Structure and reconstitution of yeast Mpp6-nuclear exosome complexes reveals that Mpp6 stimulates RNA decay and recruits the Mtr4 helicase
- Memorial Sloan Kettering Cancer Center, United States
- Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, United States
Figures
Figure 1 with 1 supplement
Mpp6 stimulates the nuclear RNA exosome and binds RNA.
(A) Mpp6 stimulates Rrp6 activities when degrading 5’ fluorescein-labeled 49 nt polyA in 10- (top) and 11- subunit exosomes (bottom). Relative positions of RNA substrate, Rrp6 products and the Dis3 …
Figure 1—figure supplement 1
Mpp6 is a RNA-binding protein cofactor of the nuclear RNA exosome that stimulates exosome exoribonuclease activities.
(A) Mpp6 binds various sequences of single-stranded RNA. Fluorescence polarization of Mpp6 bound to 5’ fluorescein-labeled polyA 37 nt, AU-rich 36 nt, and polyU 36 nt RNA. Results of triplicate …
Figure 2 with 1 supplement
Structure of the 12-subunit Mpp6 nuclear exosome.
(A) Global view of Exo12Dis3exo-endo-/Rrp6exo-/Mpp6Min bound to a 3’−3’ RNA. Mpp6Minimal interacts with an extended surface across the S1/KH subunit, Rrp40. View from side (left) and top (right). …
Figure 2—figure supplement 1
Rrp40 is necessary, but not sufficient for Mpp6 association with the exosome.
(A) Electron densities for Mpp6. On the left, feature enhanced map (blue mesh) contoured at 1 σ covering the Mpp6 peptide. The anomalous peak (red mesh) for selenomethionine substituted Mpp6 (I94M) …
Figure 3 with 1 supplement
Conserved features of Mpp6 interaction with the S1/KH subunit, Rrp40.
(A) Top: revised domain structure of Mpp6Minimal based on the crystal structure of Exo12Dis3exo-endo-/Rrp6exo-/Mpp6Min. Bottom: sequence alignment among S. cerevisiae Mpp6Minimal and other …
Figure 3—figure supplement 1
Conserved residues in Mpp6 interact with Rrp40.
Top: Sequence of Mpp6Minimal as shown in Main Figure 3A. Bottom: Representative RNA degradation assays of 5’ fluorescein-labeled 49 nt polyA RNA and Exo10Rrp6 with Mpp6Minimal WT and arginine …
Figure 4 with 1 supplement
Mpp6 cooperates with the Rrp6 lasso to stimulate exosome activities.
(A) Close-up view to show the C-terminal residue (Phe618) of the Rrp6 EAR (teal), the Rrp43 loop (residues 321–326 below Rrp6) and N-terminal residue (Thr9) (gray), and the N-terminal residue …
Figure 4—figure supplement 1
Stimulation by Mpp6 depends on the Rrp6 lasso.
(A) Anomalous Fourier difference map derived from crystals of Exo12Dis3exo-endo-/Rrp6exo-/Mpp6Min reconstituted with selenomethionine containing Rrp46/Rrp43. Anomalous peaks corresponding to SeMet …
Figure 5 with 1 supplement
Mpp6 and Rrp47 stimulate Rrp6 activity and contribute to Mtr4 recruitment.
(A) Both Mpp6 and Rrp47 stimulate Rrp6 activity in degradation of a single-stranded 5’ fluorescein-labeled 49 nt polyA RNA in Exo11Dis3/Rrp6. Initial rates from triplicate experiments shown, with …
Figure 5—figure supplement 1
Superposition of Exo12Dis3exo-endo-/Rrp6exo-/Mpp6Minonto Exo11Dis3exo-endo-/Rrp6exo- (PDB 5K36) and Mtr4 interactions with exosomes as analyzed by gel filtration and SDS-PAGE.
(A) Overall structures with the S1/KH ring in orange (Rrp40) and wheat (Csl4 and Rrp4), the PH-like ring in grey, Dis3 in pink or red, and Rrp6 in teal or cyan. Select subunits are labeled. (B) …
Figure 6
Mtr4-dependent RNA degradation requires either Mpp6 or Rrp47.
Urea-PAGE analysis of RNA decay products by indicated exosome complexes and cofactors. In reactions labeled ‘Rrp6Exo-’, Rrp6 contains a D238N mutation to render its exonuclease site catalytically …
Figure 7 with 1 supplement
Optimal cell growth depends on unique domains in Rrp6 and Mpp6.
(A) Growth analysis of Saccharomyces cerevisiae strains carrying viable combinations of Rrp6 and Mpp6 alleles. Ten-fold dilutions series of the rrp6Δ mpp6Δ strains transformed with the indicated …
Figure 7—figure supplement 1
Selection of viable yeast strains carrying different combinations of pRS415 mpp6 and pRS413 rrp6 plasmids.
(A) After transforming the rrp6 mpp6Δ + pRS426 RRP6 strain with the indicated pRS415 mpp6 and pRS413 rrp6 plasmids, single clonal yeast cells were isolated and streaked either on SD-Ura-His-Leu …
Tables
Table 1
Crystallographic data and refinement statistics.
One crystal was used. Highest resolution shell is shown in parenthesis.
| Exo12Dis3exo-endo-/Rrp6exo-/RNA/Mpp6Min | |
|---|---|
| Data collection | |
| X-ray Source | APS GM/CA 23IDD |
| Space group | P212121 |
| Cell dimensions | |
| a, b, c (Å) | 141.1, 213.6, 225.9 |
| α, β, γ (°) | 90.0, 90.0, 90.0 |
| Wavelength (Å) | 1.0332 |
| Resolution (Å) | 44.3–3.3 (3.42–3.3) * |
| Rmerge | 0.086 (0.613) |
| I/σI | 9.1 (1.7) |
| CC1/2 | 0.997 (0.161) |
| Completeness (%) | 97.0 (95.0) |
| Redundancy | 3.4 (2.6) |
| Wilson B factor (Å2) | 99.7 |
| Refinement | |
| Resolution (Å) | 44.3–3.3 |
| No. reflections observed | 341339 |
| No. unique reflections | 100440 |
| Rwork/Rfree | 0.217/0.266 |
| No. atoms | 29498 |
| Protein | 29147 |
| RNA | 249 |
| Ligands | 38 |
| Water | 64 |
| Average B-factors | |
| Protein | 138 |
| RNA | 139 |
| Ligands | 141 |
| Water | 61 |
| R.m.s deviations | |
| Bond lengths (Å) | 0.001 |
| Bond angles (°) | 0.41 |
| Ramachandran plot | |
| % favored | 93.5 |
| % allowed | 6.5 |
| % outliers | 0 |
| Molprobity | |
| Clashscore/Percentile | 5.44/100th |
| MolProbity Score/Percentile | 1.72/100th |
