Little is known about the spliceosome's structure before its extensive remodeling into a catalytically active complex. Here, we report a 3D cryo-EM structure of a pre-catalytic human spliceosomal B complex. The U2 snRNP-containing head domain is connected to the B complex main body via three main bridges. U4/U6.U5 tri-snRNP proteins, which are located in the main body, undergo significant rearrangements during tri-snRNP integration into the B complex. These include formation of a partially closed Prp8 conformation that creates, together with Dim1, a 5' splice site (ss) binding pocket, displacement of Sad1, and rearrangement of Brr2 such that it contacts its U4/U6 substrate and is poised for the subsequent spliceosome activation step. The molecular organization of several B-specific proteins suggests that they are involved in negatively regulating Brr2, positioning the U6/5'ss helix, and stabilizing the B complex structure. Our results indicate significant differences between the early activation phase of human and yeast spliceosomes.

More than 200 proteins associate with human spliceosomes, but little is known about their relative abundances in a given spliceosomal complex. Here we describe a novel two-dimensional (2D) electrophoresis method that allows separation of high-molecular-mass proteins without in-gel precipitation and thus without loss of protein. Using this system coupled with mass spectrometry, we identified 171 proteins altogether on 2D maps of stage-specific spliceosomal complexes. By staining with a fluorescent dye with a wide linear intensity range, we could quantitate and categorize proteins as present in high, moderate, or low abundance. Affinitypurified human B, B act , and C complexes contained 69, 63, and 72 highly/moderately abundant proteins, respectively. The recruitment and release of spliceosomal proteins were followed based on their abundances in A, B, B act , and C spliceosomal complexes. Staining with a phospho-specific dye revealed that approximately one-third of the proteins detected in human spliceosomal complexes by 2D gel analyses are phosphorylated. The 2D gel electrophoresis system described here allows for the first time an objective view of the relative abundances of proteins present in a particular spliceosomal complex and also sheds additional light on the spliceosome's compositional dynamics and the phosphorylation status of spliceosomal proteins at specific stages of splicing.The spliceosome is a highly complex and dynamic megadalton RNP machine. It is comprised of the five snRNPs U1, U2, U4, U5, and U6 and a large number of non-snRNP protein factors (reviewed in reference 42). Spliceosomes assemble de novo in a stepwise manner on each new intron to be spliced and thus pass through a series of distinct complexes (42). Initially, the U1 snRNP binds the pre-mRNA, forming the E complex, and after stable U2 snRNP interaction, the A complex is generated. Subsequently, the U4/U6 and U5 snRNPs associate, as part of the U4/U6.U5 tri-snRNP, and the precatalytic B complex is formed. Through a series of compositional and structural rearrangements, the B complex is activated, first yielding the B act complex. After the action of the DEXH box protein Prp2, the B* complex is formed, which catalyzes step 1 of splicing. This involves cleavage at the 5Ј splice site (ss) of the pre-mRNA and the ligation of the 5Ј end of the intron to the so-called branch site to form a lariat-like structure. After the first step, the spliceosomal C complex is formed, and it catalyzes the step 2 of splicing, during which the intron is excised and the exons are ligated together to form mRNA.Mass spectrometry (MS) analyses have shown that more than 200 proteins copurify with mixtures of human spliceosomal complexes (31, 47). Individual spliceosomal complexes contain many fewer proteins (e.g., ϳ125 for B, B act , and C complexes) and differ from each other considerably in composition (3,6,7,10). However, the relative abundances of all of the proteins present within a given spliceosomal complex are presently not clear. Spliceosomes contain...

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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 U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) is a major spliceosome building block. We obtained a three-dimensional structure of the 1.8-megadalton human tri-snRNP at a resolution of 7 angstroms using single-particle cryo-electron microscopy (cryo-EM). We fit all known high-resolution structures of tri-snRNP components into the EM density map and validated them by protein cross-linking. Our model reveals how the spatial organization of Brr2 RNA helicase prevents premature U4/U6 RNA unwinding in isolated human tri-snRNPs and how the ubiquitin C-terminal hydrolase-like protein Sad1 likely tethers the helicase Brr2 to its preactivation position. Comparison of our model with cryo-EM three-dimensional structures of the Saccharomyces cerevisiae tri-snRNP and Schizosaccharomyces pombe spliceosome indicates that Brr2 undergoes a marked conformational change during spliceosome activation, and that the scaffolding protein Prp8 is also rearranged to accommodate the spliceosome's catalytic RNA network.