Molecular architecture of the Saccharomyces cerevisiae activated spliceosome

Abstract

The activated spliceosome (B) is in a catalytically inactive state and is remodeled into a catalytically active machine by the RNA helicase Prp2, but the mechanism is unclear. Here, we describe a 3D electron cryomicroscopy structure of the Saccharomyces cerevisiae B complex at 5.8-angstrom resolution. Our model reveals that in B, the catalytic U2/U6 RNA-Prp8 ribonucleoprotein core is already established, and the 5' splice site (ss) is oriented for step 1 catalysis but occluded by protein. The first-step nucleophile-the branchsite adenosine-is sequestered within the Hsh155 HEAT domain and is held 50 angstroms away from the 5'ss. Our structure suggests that Prp2 adenosine triphosphatase-mediated remodeling leads to conformational changes in Hsh155's HEAT domain that liberate the first-step reactants for catalysis.

SF3B1/Hsh155 HEAT motif mutations affect interaction with the spliceosomal ATPase Prp5, resulting in altered branch site selectivity in pre-mRNA splicing

“…In recent structures of the spliceosome (Rauhut et al 2016;Yan et al 2016) and of SF3B alone (Cretu et al 2016), the SF3B complex forms a "spring-loaded clamp"-like structure in which Hsh155p/SF3B1 is the "spring" and the BS-U2 duplex is "clamped" between the first and last HEAT domains of Hsh155p/SF3B1 (Supplemental Fig. S4).…”

“…This suggests that mutations in SF3B1 may have effects during spliceosomal assembly and fidelity. In the cryo-electron microscropy (cryo-EM) structures of the B act complex (before first-step catalysis), the SF3B1 HEAT motifs adopt a torus or ring-like structure through which the 3 ′ end of the intron is threaded; in addition, the first and last HEATs cradle the BS-U2 duplex (Rauhut et al 2016;Yan et al 2016). The SF3B complex is displaced upon conversion to the active spliceosomal C complex by ATPase Prp2 (Warkocki et al 2009;Lardelli et al 2010) and is not present in the cryo-EM structures of complexes immediately after first-step catalysis (Galej et al 2016;Wan et al 2016).…”

Unmasking the U2AF homology motif family: a bona fide protein–protein interaction motif in disguise

“…Structures of RRM-containing ribonucleoproteins and seminal spliceosome complexes (Yan et al 2015;Galej et al 2016;Rauhut et al 2016;Wan et al 2016a,b;Yan et al 2016) demonstrate that the RRM fold serves as a platform for multiprotein assemblies with RNA. One of the first RRM structures revealed distinct α-helical and β-sheet surfaces of the U2B ′′ RRM bound to the U2A ′ protein and the U2 small nuclear (sn) RNA (Price et al 1998).…”

Functions and regulation of the Brr2 RNA helicase during splicing

“…As the RH domain can interact with the NTR of Brr2 14 as well as with U4/U6 87 and can dramatically change its position during splicing, [100][101][102][103] it may intermittently contact U4/U6 during priming of a pre-catalytic spliceosome for Brr2-mediated activation, thus guiding the core of Brr2 to its entry site on U4 snRNA. Interestingly, the Prp8 Jab1 domain resembles corresponding domains in deubiquitinating enzymes.…”

“…5A). 100,101 In B act , the Brr2 NTR adopts its autoinhibited state, wrapped around the Brr2 helicase cassettes, as in the Brr2-Jab1 complexes and as in the human tri-snRNP structure. Additionally, the globular part of the Prp8 Jab1 domain remains bound to the Brr2 NC and the Jab1 C-terminal tail is occupying the Brr2 RNA-binding tunnel, indicating that the NTR and Jab1 domain together shut Brr2 down after spliceosome activation (Fig.…”