Explore the vast range of titles available.

Key Points Analysis of the recent atomic-resolution cryo-electron microscopy structures of the spliceosome reveals that during splicing all spliceosomal complexes share a rigid core of 20 RNAs and proteins. The active site of the spliceosome is positioned in the catalytic cavity of pre-mRNA-splicing factor 8 (Prp8), is stabilized by surrounding proteins and remains static throughout the two steps of transesterification. The catalytic metals in the active site are coordinated by U6 small nuclear RNA and the catalytic triplex. The interconversion of the various spliceosomal complexes is driven by eight conserved, RNA-dependent ATPase/helicases. The spliceosome is a protein-directed metalloribozyme. Atomic-resolution structures have recently been obtained for the intact spliceosome at different stages of the splicing cycle. These structural data have proved that the spliceosome is a protein-directed metalloribozyme and have increased our understanding of pre-mRNA splicing mechanisms, explaining a large body of existing genetic and biochemical data. Precursor messenger RNA (pre-mRNA) splicing is an essential step in the flow of information from DNA to protein in all eukaryotes. Research over the past four decades has molecularly delineated the splicing pathway, including characterization of the detailed splicing reaction, definition of the spliceosome and identification of its components, and biochemical analysis of the various splicing complexes and their regulation. Structural information is central to mechanistic understanding of pre-mRNA splicing by the spliceosome. X-ray crystallography of the spliceosomal components and subcomplexes is complemented by electron microscopy of the intact spliceosome. In this Review, I discuss recent atomic-resolution structures of the intact spliceosome at different stages of the splicing cycle. These structures have provided considerable mechanistic insight into pre-mRNA splicing and have corroborated and explained a large body of genetic and biochemical data. Together, the structural data have proved that the spliceosome is a protein-directed metalloribozyme.

The cryo-electron microscopy structure of a yeast spliceosome stalled before mature RNA formation provides insight into the mechanism of exon ligation. Structure of the spliceosomal C* complex Recent years have seen substantial progress in understanding the structure of various intermediates of the splicing process. Two groups, led by Reinhard Lührmann and Kiyoshi Nagai, now describe the cryo-electron microscopy structures (from human and yeast cells, respectively) of the splicing intermediate known as the C* complex. The notable feature observed in this complex, relative to the preceding catalytic intermediate (the C complex), is a remodelling that positions the branch-site adenosine and the branched intron out of the catalytic core, opening up space for the 3′ exon to dock in preparation for exon ligation. The spliceosome excises introns from pre-mRNAs in two sequential transesterifications—branching and exon ligation 1 —catalysed at a single catalytic metal site in U6 small nuclear RNA (snRNA) 2 , 3 . Recently reported structures of the spliceosomal C complex 4 , 5 with the cleaved 5′ exon and lariat–3′-exon bound to the catalytic centre revealed that branching-specific factors such as Cwc25 lock the branch helix into position for nucleophilic attack of the branch adenosine at the 5′ splice site. Furthermore, the ATPase Prp16 is positioned to bind and translocate the intron downstream of the branch point to destabilize branching-specific factors and release the branch helix from the active site 4 . Here we present, at 3.8 Å resolution, the cryo-electron microscopy structure of a Saccharomyces cerevisiae spliceosome stalled after Prp16-mediated remodelling but before exon ligation. While the U6 snRNA catalytic core remains firmly held in the active site cavity of Prp8 by proteins common to both steps, the branch helix has rotated by 75° compared to the C complex and is stabilized in a new position by Prp17, Cef1 and the reoriented Prp8 RNase H-like domain. This rotation of the branch helix removes the branch adenosine from the catalytic core, creates a space for 3′ exon docking, and restructures the pairing of the 5′ splice site with the U6 snRNA ACAGAGA region. Slu7 and Prp18, which promote exon ligation, bind together to the Prp8 RNase H-like domain. The ATPase Prp22, bound to Prp8 in place of Prp16, could interact with the 3′ exon, suggesting a possible basis for mRNA release after exon ligation 6 , 7 . Together with the structure of the C complex 4 , our structure of the C* complex reveals the two major conformations of the spliceosome during the catalytic stages of splicing.

Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2–U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 Å, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains. RNA helicase PRP22 is located about 100 Å from the catalytic centre, suggesting that it destabilizes the spliced mRNA after step two from a distance. Comparison of the structure of the yeast C and human C* complexes reveals numerous RNP rearrangements that are likely to be facilitated by PRP16, including a large-scale movement of the U2 small nuclear RNP. The cryo-EM structure of the splicing intermediate known as the C* complex from human. Structure of the spliceosomal C* complex Recent years have seen substantial progress in understanding the structure of various intermediates of the splicing process. Two groups, led by Reinhard Lührmann and Kiyoshi Nagai, now describe the cryo-electron microscopy structures (from human and yeast cells, respectively) of the splicing intermediate known as the C* complex. The notable feature observed in this complex, relative to the preceding catalytic intermediate (the C complex), is a remodelling that positions the branch-site adenosine and the branched intron out of the catalytic core, opening up space for the 3′ exon to dock in preparation for exon ligation.

Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariat–intron intermediate. Here we present the 3.8 Å cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5′-splice site is cleaved but remains close to the catalytic Mg 2+ site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5′-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2′OH. The 5′-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-Watson–Crick interactions between the branch helix and 5′-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAG AGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation. Cryo-EM reveals the configuration of substrate pre-mRNA within the active spliceosome and suggests how remodelling occurs prior to exon ligation. Structure of the branched splicing complex The excision of introns from RNA is not a concerted process, but is rather an ordered one involving two transesterification reactions by the spliceosome. In the first step, the 5′-splice site is cleaved and the intron end is joined to make a lariat structure. Kiyoshi Nagai and colleagues have captured the Saccharomyces cerevisiae spliceosome stalled immediately after this first transesterification (branching) reaction by cryo-electron microscopy single-particle reconstruction at an overall resolution of 3.8 Å. The configuration of the RNA within the complex suggests that remodelling occurs before the second step, exon ligation.

The molecular mechanisms of exon definition and back-splicing are fundamental unanswered questions in pre-mRNA splicing. Here we report cryo-electron microscopy structures of the yeast spliceosomal E complex assembled on introns, providing a view of the earliest event in the splicing cycle that commits pre-mRNAs to splicing. The E complex architecture suggests that the same spliceosome can assemble across an exon, and that it either remodels to span an intron for canonical linear splicing (typically on short exons) or catalyses back-splicing to generate circular RNA (on long exons). The model is supported by our experiments, which show that an E complex assembled on the middle exon of yeast EFM5 or HMRA1 can be chased into circular RNA when the exon is sufficiently long. This simple model unifies intron definition, exon definition, and back-splicing through the same spliceosome in all eukaryotes and should inspire experiments in many other systems to understand the mechanism and regulation of these processes. The cryo-electron microscopy structures of an early spliceosome complex in yeast reveal a unified mechanism for defining introns and exons and also for back-splicing to generate circular RNA.

The spliceosome catalyses the excision of introns from pre-mRNA in two steps, branching and exon ligation, and is assembled from five small nuclear ribonucleoprotein particles (snRNPs; U1, U2, U4, U5, U6) and numerous non-snRNP factors 1 . For branching, the intron 5′ splice site and the branch point sequence are selected and brought by the U1 and U2 snRNPs into the prespliceosome 1 , which is a focal point for regulation by alternative splicing factors 2 . The U4/U6.U5 tri-snRNP subsequently joins the prespliceosome to form the complete pre-catalytic spliceosome. Recent studies have revealed the structural basis of the branching and exon-ligation reactions 3 , however, the structural basis of the early events in spliceosome assembly remains poorly understood 4 . Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae prespliceosome at near-atomic resolution. The structure reveals an induced stabilization of the 5′ splice site in the U1 snRNP, and provides structural insights into the functions of the human alternative splicing factors LUC7-like (yeast Luc7) and TIA-1 (yeast Nam8), both of which have been linked to human disease 5 , 6 . In the prespliceosome, the U1 snRNP associates with the U2 snRNP through a stable contact with the U2 3′ domain and a transient yeast-specific contact with the U2 SF3b-containing 5′ region, leaving its tri-snRNP-binding interface fully exposed. The results suggest mechanisms for 5′ splice site transfer to the U6 ACAGAGA region within the assembled spliceosome and for its subsequent conversion to the activation-competent B-complex spliceosome 7 , 8 . Taken together, the data provide a working model to investigate the early steps of spliceosome assembly. The cryo-electron microscopy structure of the Saccharomyces cerevisiae prespliceosome provides insights into splice-site selection and early spliceosome assembly events.

Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome’s catalytic centre. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae pre-catalytic B complex spliceosome at near-atomic resolution. The mobile U2 small nuclear ribonucleoprotein particle (snRNP) associates with U4/U6.U5 tri-snRNP through the U2/U6 helix II and an interface between U4/U6 di-snRNP and the U2 snRNP SF3b-containing domain, which also transiently contacts the helicase Brr2. The 3′ region of the U2 snRNP is flexibly attached to the SF3b-containing domain and protrudes over the concave surface of tri-snRNP, where the U1 snRNP may reside before its release from the pre-mRNA 5′ splice site. The U6 ACAGAGA sequence forms a hairpin that weakly tethers the 5′ splice site. The B complex proteins Prp38, Snu23 and Spp381 bind the Prp8 N-terminal domain and stabilize U6 ACAGAGA stem–pre-mRNA and Brr2–U4 small nuclear RNA interactions. These results provide important insights into the events leading to active site formation. The cryo-electron microscopy structure of the yeast spliceosome in a pre-catalytic state provides insights into the molecular events leading to formation of the spliceosome active site. Visualization of a poised spliceosome Protein-coding regions of DNA can be interrupted by non-coding regions, or introns. A large multisubunit complex, the spliceosome, is used to excise introns from the messenger RNA before it is translated into protein. Formation of an active spliceosome complex on an intron requires stepwise assembly of subcomplexes, followed by their rearrangement and the loss of some factors. Kiyoshi Nagai and colleagues have solved the structure of the B complex spliceosome, poised in a pre-catalytic state. The detection of several factors that were not visualized in previous spliceosome structures provides new insights regarding the process by which the complex is activated.

Splicing of precursor messenger RNA (pre-mRNA) in yeast is executed by the spliceosome, which consists of five small nuclear ribonucleoproteins (snRNPs), NTC (nineteen complex), NTC-related proteins (NTR), and a number of associated enzymes and cofactors. Here, we report the three-dimensional structure of a Schizosaccharomyces pombe spliceosome at 3.6-angstrom resolution, revealed by means of single-particle cryogenic electron microscopy. This spliceosome contains U2 and U5 snRNPs, NTC, NTR, U6 small nuclear RNA, and an RNA intron lariat. The atomic model includes 10,574 amino acids from 37 proteins and four RNA molecules, with a combined molecular mass of approximately 1.3 megadaltons. Spp42 (Prp8 in Saccharomyces cerevisiae), the key protein component of the U5 snRNP, forms a central scaffold and anchors the catalytic center. Both the morphology and the placement of protein components appear to have evolved to facilitate the dynamic process of pre-mRNA splicing. Our near-atomic-resolution structure of a central spliceosome provides a molecular framework for mechanistic understanding of pre-mRNA splicing.

Catalyzed by the spliceosome, precursor mRNA splicing proceeds in two steps: branching and exon ligation. Transition from the C (catalytic post-branching spliceosome) to the C* (catalytic pre-exon ligation spliceosome) complex is driven by the adenosine triphosphatase/helicase Prp16. Zhan et al. report the cryo-electron microscopy structure of the human C complex, showing that two step I splicing factors stabilize the active site and link it to Prp16. Science , this issue p. 537 The cryo–electron microscopy structure of the human C complex spliceosome reveals mechanistic insights into ribonucleoprotein remodeling. Splicing by the spliceosome involves branching and exon ligation. The branching reaction leads to the formation of the catalytic step I spliceosome (C complex). Here we report the cryo–electron microscopy structure of the human C complex at an average resolution of 4.1 angstroms. Compared with the Saccharomyces cerevisiae C complex, the human complex contains 11 additional proteins. The step I splicing factors CCDC49 and CCDC94 (Cwc25 and Yju2 in S. cerevisiae , respectively) closely interact with the DEAH-family adenosine triphosphatase/helicase Prp16 and bridge the gap between Prp16 and the active-site RNA elements. These features, together with structural comparison of the human C and C* complexes, provide mechanistic insights into ribonucleoprotein remodeling and allow the proposition of a working mechanism for the C-to-C* transition.

The prespliceosome, comprising U1 and U2 small nuclear ribonucleoproteins (snRNPs) bound to the precursor messenger RNA 5ʹ splice site (5ʹSS) and branch point sequence, associates with the U4/U6.U5 tri-snRNP to form the fully assembled precatalytic pre–B spliceosome. Here, we report cryo–electron microscopy structures of the human pre–B complex captured before U1 snRNP dissociation at 3.3-angstrom core resolution and the human tri-snRNP at 2.9-angstrom resolution. U1 snRNP inserts the 5ʹSS–U1 snRNA helix between the two RecA domains of the Prp28 DEAD-box helicase. Adenosine 5ʹ-triphosphate–dependent closure of the Prp28 RecA domains releases the 5ʹSS to pair with the nearby U6 ACAGAGA-box sequence presented as a mobile loop. The structures suggest that formation of the 5ʹSS-ACAGAGA helix triggers remodeling of an intricate protein-RNA network to induce Brr2 helicase relocation to its loading sequence in U4 snRNA, enabling Brr2 to unwind the U4/U6 snRNA duplex to allow U6 snRNA to form the catalytic center of the spliceosome.