No abstract

We have determined the crystal structure of a spliceosomal RNP complex comprising the 15.5kD protein of the human U4/U6.U5 tri-snRNP and the 5' stem-loop of U4 snRNA. The protein interacts almost exclusively with a purine-rich (5+2) internal loop within the 5' stem-loop, giving an unusual RNA fold characterized by two tandem sheared G-A base pairs, a high degree of purine stacking, and the accommodation of a single RNA base, rotated out of the RNA chain, in a pocket of the protein. Apart from yielding the structure of an important entity in the pre-mRNA splicing apparatus, this work also implies a model for the complex of the 15.5kD protein with box C/D snoRNAs. It additionally suggests a general recognition principle in a novel family of RNA binding proteins.

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...

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.