which one of the substrates was internally labeled and the other was unlabeled.21 (B) Assays with 0.3 pmol of the Csl4-exosome and the substrates described in (A). (C) Assays with the DnaG-Csl4-exosome (0.3 pmol) and the Csl4-exosome (0.3 pmol) using 0.5 pmol labeled A-rich transcript (59 nt). (D) Assays with the DnaG-Csl4-exosome (0.3 pmol) and the Csl4-exosome (0.3 pmol) using 0.2 pmol labeled A-rich transcript (59 nt) and 0.2 pmol unlabeled MCS-RNA (97 nt). Graphs represent results from three independent experiments in (A and B), and two independent experiments in (C and D). His-tagged proteins were used for the experiments in this figure.
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Figure 4: Figure 4. DnaG enhances the interaction between heteropolymeric, adenine-rich transcripts and the Csl4-exosome. The relative amount of the remaining labeled substrate (in %) is plotted over time (in min). (A) Degradation assays with 0.3 pmol of the DnaG-Csl4-exosome, 0.2 pmol MCS-RNA (97 nt) and 0.2 pmol MCS-RNA carrying a heteropolymeric, adenine-rich tail of 19 nt (MCS-RNA19hetero). The two substrates were present together in reaction mixtures, in which one of the substrates was internally labeled and the other was unlabeled.21 (B) Assays with 0.3 pmol of the Csl4-exosome and the substrates described in (A). (C) Assays with the DnaG-Csl4-exosome (0.3 pmol) and the Csl4-exosome (0.3 pmol) using 0.5 pmol labeled A-rich transcript (59 nt). (D) Assays with the DnaG-Csl4-exosome (0.3 pmol) and the Csl4-exosome (0.3 pmol) using 0.2 pmol labeled A-rich transcript (59 nt) and 0.2 pmol unlabeled MCS-RNA (97 nt). Graphs represent results from three independent experiments in (A and B), and two independent experiments in (C and D). His-tagged proteins were used for the experiments in this figure.

Mentions: Since S. solfataricus does not harbor homopolymeric poly(A)-tails, we assumed that the function of DnaG in the RNA-binding cap of the S. solfataricus exosome is to ensure an efficient interaction with adenine-rich substrates.22 To test this, we compared the degradation of MCS-RNA with the degradation of its tailed variant MCS-RNA19hetero by the Csl4-exosome and by the DnaG-Csl4-exosome. The latter substrate contains a heteromeric, adenine-rich tail of 19 nt (47% A-content of the tail), which was originally detected at the 3′-end of a 16S rRNA fragment in S. solfataricus.21,22 We found that the DnaG-Csl4-exosome degrades MCS-RNA19hetero with higher efficiency than MCS-RNA (Fig. 4A). Conversely, the Csl4-exosome even tended to prefer the MCS-RNA as substrate (Fig. 4B). In previous assays, the Csl4-exosome did not show clear preference for one of these substrates.21 Thus, the DnaG-Csl4-exosome can better degrade the substrate with the adenine-rich tail than the non-tailed substrate, similarly to what was previously shown for the Rrp4-exosome.21


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 4. DnaG enhances the interaction between heteropolymeric, adenine-rich transcripts and the Csl4-exosome. The relative amount of the remaining labeled substrate (in %) is plotted over time (in min). (A) Degradation assays with 0.3 pmol of the DnaG-Csl4-exosome, 0.2 pmol MCS-RNA (97 nt) and 0.2 pmol MCS-RNA carrying a heteropolymeric, adenine-rich tail of 19 nt (MCS-RNA19hetero). The two substrates were present together in reaction mixtures, in which one of the substrates was internally labeled and the other was unlabeled.21 (B) Assays with 0.3 pmol of the Csl4-exosome and the substrates described in (A). (C) Assays with the DnaG-Csl4-exosome (0.3 pmol) and the Csl4-exosome (0.3 pmol) using 0.5 pmol labeled A-rich transcript (59 nt). (D) Assays with the DnaG-Csl4-exosome (0.3 pmol) and the Csl4-exosome (0.3 pmol) using 0.2 pmol labeled A-rich transcript (59 nt) and 0.2 pmol unlabeled MCS-RNA (97 nt). Graphs represent results from three independent experiments in (A and B), and two independent experiments in (C and D). His-tagged proteins were used for the experiments in this figure.
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Related In: Results  -  Collection

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Figure 4: Figure 4. DnaG enhances the interaction between heteropolymeric, adenine-rich transcripts and the Csl4-exosome. The relative amount of the remaining labeled substrate (in %) is plotted over time (in min). (A) Degradation assays with 0.3 pmol of the DnaG-Csl4-exosome, 0.2 pmol MCS-RNA (97 nt) and 0.2 pmol MCS-RNA carrying a heteropolymeric, adenine-rich tail of 19 nt (MCS-RNA19hetero). The two substrates were present together in reaction mixtures, in which one of the substrates was internally labeled and the other was unlabeled.21 (B) Assays with 0.3 pmol of the Csl4-exosome and the substrates described in (A). (C) Assays with the DnaG-Csl4-exosome (0.3 pmol) and the Csl4-exosome (0.3 pmol) using 0.5 pmol labeled A-rich transcript (59 nt). (D) Assays with the DnaG-Csl4-exosome (0.3 pmol) and the Csl4-exosome (0.3 pmol) using 0.2 pmol labeled A-rich transcript (59 nt) and 0.2 pmol unlabeled MCS-RNA (97 nt). Graphs represent results from three independent experiments in (A and B), and two independent experiments in (C and D). His-tagged proteins were used for the experiments in this figure.
Mentions: Since S. solfataricus does not harbor homopolymeric poly(A)-tails, we assumed that the function of DnaG in the RNA-binding cap of the S. solfataricus exosome is to ensure an efficient interaction with adenine-rich substrates.22 To test this, we compared the degradation of MCS-RNA with the degradation of its tailed variant MCS-RNA19hetero by the Csl4-exosome and by the DnaG-Csl4-exosome. The latter substrate contains a heteromeric, adenine-rich tail of 19 nt (47% A-content of the tail), which was originally detected at the 3′-end of a 16S rRNA fragment in S. solfataricus.21,22 We found that the DnaG-Csl4-exosome degrades MCS-RNA19hetero with higher efficiency than MCS-RNA (Fig. 4A). Conversely, the Csl4-exosome even tended to prefer the MCS-RNA as substrate (Fig. 4B). In previous assays, the Csl4-exosome did not show clear preference for one of these substrates.21 Thus, the DnaG-Csl4-exosome can better degrade the substrate with the adenine-rich tail than the non-tailed substrate, similarly to what was previously shown for the Rrp4-exosome.21

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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