Cryo-electron microscopy snapshots of the spliceosome: structural insights into a dynamic ribonucleoprotein machine

Abstract

The spliceosome excises introns from pre-messenger RNAs using an RNA-based active site that is cradled by a dynamic protein scaffold. A recent revolution in cryo-electron microscopy (cryo-EM) has led to near-atomic-resolution structures of key spliceosome complexes that provide insight into the mechanism of activation, splice site positioning, catalysis, protein rearrangements and ATPase-mediated dynamics of the active site. The cryo-EM structures rationalize decades of observations from genetic and biochemical studies and provide a molecular framework for future functional studies.

Figure 1. A functional view of the splicing cycle.

a, Two step mechanism of pre-mRNA splicing. b, Assembly and catalytic cycle of the spliceosome.

Figure 2. A structural view of the splicing cycle.

Complexes for which high-resolution structures were solved by cryo-EM are shown in surface representation. Key features are indicated for each complex (e.g. the location of the active site). Major sub-complexes are coloured as follows: U5 snRNP, blue; U6 snRNP, red; U4 snRNP and U4/U6 proteins, yellow; U2 snRNP, green; NTC and NTC-associated factors, orange; trans-acting protein factors, magenta. The following PDB entries were used: 5GAN (U4/U6•U5 tri-snRNP); 5NRL (B complex); 5GM6 (B^act complex); 5LJ5 (C complex); 5MQ0 (C* complex); 3JB9 (ILS).

Figure 3. Activation of the spliceosome.

A fully assembled spliceosome, pre-B complex, is converted to stable B complex upon release of U1 snRNP by the action of the ATPase Prp28. Within the B complex, the single-stranded region of U4 snRNA is already bound to the active site of the Brr2 helicase, ready to translocate along U4 snRNA and free U6 snRNA from U4 snRNA and U4/U6 di-snRNP proteins. U6 snRNA folds, pairs with U2 snRNA and interacts with NTC and NTR proteins. During this process, U2, U5 and U6 snRNAs, together with the pre-mRNA substrate, forms the catalytic RNA core, reminiscent of the group II intron active site (see Fig. 4).

Figure 4. The active site of the spliceosome and its interaction with substrate.

a, The RNA interaction network prior to the first trans-esterification reaction is shown. U6 snRNA forms the intra-molecular stem-loop (ISL) and helices Ia and Ib with U2 snRNA. The three nucleotides in cyan (catalytic triad) form three consecutive triple base-pairs with U80, U52 and U53 and U2 snRNA nucleotides (catalytic triplex). The first six nucleotides of the intron, GUAUGU, are base-paired with the ACAGAGA box in U6 snRNA. The conserved UACUAAC (where A represents the branch point adenosine)_sequence in the intron basepairs with U2 snRNA to form the branch helix from which the branch point adenosine bulges out. The hydroxyl group of this adenosine functions as a nucleophile that attacks the 5’ splice site. RNAs are colour coded: red, U6 snRNA; cyan, catalytic triad of U6 snRNA; green, U2 snRNA; blue, U5 snRNA; orange, 5’exon; black, intron. b, three-dimensional structure of the active site RNA in C complex. Magnesium ions are represented by two yellow spheres located between the backbone of the catalytic triad and the highly twisted backbone at the bulge in ISL. c, the 5’-exon and branched intron bound to the active site (overlaid on b). The base of the branch point adenosine is shown in magenta and sphere representation. The 5’ phosphate of the first intron nucleotide (G+1) forms a 2’-5’ phosphodiester bond with the 2’hydroxyl of the branch-point adenosine. The branch helix is not depicted, for clarity. d-f, Interaction of the catalytic core of the spliceosome and movement of the branch helix in B^act, C and C* complexes. RNAs are colour-coded as above. Domains of Prp8 are colour-coded as follows: light blue, the N-terminal domain; marine, the Reverse transcriptase domain; white, Linker domain; light yellow, endonuclease domain; deep blue, RNaseH domain. The branch-point adenosine is coloured in magenta. Note that the active site RNAs remain unchanged but the branch helix shows large movements between B^act, C and C* complexes. The branch helix is stablised by SF3b (B^act), step 1 factors (C) and step 2 factors (C*), respectively (See Figure 5). Yellow arrow indicates the active site metals.

Figure 5. Movement of the Prp8 RNaseH-like domain and its interaction with active site elements.

a-e, Surface representation of the position and key interacting partners of the Prp8 RNaseH-like domain (Prp8^RH) in specific spliceosomal complexes, relative to the Prp8 Large domain (Prp8^L). The insets show the relative movement of the domain; RH, RNaseH domain; L, Large domain. The RNaseH-like domain of Prp8 rotates during the catalytic phase of splicing and mediates conformational changes in the active site.

Figure 6. Binding of DEAH-box ATPases to specific spliceosomal complexes.

a-c, Surface representation of the positions and key interacting partners of DEAH-box ATPases in specific spliceosomal complexes, relative to Prp8. Key RNA components are shown in cartoon representation. The likely paths of the intron 3’ of the BP in B^act and C, and of the 3’-exon in C* are indicated as dashed lines. d, Different conformations of Prp2 and Prp22 observed in the cryo-EM maps of Bact and C* complexes. Note that for Prp2 no bound RNA was modelled and the RecA cassettes are present in a closed conformation, while for Prp22, bound RNA could be observed and the RecA cassettes are open as a result of a downward movement of RecA2 relative to the RecA1 domain. The open and closed conformations are shown schematically in the lower diagram, where bound RNA is coloured.