Structure of a yeast activated spliceosome at 3.5 Å resolution

Abstract

Pre-messenger RNA (pre-mRNA) splicing is carried out by the spliceosome, which undergoes an intricate assembly and activation process. Here, we report an atomic structure of an activated spliceosome (known as the B(act) complex) from Saccharomyces cerevisiae, determined by cryo-electron microscopy at an average resolution of 3.52 angstroms. The final refined model contains U2 and U5 small nuclear ribonucleoprotein particles (snRNPs), U6 small nuclear RNA (snRNA), nineteen complex (NTC), NTC-related (NTR) protein, and a 71-nucleotide pre-mRNA molecule, which amount to 13,505 amino acids from 38 proteins and a combined molecular mass of about 1.6 megadaltons. The 5' exon is anchored by loop I of U5 snRNA, whereas the 5' splice site (5'SS) and the branch-point sequence (BPS) of the intron are specifically recognized by U6 and U2 snRNA, respectively. Except for coordination of the catalytic metal ions, the RNA elements at the catalytic cavity of Prp8 are mostly primed for catalysis. The catalytic latency is maintained by the SF3b complex, which encircles the BPS, and the splicing factors Cwc24 and Prp11, which shield the 5' exon-5'SS junction. This structure, together with those determined earlier, outlines a molecular framework for the pre-mRNA splicing reaction.

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

“…The N-terminal SAP49 RRM engages a neighboring Cus1 subunit in the activated yeast spliceosome (Yan et al 2016) via a tryptophan-mediated interaction that is qualitatively similar to a UHM. In addition to the SAP49-Cus1 complex (Yan et al 2016), new examples of tryptophan-mediated interactions with RRM-like domains have emerged outside the UHM family, including eIF3b-eIF3j in the translation initiation complex (ElAntak et al 2010), Snu17p-Bud13p in the RES complex (Tripsianes et al 2014), mRNA export complexes of viral proteins with AlyREF (Tunnicliffe et al 2011;Tunnicliffe et al 2014), and an intramolecular interface of CPEB1 (Afroz et al 2014). The exact conformations of the tryptophan-containing ligands differ among these atypical RRMs, which also lack the RXF motif, acidic α1, and C-terminal α-helix of UHMs.…”

“…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). Subsequent structures, including the complex of alternative splicing factors PTB with Raver1 (Rideau et al 2006;Joshi et al 2011), intramolecular CPEB1 contacts for polyadenylation (Afroz et al 2014), and the Snu17p RRM bound to Bud13p in the pre-mRNA retention and splicing complex (RES) (Tripsianes et al 2014;Yan et al 2016), establish that separate α-helical and RNP surfaces of the RRMs often bind protein and RNA partners simultaneously.…”

“…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

“…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.…”

“…State of Brr2 in different spliceosomal contexts and interaction with other spliceosomal helicases. Views of (A) a yeast spliceosomal B act complex (PDB ID 5GM6) 100 and (B) a yeast spliceosomal C complex (PDB ID 5LJ5). 102 Brr2 and proteins interacting with the Brr2 NTR, and the Prp2 and Prp16 helicases are highlighted by colors.…”