Structural studies of the spliceosome: Bridging the gaps

Abstract

The spliceosome is a multi-megadalton RNA-protein complex responsible for the removal of non-coding introns from pre-mRNAs. Due to its complexity and dynamic nature, it has proven to be a very challenging target for structural studies. Developments in single particle cryo-EM have overcome these previous limitations and paved the way towards a structural characterisation of the splicing machinery. Despite tremendous progress, many aspects of spliceosome structure and function remain elusive. In particular, the events leading to the definition of exon-intron boundaries, alternative and non-canonical splicing events, and cross-talk with other cellular machineries. Efforts are being made to address these knowledge gaps and further our mechanistic understanding of the spliceosome. Here, we summarise recent progress in the structural and functional analysis of the spliceosome.

Figure 1

Stepwise assembly of yeast (Saccharomyces cerevisiae) and human spliceosomes from snRNPs and trans-acting factors. For simplicity, only factors relevant to this review are indicated. Cartoon shapes of splicing complexes are based on yeast structures, except the 17S U2 snRNP and pre-B^act complex, for which only human structures are available. Please note, that there are some differences between yeast and human splicing pathways which were not depicted here.

Figure 2

Structural insights into early splicing events. (a) Proposed order of events during branch site recognition. (b) U2 snRNP 5′-domain structures from recently reported early splicing complexes with highlighted key protein factors and RNA elements [17,18,25,31]. (c) Schematics of RNA secondary structures from the corresponding complexes showing the key transitions and interactions between U2 snRNA and the pre-mRNA substrate. (d) The structure of the U1 snRNP-RNAPII complex determined by cryo-EM, showing the stable interfaces between components of the two complexes [42]. (e) Proposed intron looping model for co-transcriptional splicing and formation of the spliceosomal A complex.

Figure 3

Spliceosome activation and the pre-Bact complexes. (a) Schematics depicting the key proteins and RNAs recruited to or displaced from the spliceosome during activation, based on the structures of human B [46,47], pre-B^act-1 and pre-B^act-2 [45] and B^act [50,51] complexes. (b) Schematic representation of RNA secondary structure in the corresponding complexes highlighting progressive formation of the U2/U6 catalytic core. (c) Molecular models of the B, pre-B^act-1, pre-B^act-2 and Bact complexes showing large scale global conformational changes in the corresponding complexes.

Figure 4

Structure of the minor spliceosome. (a) Overall architecture of the major spliceosome B^act complex with RNA elements highlighted in the foreground [67]. (b) Human minor spliceosome B^act complex [50]. (c) and (d) Structures of the RNAs in the major and minor B^act complexes showing remarkable structural similarity despite divergent sequences. (e) and (f) Schematics of the RNA secondary structure elements present in the major and minor B^act complexes. Elements exclusive to the major or minor spliceosome are indicated with ∗ and ∗∗, respectively. (g) and (h) Distinct proteins likely serve similar functions in the major and minor splicing systems.