Postcatalytic spliceosome structure reveals mechanism of 3'-splice site selection

Abstract

Introns are removed from eukaryotic messenger RNA precursors by the spliceosome in two transesterification reactions-branching and exon ligation. The mechanism of 3'-splice site recognition during exon ligation has remained unclear. Here we present the 3.7-angstrom cryo-electron microscopy structure of the yeast P-complex spliceosome immediately after exon ligation. The 3'-splice site AG dinucleotide is recognized through non-Watson-Crick pairing with the 5' splice site and the branch-point adenosine. After the branching reaction, protein factors work together to remodel the spliceosome and stabilize a conformation competent for 3'-splice site docking, thereby promoting exon ligation. The structure accounts for the strict conservation of the GU and AG dinucleotides at the 5' and 3' ends of introns and provides insight into the catalytic mechanism of exon ligation.

Figure 1. P complex structure.

A, Overview of the P complex spliceosome. NTC, NineTeen Complex. B, The same view of P complex with the path of the substrate intron and exons shown. Dotted lines indicate the path of nucleotides not visible in the density. C, Binding of substrate at the core of P complex. U6 snRNA and NTC/NTR proteins are omitted for clarity. 3'SS, 3' splice site; RT, Prp8 reverse transcriptase domain; CR, Prp18 conserved region.

Figure 2. Structure of the RNA catalytic core.

A, Key RNA elements at the active site of the C complex spliceosome. ISL, internal stem-loop; M1 and M2, catalytic metal ions one and two. B, Equivalent view to A of the active site of the P complex spliceosome. M1 was not visible in the density and its position is inferred from C and C* complexes. C, Non-Watson-Crick RNA-RNA interactions mediate recognition of the 3' splice site. Putative hydrogen bonds are shown with dotted blue lines. Branch point adenosine and U6 snRNA A51 are highlighted. D, CryoEM density around the exon junction for the 5'-exon, 3'-exon and 3' splice site. E, Base-pairing scheme of the P complex active site. Watson-Crick pairing is indicated with lines, other base pairs with dotted lines. Ψ, pseudo-uridine. F, Details of the pairing that mediates 3' splice site (3'SS) recognition. 5'SS, 5' splice site; BP A, branch point adenosine.

Figure 3. Proteins at the active site.

A, The Prp8 α-finger and β-hairpin clamp around the active site, with Prp18 bound on the outer face of the Prp8 RNaseH domain. CR, Prp18 conserved region; RH, Prp8 RNaseH domain. B, The Prp8 α-finger contacts both 3'-exon and 3' splice site; Prp18 CR loop contacts the 3'SS from the opposite side. C, Residues of the Prp8 α-finger that contact the 3' splice site. D, In vitro splicing reaction with wild-type (WT) and Arg1604 to Ala (R1604A) mutant Prp8. RNA species found in Prp8-immunoprecipitated spliceosomes are labelled schematically. R1604A causes a second-step defect, evidenced by accumulation of lariat intron-3'-exon intermediate.

Figure 4. Docking of the 3' splice site is associated with binding of exon ligation factors.

A, The binding of exon ligation factors Slu7 and Prp18 to the surface of P complex. Disordered segments of Slu7 are shown with dotted lines. Domains of Prp8 are colored as indicated. N, Prp8 N-terminal domain; RT, Prp8 reverse transcriptase domain; EN, Prp8 endonuclease domain; ZnK, Slu7 zinc knuckle domain; CR, Prp18 conserved region. B, C, Cryo-EM density maps for P complex with the 3'SS docked and undocked. Maps were filtered to 5 Å resolution to aid visualization. Movements of the branch helix, Prp17, and the Prp8 endonuclease domain when changing into the docked conformation are indicated.

Figure 5. Model for the action of exon ligation factors.

Following Prp16-mediated remodeling of C complex the branching factors Cwc25, Yju2 N-domain and Isy1 (not depicted) are removed. The undocked branch helix is then locked in a conformation competent for second step catalysis by the binding of exon ligation factors Prp18 and Slu7 and the C-domain of Yju2. The 3'SS docked and undocked conformations may be in equilibrium due to flexibility of the branch helix and Prp8 Endonuclease domain in the C* state.