Molecular Mechanisms of pre-mRNA Splicing through Structural Biology of the Spliceosome

Abstract

Precursor messenger RNA (pre-mRNA) splicing is executed by the spliceosome. In the past 3 years, cryoelectron microscopy (cryo-EM) structures have been elucidated for a majority of the yeast spliceosomal complexes and for a few human spliceosomes. During the splicing reaction, the dynamic spliceosome has an immobile core of about 20 protein and RNA components, which are organized around a conserved splicing active site. The divalent metal ions, coordinated by U6 small nuclear RNA (snRNA), catalyze the branching reaction and exon ligation. The spliceosome also contains a mobile but compositionally stable group of about 13 proteins and a portion of U2 snRNA, which facilitate substrate delivery into the splicing active site. The spliceosomal transitions are driven by the RNA-dependent ATPase/helicases, resulting in the recruitment and dissociation of specific splicing factors that enable the reaction. In summary, the spliceosome is a protein-directed metalloribozyme.

Figure 1.

The precursor messenger RNA (pre-mRNA) splicing cycle and the major structures of the spliceosome. (A) The pre-mRNA splicing cycle. Each cycle includes three phases: assembly and activation of the spliceosome, execution of the splicing reaction, and disassembly of the spliceosome. The pre-B complex represents the first fully assembled spliceosome, in which all five snRNPs are present and the 5′SS is still recognized by U1 snRNP. In addition to the pre-B complex, the assembled spliceosome exists in seven compositionally distinct states: B, B^act, B*, C, C*, P, and intron lariat spliceosome (ILS). The unidirectional conversion of each of these neighboring spliceosomal complexes to the next is driven by the conserved ATPases/helicases Brr2 (B-to-B^act), Prp2 (B^act-to-B*), Prp16 (C-to-C*), and Prp22 (P-to-ILS). The splicing factors Cwc25 and Yju2 facilitate the branching reaction, whereas Prp18 and Slu7 assist exon ligation. Disassembly of the ILS complex is executed by the ATPase/helicase Prp43. (B) The major cryoelectron microscopy (cryo-EM) structures of the spliceosome since 2015. The time axis is drawn to scale, with each white dot representing a month. The first atomic model of an intact spliceosome was elucidated for the ILS complex from Saccharomyces pombe on the basis of an EM density map at an average resolution of 3.6 Å (Hang et al. 2015; Yan et al. 2015). The 18 cryo-EM structures of the spliceosome come from four independent laboratories: nine from the Shi group (shaded light purple), four from the Nagai group (shaded yellow), four from the Luhrmann/Stark group (shaded cyan), and one from the Zhao/Zhou group (shaded light brown). (C) The cryo-EM structure of the ILS complex from S. pombe (PDB code 3JB9) (Hang et al. 2015; Yan et al. 2015). The color-coded structure is shown on the left, and the individual components resolved in the structure are tabulated on the right.

Figure 2.

Shared structural features of the spliceosome during the splicing reaction. (A) The RNA elements at the conserved splicing active site. The active site comprises the intramolecular stem loop (ISL) of U6 small nuclear RNA (snRNA) and associated metal ions, the U2/U6 catalytic triplex, helix I of the U2/U6 duplex, and loop I of U5 snRNA. Although the active site shown here is derived from the Saccharomyces cerevisiae C complex (PDB code 5GMK) (Wan et al. 2016a), the overall conformation is identical in the B^act through intron lariat spliceosome (ILS) complexes. (B) Structure of the conserved catalytic triplex. The original electron microscopy (EM) density map, shown for the S. cerevisiae C complex (Wan et al. 2016a), allows unambiguous identification of the metal ions and the nucleobases. (C) U5 and U6 snRNAs along their entire lengths and the 5′ 30 nucleotides of U2 snRNA remain largely static in the B^act through ILS complexes. The RNA elements in the S. cerevisiae C complex are color coded; whereas those from the B^act, C*, P, and ILS complexes are each displayed in a single color. The PDB codes for the B^act, C, C*, P, and ILS complexes are 5GM6, 5GMK, 5WSG, 5YLZ, and 5Y88, respectively. (D) The conserved splicing active site RNA elements are anchored in the catalytic cavity of Prp8. The cavity is formed at the interface between the N-domain and the Prp8 core and is enriched in positively charged residues. (E) Sixteen additional protein components, for their partial or entire lengths, remain largely static in the B^act through ILS complexes. These proteins, together with Prp8, maintain the rigid conformation of the snRNA elements (U5 and U6 along their entire lengths and the 5′ 30 nucleotides of U2 snRNA). These proteins and the snRNAs from the S. cerevisiae C complex are color coded here. (F) A compositionally constant group of 13 proteins in the B^act through ILS complexes. These proteins constitute two groups: nine in the U2 snRNP (the heptameric Sm complex, Lea1, and Msl1) and four in the NTC (Prp19, Snt309, Cef1, Syf1, and Clf1). Each group rigidly maintains its collective structure but is translocated during the two steps of the splicing reaction. These proteins, together with the other 20 rigid components, give rise to the characteristic appearance of the spliceosome.

Figure 3.

The spliceosome is a metalloribozyme. (A) Coordination of the catalytic metals M1 and M2. Shown here is metal coordination in the Saccharomyces cerevisiae C complex (Wan et al. 2016a). M1 is recognized by four ligands arranged in a planar fashion. The original electron microscopy (EM) density map is shown in panels A, B, and D. PDB code 5GMK and EMDB code EMD-9525 for the C complex; PDB code 5YLZ and EMDB code EMD-6839 for the P complex. (B) Coordination of M1 and M2 in the S. cerevisiae P complex (Bai et al. 2017). M2 is bound by five potential ligands. (C) Choreography of the catalytic metals during the splicing reaction. The coordination of M1 and M2 is shown for the spliceosomal B^act, B* (predicted), C, C*, P, and intron lariat spliceosome (ILS) complexes. Elements of the U6 small nuclear RNA (snRNA) are shown in the same orientation in the six complexes to allow meaningful comparison. Metal coordination in the B* complex is predicted based on the C complex and knowledge of the splicing reaction. (D) The structural metals are defined by an excellent EM density map (shown here for the S. cerevisiae C complex; Wan et al. 2016a) and presumably stabilize the intramolecular stem loop (ISL) fold by neutralizing the negative charges of the RNA phosphodiester backbone.

Figure 4.

Protein components and catalytic motifs of the spliceosome. (A) The spring-like proteins, exemplified by Syf1 and Clf1 in Saccharomyces cerevisiae, allow large conformational rearrangements of the spliceosome while maintaining their structural integrity. The S. cerevisiae C complex (Wan et al. 2016a) is used for illustration in panels A–C. Syf1 and Clf1 are colored yellow and cyan, respectively. (B) The rope-like proteins, represented by Prp45 and Cwc15, stabilize spliceosomal organization through direct interactions with multiple proteins and RNA elements. Prp45, colored red and displayed in surface representation, simultaneously interacts with at least nine proteins and two small nuclear RNA (snRNA) elements (U2 and U6). (C) The step I factors Cwc25 and Yju2 constitute an integral part of the splicing active site during the branching reaction. The amino termini of both proteins reach into the center of the active site RNA elements and stabilize their conformations. (D) The step II factors Prp17 and Prp18 stabilize the active site conformation. Shown here is the S. cerevisiae C* complex (Yan et al. 2017). (E) The splicing factor Cwc24 protects the guanine nucleotide at the 5′-end of the 5′SS in the S. cerevisiae B^act complex (Yan et al. 2016). Two aromatic residues Tyr155 and Phe161 sandwich the guanine nucleobase. (F) The splicing factor Cwc21 stabilizes the binding of the 5′-exon to loop I of U5 snRNA. Cwc21 simultaneously interacts with nucleotides of the 5′-exon and the switch loop of Prp8 in the B^act through P complexes. The interactions, shown here for the S. cerevisiae B^act complex (Yan et al. 2016), are identically preserved in the C, C*, and P complexes. (G) The 1585-loop of Prp8 in the S. cerevisiae B^act (Yan et al. 2016) (left panel) and P (Bai et al. 2017) (right panel) complexes. (H) The β-finger (from the RNaseH-like domain of Prp8) in the S. cerevisiae C (Wan et al. 2016a) (left panel) and P (Bai et al. 2017) (right panel) complexes.

Figure 5.

Spliceosomal transitions are driven by the ATPase/helicases. (A) The B-to-B^act transition is propelled by Brr2. Shown here is a surface view of the Saccharomyces cerevisiae B complex (Plaschka et al. 2017). Brr2 is thought to pull on the single-stranded U4 small nuclear RNA (snRNA) sequences, triggering the unwinding of the U4/U6 duplex and subsequent changes. (B) The B^act-to-B* transition is mediated by Prp2. Shown here is a surface view of the S. cerevisiae B^act complex (Yan et al. 2016). Prp2 likely pulls on the 3′-end sequences of the pre-mRNA, leading to dissociation of the retention and splicing complex (RES) complex, the SF3b and SF3a complexes, and the splicing factor Cwc24. (C) The C-to-C* transition is executed by Prp16. Shown here is a surface view of the S. cerevisiae C complex (Wan et al. 2016a). Prp16 presumably pulls on the 3′-end sequences of the intron lariat-3′-exon intermediate. (D) The P-to-intron lariat spliceosome (ILS) transition is driven by Prp22. Shown here is a surface view of the S. cerevisiae P complex (Bai et al. 2017). Dissociation of the ligated exon is mediated by Prp22 pulling on its 3′-end sequences. (E) The disassembly of the ILS complex is performed by Prp43. Shown here is a surface view of the S. cerevisiae ILS complex (Wan et al. 2017). Prp43 may function by binding to and pulling on either the 3′ end of U6 snRNA or the intron lariat. The PDB codes for the B, B^act, C, P, and ILS complexes are 5NRL, 5GM6, 5GMK/5LJ5, 5YLZ, and 5Y88, respectively.

Figure 6.

Choreography of the protein and RNA components during the splicing reaction. (A) Recognition of the AG dinucleotide at the 3′SS by the lariat junction in the P complex (Bai et al. 2017; Liu et al. 2017; Wilkinson et al. 2017). Notably, the AG nucleobases at the 3′SS form noncanonical Watson–Crick base-pairing interactions with two consecutive AG nucleobases of the lariat junction. (B) Recognition of the nucleophile-containing adenine nucleotide of the branch point sequence (BPS) by Hsh155. The adenine nucleobase is already flipped out of the registry of the intron/U2 duplex and is in close contact with a few residues of Hsh155 and Rds3. (C) A cartoon diagram depicting the activation of the B^act complex, the branching and exon ligation, and the disassembly of the intron lariat spliceosome (ILS) complex.