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B-act complex; Electron microscopy; Immunolabeling; Protein localization; Spliceosome
Abstract:
We have elucidated the spatial arrangement of proteins and snRNP subunits within the purified spliceosomal Bact complex from Saccharomyces cerevisiae, using negative-stain immunoelectron microscopy. The Bact spliceosome exhibits a mushroom-like shape with a main body connected to a foot and a steep and a shallow slope. The U5 core components, including proteins Snu114 and Prp8, are located in the main body and foot, while Brr2 is on the shallow slope. U2 snRNP components and the RNA helicase Prp2 were predominantly located in the upper regions of both slopes. While several proteins of the “nineteen complex” are located on the steep slope, Prp19, Cef1, and the U6 snRNA-binding protein Cwc2 are on the main body. Our results also indicate that the catalytic core RNP of the spliceosome resides in its main body. We thus assign distinct domains of the Bact complex to its snRNP and protein components, and we provide first structural insights into the remodeling events at the spliceosome during its transformation from the B to the Bact complex.
