Structures of the human spliceosomes before and after release of the ligated exon

Abstract

Pre-mRNA splicing is executed by the spliceosome, which has eight major functional states each with distinct composition. Five of these eight human spliceosomal complexes, all preceding exon ligation, have been structurally characterized. In this study, we report the cryo-electron microscopy structures of the human post-catalytic spliceosome (P complex) and intron lariat spliceosome (ILS) at average resolutions of 3.0 and 2.9 Å, respectively. In the P complex, the ligated exon remains anchored to loop I of U5 small nuclear RNA, and the 3'-splice site is recognized by the junction between the 5'-splice site and the branch point sequence. The ATPase/helicase Prp22, along with the ligated exon and eight other proteins, are dissociated in the P-to-ILS transition. Intriguingly, the ILS complex exists in two distinct conformations, one with the ATPase/helicase Prp43 and one without. Comparison of these three late-stage human spliceosomes reveals mechanistic insights into exon release and spliceosome disassembly.

Fig. 1

Cryo-EM structures of the human post-catalytic spliceosome (P complex) and the human intron lariat spliceosome (ILS complex). a Structure of the human spliceosomal P complex at an average resolution of 3.0 Å. Two perpendicular views are shown. Components of the P complex are color-coded and tabulated below the structure. NTC: NineTeen complex; NTR: NTC-related complex. b Structure of the human ILS complex 1 (ILS1) at an average resolution of 2.91 Å. The ATPase/helicase Prp43 is yet to be loaded in the ILS1 complex. c Structure of the human ILS complex 2 (ILS2) at an average resolution of 2.86 Å. Prp43 is present in the ILS2 complex, ready to disassemble the spliceosome

Fig. 2

Structural features of the human P complex. a Structure of the RNA elements in the P complex. An overall cartoon representation of the RNA map is shown on the left panel, and a close-up view on the splicing active site center is displayed in the right panel. Disordered RNA sequences in the intron lariat and the 3′-exon are represented by magenta and red dotted lines, respectively. The catalytic and structural metals are shown in magenta and gray spheres, respectively. The 3′-splice site (3′SS) is highlighted in black. Putative hydrogen bonds and van der Waals interactions that mediate recognition of the 3′SS are represented by blue and black dotted lines, respectively. b Specific coordination of the catalytic metal ions in the human P complex. c Structural rearrangements of the spliceosomal components at the active site during the C-to-P transition. The human C complex (left panel) and P complex (right panel) are shown in the same orientation as determined by the core of Prp8 and U5 snRNA. Shown here are the proteins and RNA elements around the catalytic center. d A cartoon diagram of the 3′SS recognition. Remodeling of the C complex by Prp16 results in dissociation of the NTC component Isy1 and the step I factors CCDC49 and CCDC94/YJU2. The Linker domain of Prp8 is rotated away from the U2/BPS duplex. Consequently, the 1585-loop which binds the 3′-tail of the intron loads the 3′SS into the splicing active site center. The RNaseH-like domain is translocated towards the active site, pushing the β-finger towards the lariat junction. The WD40 domain of Prp17 is also translocated toward the branch helix; together with the splicing factor PRKRIP1 stabilizes the new conformation of RNaseH-like domain and branch helix. The intron lariat junction is sandwiched by the β-finger and the 1585-loop. Slu7 adopts an extended conformation and binds the RNaseH-like and Linker domains of Prp8, stabilizing the local conformation

Fig. 3

Structural rearrangements during the P-to-ILS1 transition. a Overall structural changes during the P-to-ILS1 transition. All RNA elements are shown. The protein components that remain unchanged are not shown. SRm300 is shown in a surface representation. EJC exon junction complex. b Conformational changes of Prp8 during release of the ligated exon. During the P-to-ILS1 transition, the step II factor Slu7 and SRm300 is dissociated and the Switch loop that binds SRm300 is flipped by about 180 degrees. The RNaseH-like domain (colored wheat) and the 3′SS are moved away from the active site in the P complex. c A cartoon diagram for the release of ligated exon. Driven by Prp22-mediated pulling, the β-finger (colored cyan) of the RNaseH-like domain is unbolted from the active site of the P complex; the Switch loop along with SRm300 and the 5′-exon are rotated 180°, releasing the ligated exon through the tunnel formed by RNaseH-like, the Linker/Endo and N-domain of Prp8. In the P complex, the 1585-loop binds the 3′SS next to the exon junction, and this interaction must be disrupted prior to the release of the ligated exon. The 3′SS is flipped out of the catalytic center and becomes flexible during this process. The putative gate of exon release between intron lariat and four domains of Prp8 is marked by black dotted lines in the ILS1 complex

Fig. 4

Cwf19L2 may play an important role during spliceosome disassembly and intron lariat RNA debranching. a In the ILS1 complex, Cwf19L2 interacts closely with the lariat junction at the splicing active site and directly contacts the RNaseH-like (wheat), Linker/Endo (light blue), and N-domain (gray) of Prp8. Shown in the left is a cartoon representation of key components at the active site. Shown in the right is a surface representation of these components in a related view. b A close-up view on the splicing active site. Prp8 is hidden except the 1585-loop (colored blue), which is located beneath the “clamp” of Cwf19L2. c Cwf19L2 employs a positively charged surface to interact with the RNA elements. The electrostatic surface potential is shown for Cwf19L2. Two perpendicular views are presented

Fig. 5

Structural rearrangements during the ILS1-to-ILS2 transition. a Overall structural changes during the ILS1-to-ILS2 transition. The mobile protein components in the transition are color-coded to highlight the structural changes. Immobile proteins are shown in gray. In the transition, Prp43 is recruited into ILS2. b A close-up view on select regions of the ILS1 and ILS2 complexes. Two orientations are shown. c A close-up view on the transition from ILS1 to ILS2

Fig. 6

Structural comparison of the ILS complexes between yeast and human. a Comparison of the overall structures of the ILS complex from S. pombe (left panel), human (middle panel), and S. cerevisiae (right panel). Components of the ILS complex are shown in surface representation. The components identically shared among the three complexes are shown in gray. Only those proteins that are different between the human and yeast complexes are color-coded. The S. pombe ILS complex resembles human ILS1; S. pombe Cwf19 (human Cwf19L2) closely interacts with Prp8 and the intron lariat, whereas Prp43 is yet to be loaded. The S. cerevisiae ILS lacks Cwf19 but contains 3 more proteins (Spp382/Ntr1, Ntr2, and Cwc23) that may participate in spliceosome disassembly. Notably, a large proportion of the RNA sequences in the intron lariat can be traced only in the human ILS complexes, but not in the yeast complexes. In the human ILS2 complex, the intron sequences upstream of the U2/BPS duplex is sequentially bound by CypE and Aquarius. The intron lariat traverses through a cavity formed by RBM22 in human. b Comparison of the RNA elements and specific protein components in the ILS complex from S. pombe (left panel), human (middle panel), and S. cerevisiae (right panel). The RNaseH-like domain is colored wheat; arrow marks the β-finger (left/right panel) or Cwf19L2 (middle panel)