Helicase-Dependent RNA Decay Illuminated by a Cryo-EM Structure of a Human Nuclear RNA Exosome-MTR4 Complex

Abstract

The ribonucleolytic RNA exosome interacts with RNA helicases to degrade RNA. To understand how the 3' to 5' Mtr4 helicase engages RNA and the nuclear exosome, we reconstituted 14-subunit Mtr4-containing RNA exosomes from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human and show that they unwind structured substrates to promote degradation. We loaded a human exosome with an optimized DNA-RNA chimera that stalls MTR4 during unwinding and determined its structure to an overall resolution of 3.45 Å by cryoelectron microscopy (cryo-EM). The structure reveals an RNA-engaged helicase atop the non-catalytic core, with RNA captured within the central channel and DIS3 exoribonuclease active site. MPP6 tethers MTR4 to the exosome through contacts to the RecA domains of MTR4. EXOSC10 remains bound to the core, but its catalytic module and cofactor C1D are displaced by RNA-engaged MTR4. Competition for the exosome core may ensure that RNA is committed to degradation by DIS3 when engaged by MTR4.

Keywords: RNA degradation; RNA-protein complex; SF2; exoribonuclease; exosome; helicase; hydrolase; nuclear; ribonuclease; translocase.

Figure 1.. Mtr4 unwinding and translocation activities in RNA exosomes.

(A) Unwinding time-course assays using a 3′ A₂₀ tailed RNA duplex substrate. Marker indicates position of the fluorescein-labeled (*) displaced strand captured by a DNA trap oligo (gray line). Cartoons represent composition of complexes used. Legend applies to all panels. (B) Comparative unwinding assays for human complexes using the RNA (top) or DNA-RNA chimera (bottom) translocation strand. Substrate RNA or DNA is indicated by black and blue lines, respectively. (C and D) UV-induced crosslinking of human exosome complexes to tripartite substrates in the presence of AMPPNP or ATP. Substrates schematically represented (top). Each substrate contains a 5′ fluorescein (*) and a single 4-thiouridine (4SU) at indicated positions in the DNA-RNA chimera translocation strand. Markers shown for indicated subunits (right) with schematics below each gel indicating qualitative changes in crosslinking patterns (bottom). Gel images in panels A-D are representative of three independent experiments. See also Figure S1.

Figure 2.. Crosslinking to human MTR4-exosomes and helicase-dependent degradation.

(A) Schematic representation of tripartite substrates (A-C) used for crosslinking in panel B. Legend applies to all panels. Substrates include a DNA-RNA chimera translocation strand and two complementary strands composed of RNA (black) complementary to the DNA portion of the translocation strand, and DNA (blue) complementary to a portion of the RNA translocation strand. Each substrate includes a 5′ fluorescein and one 4SU at position −2 in the DNA-RNA chimera. (B) Crosslinking patterns for H. sapiens Exo14^{DIS3exo-endo-/EXOSC10exo-/C1D/MPP6/MTR4} using substrates in (A) in the presence of ATP. Gel image is representative of three independent experiments. Markers for DIS3 adducts with individual substrates next to each sample. (C) RNA decay assays using a substrate with RNA for the entire translocation strand. Time-course assays with indicated complexes in the presence of a non-hydrolyzable ATP analogue AMPPNP (left) or ATP (right). Gel images are representative of two independent experiments for S. cerevisiae and three independent experiments for S. pombe and human. See also Figure S1.

Figure 3.. Electron densities and overall structure.

(A) Electron densities from the overall reconstruction. (B) Composite map generated by combining each map from focused refinement. (C) Overall structure of the human nuclear exosome. Subunits labeled and uniquely colored. See also Figure S2–S6 and Table S1.

Figure 4.. Electron densities and overall path for RNA.

Electron densities (blue mesh) shown superposed on the model for (A) the DNA/RNA duplex and MTR4, (B) the overall model with electron density for RNA as an orange surface after low pass filtering the overall map to 4.0 A resolution. (C) densities for single stranded RNA within and below the MTR4 helicase, (D) the base of the Exo9 core and (E) entering the DIS3 RNB domain. Densities for the overall path in panel B were obtained by segmenting in Chimera. Electron densities for regions in panels B-E shown from maps as indicated in each panel. Protein chains are labeled and depicted as cartoon or ribbon with RNA as sticks with select residues labeled. See also Figures S4 and S7.

Figure 5.. Contacts to RNA after duplex unwinding, comparison to HEL308 and conformations of MTR4.

(A) Schematic representation of RNA and protein interactions. MTR4, EXOSC2 and EXOSC3 residues uniquely colored and labeled with polar and stacking interactions indicated by dashed or solid lines. DNA residues lacking the 2′ OH in the DNA-RNA chimera indicated with a lower case d. (B-D) Stick representation of the structure showing nucleic acid residues and proximal side chains from MTR4, EXOSC2 and EXOSC3 with side chains colored and labeled as in panel A. Electron densities shown to the right overlaying the corresponding areas with blue mesh for RNA and grey mesh for protein. (E) Ribbon diagram of MTR4 engaged in unwinding RNA in a DNA-RNA chimera (F) Ribbon diagram of HEL308 engaged in unwinding DNA (G) Reconstruction shown for one class containing the MTR4 KOW domain in an open conformation. (H) Reconstruction shown for one class containing the MTR4 KOW domain in a closed conformation. See also Figure S5.

Figure 6.. MTR4 contacts to the exosome core and comparison to yeast Rrp6-core interactions.

(A) Overall structure of the human MTR4 RNA exosome complex with a black box indicating the region of interactions with EXOSC2 shown in panels C-F. (B) Rotated view of panel A to display the EXOSC2 surface that contacts MTR4 with MTR4 removed. Amino acids that engage in direct contact to MTR4 are highlighted in red. (C-F) Contacts between MTR4 and EXOSC2 N-terminal domain (C) and S1 domain (D-F) with subunits, domains, and side chains labeled and colored as in Figure 3 in ribbon representation with side chains as sticks. Corresponding electron densities (blue mesh) are shown below (D-E) or beside (F) respective panels. (G) Overall structure of the S. cerevisiae RNA exosome complex (PDBID 5K36). (H) Rotated view of panel G to display the Rrp4 surface that contacts Rrp6 with Rrp6 removed. Amino acids that engage in direct contact to Rrp6 are highlighted in red. See also Figure S7G,H.

Figure 7.. MPP6 interacts with MTR4 and the exosome core and comparison to ZCCHC8 contacts to MTR4.

(A) Contacts between MPP6, EXOSC1 and EXOSC3. Overall structure shown to the right colored and labeled as in Figure 3 with boxes indicating three regions of interaction. Specific interactions are shown in individual panels in stick representation with subunits and amino acids labeled. Electron densities (blue mesh) covering a region that includes MPP6 Arg74 adjacent to the corresponding schematic. (B) Overall structure of MTR4 highlighting the surface that binds to the N-terminal portion of MPP6 colored and labeled as in Figure 3. (C) Orthogonal views of interactions between MPP6 and MTR4 with MTR4 depicted in cartoon with different shades of blue to indicate the labeled domains. MPP6 is shown in ribbon representation. Side chains are shown in stick representation with select amino acids labeled. (D) Stereo view of electron density (blue mesh) for MPP6 amino acids that contact MTR4. (E) Close-up of interactions between a ZCCHC8 C-terminal domain and MTR4 (PDBID: 6C90) in the same orientation for MTR4/MPP6 as in panel C. (F) The ZCCHC8 C-terminal domain binds to MTR4 through interfaces that are mutually exclusive to those used by MTR4 to interact with MPP6 and EXOSC2. See also Figure S7.