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

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.

Keywords: DnaG; RNA binding; archaea; exosome; poly(A) binding.

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.

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. 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.

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.

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-RNA_19hetero). The two substrates were present together in reaction mixtures, in which one of the substrates was internally labeled and the other was unlabeled. (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.

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.