Protein Composition and Electron Microscopy Structure of Affinity-Purified Human Spliceosomal B Complexes Isolated under Physiological Conditions

Abstract

The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including ϳ50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 Å and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.Pre-mRNA splicing is catalyzed by a large RNP molecular machine, termed the spliceosome, which consists of the U1, U2, U4/U6, and U5 snRNPs and a multitude of non-snRNP proteins (reviewed in reference 48). The active site(s) responsible for the catalysis of pre-mRNA splicing is not preformed but, rather, is created anew during the highly dynamic process of spliceosome assembly. The latter is an ordered process during which several intermediates, termed E, A, B, and B*, can be detected in vitro (reviewed in reference 48). Assembly is initiated by the ATP-independent interaction of the U1 snRNP with the conserved 5Ј splice site of the pre-mRNA, forming the E complex. At this stage, the U2 snRNP is loosely associated with the pre-mRNA (11). In a subsequent step requiring ATP, the U2 snRNP stably interacts with the pre-mRNA's branch site, leading to formation of the A complex (also called the prespliceosome). Spliceosome assembly culminates with the formation of the spliceosomal B complex, during which the preformed U4/U6.U5 tri-snRNP particle interacts with the A complex. The B complex thus contains a full set of U snRNAs in a precatalytic state. It subsequently undergoes major rearrangements, including destabilization or loss of the U1 and U4 snRNPs, leading to catalytic activation and the formation of the so-called activated spliceosome (B* complex).Splicing catalysis subsequently proceeds by a two-step mechanism (reviewed in reference 29). During the first step the 5Ј splice site is cleaved, and the 5Ј end of the intron is covalen...

Mutations in the PQBP1 gene prevent its interaction with the spliceosomal protein U5–15kD

“…9), whereas the C-terminal YxxPxxVL motif binds to the spliceosomal protein U5-15kD [6][7][8] . PQBP1, WBP11 and U5-15kD are all components of the precatalytic B complex 4,5 , and thus PQBP1 probably binds to both WBP11 and U5-15kD in the B complex.…”

“…PQBP1 is a component of the Prp19 complex, whereas U5-15kD and U5-52K are both U5 snRNP components 4,5,35 . Hoskins et al 36 have shown that the PrP19 complex joins the spliceosome after the U4/U6.U5 tri-snRNP recruitment.…”

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“…The recruitment of U2 snRNP leads then to formation of the A complex, which is also called the pre-spliceosome [ 27 , 28 ]. Upon integration of the U4/U6.U5 tri-snRNP and additional splicing factors, called B specifi c proteins, the pre-catalytic spliceosome (B complex) is developed [ 29 ]. Structural RNA and protein rearrangements within the B complex induced by RNA helicases Brr2 (U5-200K) and Snu114 (U5-116K) cause the dissociation of U1 and U4 snRNPs.…”

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“…Components of the splicing complex include several snRNPs, numerous serine/arginine-rich (SR) proteins and other non-snRNP proteins. [60][61][62] The spliceosome is highly dynamic during splicing progression, guided by consensus sequences in the premRNA to form sequential complexes. 63 As in other eukaryotes, alternative splicing is an essential mechanism for modulating gene expression and increasing transcriptome plasticity and proteome diversity in plants.…”