The RNA exosome complex central channel controls both exonuclease and endonuclease Dis3 activities in vivo and in vitro

Abstract

The RNA exosome is an essential ribonuclease complex involved in RNA processing and decay. It consists of a 9-subunit catalytically inert ring composed of six RNase PH-like proteins forming a central channel and three cap subunits with KH/S1 domains located at the top. The yeast exosome catalytic activity is supplied by the Dis3 (also known as Rrp44) protein, which has both endo- and exoribonucleolytic activities and the nucleus-specific exonuclease Rrp6. In vitro studies suggest that substrates reach the Dis3 exonucleolytic active site following passage through the ring channel, but in vivo support is lacking. Here, we constructed an Rrp41 ring subunit mutant with a partially blocked channel that led to thermosensitivity and synthetic lethality with Rrp6 deletion. Rrp41 mutation caused accumulation of nuclear and cytoplasmic exosome substrates including the non-stop decay reporter, for which degradation is dependent on either endonucleolytic or exonucleolytic Dis3 activities. This suggests that the central channel also controls endonucleolytic activity. In vitro experiments performed using Chaetomium thermophilum exosomes reconstituted from recombinant subunits confirmed this notion. Finally, we analysed the impact of a lethal mutation of conserved basic residues in Rrp4 cap subunit and found that it inhibits digestion of single-stranded and structured RNA substrates.

Figure 1.

The Rrp41^4M mutation blocking the channel inhibits growth at elevated temperatures. (A) Architecture of the exosome complex. The position of mutated sites is indicated (mutated residues in Rrp41 responsible for the interaction with RNA shown in red, mutated residues in Rrp4 presumably responsible for the interaction with RNA shown in yellow). (B) Serial dilutions of strains: rrp41^4M, dis3^endo−, rrp41^4M dis3^endo−, dis3^exo−, rrp41^4M dis3^exo− and an isogenic wild-type (WT) control strain were placed on YPDA plates and incubated at 25, 30 and 37°C for 3 days.

Figure 2.

The Rrp41^4M mutation causes accumulation of nuclear exosome substrates. RNA from WT, rrp41^4M, dis3^endo−, rrp41^4M dis3^endo−, dis3^exo− and rrp41^4M dis3^exo− strains grown to early exponential phase was isolated and subjected to northern blot hybridizations for the following RNAs: 7S rRNA, 5.8S rRNA, 5′-ETS and CUT NEL025. The 5S rRNA was used as a loading control.

Figure 3.

The Rrp41^4M mutation strongly inhibits cytoplasmic mRNA decay and surveillance. Dcp^1–2 and dcp^1–2 rrp41^4M strains were transformed with MFA2 reporter for normal mRNA decay of short living transcript (A) WT PGK1 reporter for normal mRNA decay of long living transcript (B) PTC reporter for analysis of NMD (C) PGK1 with a stem loop structure for analysis of NGD (D) PGK1 lacking a stop codon for analysis of NGD (E) mRNA stability in analysed strains was examined by growing cells at 25°C in galactose medium followed by a temperature shift to 37°C for 1 h and transcription termination by addition of glucose to the culture medium. RNA was isolated at the times indicated followed by northern blot analysis using a polyC probe complementary to the polyG sequence present in all reporters (upper panels). Quantifications from three independent experiments are plotted as mRNA at each time after normalization using an SCR1 probe, and (F) Steady state levels of nonstop reporter in the following strains: WT, rrp41^4M, dis3^endo−, rrp41^4M dis3^endo−, dis3^exo− and rrp41^4M dis3^exo−.

Figure 4.

Exoribonucleolytic activity of Dis3 is strongly inhibited by mutations in the Rrp41 core subunit that blocks the central channel of the exosome. (A) 5′-labelled RNA substrates with a 17 nt generic sequence followed by a 34 adenosine tail [ss17-(A)₃₄] were incubated in buffer containing 100 μM magnesium with various in vitro reconstituted C. thermophilum 10-subunit exosome complexes, containing either wild-type Rrp41 or Rrp41^4M protein as a component of the central channel (designated channel^WT or channel^4M, respectively), in conjunction with different versions of the full-length Dis3 catalytic subunit (Dis3^WT, Dis3^exo−, Dis3^endo−, Dis3^{exo−/endo−}); parallel assays were done using individual Dis3^WT or Dis3^exo− and no added protein. Samples were collected at the indicated time points (minutes) and analysed by denaturing PAGE and phosphorimaging. Equal molar concentrations (0.05 μM) of exosome complexes or individual subunits were used for the assay. (B) 5′-labelled RNA substrate forming partial duplex ds17-(A)₃₄ (one strand consisting of 17 nucleotides generic sequence followed by a 34 adenosine-long tail and another oligo complementary to a generic sequence) was incubated in a buffer containing 100 μM magnesium with various in vitro reconstituted C. thermophilum 10-subunit exosome complexes and analysed further as in (A).

Figure 5.

Rrp41^4M mutation, which blocks the central exosome channel, significantly decreases endoribonucleolytic activity of Dis3 PIN domain. The 5′-labelled ss17-(A)₃₄ substrate was incubated in a buffer containing 3 mM manganese with various in vitro reconstituted C. thermophilum exosome complexes, encompassing either channel^WT or channel^4M, in conjunction with different versions of PIN domain (PIN^WT, PIN^endo−) or full-length Dis3 (Dis3^exo−, Dis3^{exo−/endo−}); parallel assays were done using individual PIN^WT, PIN^endo−, Dis3^exo− and Dis3^{exo−/endo−} proteins. Assays were performed and analysed as described in the Figure 4A legend.

Figure 6.

Rrp4^3M mutation leads to accumulation of typical nuclear exosome substrates, but does not affect assembly of the complex. (A) Northern blot analysis of 7S rRNA, 5.8S rRNA, 5′-ETS and CUT in RRP4 tet-off strains transformed with leucine-marked plasmids containing WT, the 3M version of RRP4 or empty plasmid as a control grown 20 h in medium without leucine and containing doxycyline. The 5S rRNA was used as a loading control. (B) The Rrp4^3M mutation does not impair exosome assembly. SDS–PAGE analysis of exosomes purified from the RRP4 tet-off strain transformed with plasmid containing WT or 3M version of RRP4 with protein A sequence at the C-terminus.

Figure 7.

The Rrp4^3M mutation inhibits degradation of various RNA substrates by both native (A and B) and reconstructed (C and D) exosomes. (A) Native exosome containing Rrp4^3M protein degrades single-stranded RNA substrate 2–4-fold less efficiently comparing with the wild-type exosome. The 5′-labelled ss17-(A)₁₄ substrate was incubated in buffer containing 100 μM magnesium with native yeast 10-subunit exosome complexes containing either wild-type Rrp4 or Rrp4^3M (and Dis3^WT). Samples were collected at the indicated time points (minutes) and analysed by denaturing PAGE and phosphorimaging. An equal concentration of WT or mutated exosomes and substrate was used for the assay. (B) Rrp4^3M mutation in the native yeast exosome decreases its activity towards structured RNA substrate. ds17-(A)₃₄ structured substrate substrate was incubated in buffer containing 100 μM magnesium with native yeast 10-subunit exosome complexes containing either wild-type Rrp4 or Rrp4^3M (and Dis3^WT). Samples were collected at the indicated time points (min) and analysed by denaturing PAGE and phosphorimaging. An equal concentration of WT or mutated exosomes and substrate was used for the assay. (C) The 5′-labelled ss17-(A)₃₄ substrate was incubated in buffer containing 100 μM magnesium with in vitro reconstituted C. thermophilum 10-subunit exosome complexes containing either wild-type Rrp4 or Rrp4^3M (and Dis3^WT) or in the absence of added protein. Samples were collected at the indicated time points (min) and analysed by denaturing PAGE and phosphorimaging. (D) ds17-(A)₃₄ structured substrate was incubated in buffer containing 100 μM magnesium with in vitro reconstituted C. thermophilum 10-subunit exosome complexes containing either wild-type Rrp4 or Rrp4^3M (and Dis3^WT) or in the absence of added protein. Samples were collected at the indicated time points (min) and analysed by denaturing PAGE and phosphorimaging.

Figure 8.

Analysis of Rrp4 localization and its interaction with Mtr4. (A) Rrp4^WT and Rrp^3M have the same cellular localization. RRP4 tet-off strains transformed with plasmids coding for either gfp-tagged Rrp4^WT or Rrp^3M were grown in medium with doxycycline for 20 h and localization of the fusion proteins was analysed using fluorescence microscopy. (B) The Rrp4^3M mutation does not disrupt the interaction between the exosome and Mtr4. Extracts from the RRP4 tet-off strain with gfp-tagged Mtr4 transformed with plasmids containing either WT or 3M RRP4 tagged with protein A sequence were subjected to immunoglobulin G affinity chromatography followed by western blot analysis using anti-gfp antibodies.