Structural insights into spliceosome fidelity: DHX35-GPATCH1- mediated rejection of aberrant splicing substrates

Abstract

The spliceosome, a highly dynamic macromolecular assembly, catalyzes the precise removal of introns from pre-mRNAs. Recent studies have provided comprehensive structural insights into the step-wise assembly, catalytic splicing and final disassembly of the spliceosome. However, the molecular details of how the spliceosome recognizes and rejects suboptimal splicing substrates remained unclear. Here, we show cryo-electron microscopy structures of spliceosomal quality control complexes from a thermophilic eukaryote, Chaetomium thermophilum. The spliceosomes, henceforth termed B*^Q, are stalled at a catalytically activated state but prior to the first splicing reaction due to an aberrant 5' splice site conformation. This state is recognized by G-patch protein GPATCH1, which is docked onto PRP8-EN and -RH domains and has recruited the cognate DHX35 helicase to its U2 snRNA substrate. In B*^Q, DHX35 has dissociated the U2/branch site helix, while the disassembly helicase DHX15 is docked close to its U6 RNA 3'-end substrate. Our work thus provides mechanistic insights into the concerted action of two spliceosomal helicases in maintaining splicing fidelity by priming spliceosomes that are bound to aberrant splice substrates for disassembly.

Fig. 1. Cryo-EM structures of the C. thermophilum ILS complexes.

a Cryo-EM structure of the ILS state of C. thermophilum observed after affinity purification of DHX15. Two different views of the molecular model of the ctILS state are presented, with average resolution expressed in Å. b A schematic of the domain architecture of DHX15. c In the ctILS state, the resolution as illustrated with the density map of the U6 snRNA allows the identification of the 3′-end of the U6 snRNA, which protrudes into the RNA binding tunnel of DHX15. The density of the 5’ m3G cap of the U2 snRNA is shown as an insert (down right). The density map is derived from a focused refinement map. d Summary of all modeled proteins from the ctILS structure and RNAs with color code.

Fig. 2. Cryo-EM structures of the C. thermophilum B*^Q complexes.

a, b Two different views of the molecular models (left) and cryo-EM density maps (right) of the C. thermophilum spliceosome in B*^Q1 (a) and B*^Q2 (b). The compositional change during the transition is indicated in the middle. The composite cryo-EM density maps were generated from multi-body refined maps. The IBC module is shown at different contour levels. c, d The RNA structures in B*^Q1 (c) and B*^Q2 (d) are shown together with DHX35 (tomato red), which is bound to the U2 snRNA (green). The suboptimal pre-mRNA in the active center is highlighted. A simplified scheme of the RNA interactions is illustrated in the bottom row, with the 5’ ss bulge indicated as a zigzag line. e Summary of all modeled proteins from the B*^Q structures and RNAs with color code.

Fig. 3. GPATCH1 extensively interacts with PRP8 and DHX35.

a Overview of key interaction sites between GPATCH1 (rainbow color, from purple to yellow), PRP8 (pink) and DHX35 (tomato red). GPATCH1 (rainbow) binds to multiple PRP8 domains, including the EN, RT and RH domains, and to the DHX35 helicase. Unstructured regions of PRP8 and DHX35 are indicated by dashed lines. b Schematic representation of GPATCH1 interactions. Key interaction sites along the GPATCH1 sequence are highlighted.

Fig. 4. GPATCH1 recognizes stalled splicing intermediates and anchors DHX35 to the spliceosome.

a GPATCH1 region aa 40–60 binds to the α-finger of PRP8. b W66 of GPATCH1 probes the active center downstream of the –1 position of the pre-mRNA. The density map is taken from a focused refinement map. c Comparison of the pre-mRNA in B*^Q1 with that in the B* complex (PDB: 6J6Q). The proposed G⁺¹ nucleotide of the 5’ss is about 6 Å away from the active cleavage site. The active Mg²⁺ shown are taken from 6J6Q. The movement of the G⁺¹ nucleotide is indicated by the black arrow. d GPATCH1 region aa 70–120 binds to the EN domain of PRP8. e GPATCH1 (aa 120–220) is sandwiched between the PRP8 RH domain and DHX35. f The G-patch domain (aa 152–220) of GPATCH1 binds to DHX35, which is depicted with color-coded domains (RecA domains, tomato red; WH domain, deep pink; Ratchet, magenta; OB-fold domain, dark red).

Fig. 5. DHX35 and DHX15 prime the stalled spliceosome for disassembly.

a Illustration of the position of the DHX35 (tomato red) and DHX15 (orange) helicases within the B*^Q2 complex, including the depiction of the U2 snRNA (green) that threads into DHX35 (tomato red) and the U6 snRNA (blue) located close to DHX15 (orange). The density map of the B*^Q2 complex is shown in the background, with the predicted binding path of U6 snRNA in DHX15 indicated by a dashed line. b DHX35 binds to the region of the U2 snRNA that forms a helix with the BS of the pre-mRNA in catalytic spliceosomes. The density map for the U2 snRNA is shown to illustrate the resolution and connectivity. Detailed interaction of GPATCH1 with U2 snRNA is shown with a density map as an insert. The molecular model and density map are derived from B*^Q1. c Close-up view displaying the rigid body fit of DHX15 within the focused refined cryo-EM map in B*^Q2 (focus DHX15). The predicted binding path of U6 snRNA in DHX15 is indicated by a dashed line.

Fig. 6. Structural model of the quality control of suboptimal splicing substrates involving DHX35 and DHX15.

Schematic of rearrangements and repositioning of key factors during optimal splicing processes and disassembly by DHX15 (top) and during suboptimal splicing processes (bottom) leading to stalled spliceosomes, quality control and disassembly by DHX35 and DHX15.