High-resolution structure determination of human spliceosome complexes by cryo-EM

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The eukaryotic splicing of precursors to mRNA is facilitated by a highly dynamic, multi-megadalton macromolecular machine termed the spliceosome. The underlying chemical reaction features the excision of an intron, which is followed by the re-ligation of two exons with single nucleotide precision. The spliceosome therefore actively participates in the flow of genetic information. How catalysis is mechanistically achieved and why the dynamic nature of the molecular machine is essential for its function was poorly understood in the past. This work presents the first high-resolution structures of human spliceosomes in the pre-catalytic and the catalytically activated phase of assembly. Elucidated by cryo-EM, the molecular architectures of the B and C* complex reveal significant insights into the mechanism of catalytic activation and general activity. The pre-catalytic B complex thereby shows a distinctive spatial separation of the reactive pre mRNA BS A and 5’SS elements during spliceosomal assembly. Mechanistically, the structure sheds light on the tremendous restructuring events that take place upon the integration of the tri-snRNP into the pre-spliceosomal A complex. B specific proteins like PRP38, SNU23, MFAP1 or SMU1 specifically stabilize the B complex configuration and prevent premature activation by contacting the important U6 snRNA ACAGA box helix and RNA helicase BRR2. Intriguingly, a detailed comparison between the yeast and human pre-catalytic spliceosome structures unexpectedly reveals a potentially different catalytic activation pathway in higher and lower eukaryotes. The molecular architecture of the C* complex highlights a profound conservation of the catalytic core of the assembly between species once the spliceosome is catalytically activated. Unexpected differences nonetheless exist between the structural organization of yeast and human spliceosomes: for example, the ACAGA box and BSH helices are characteristically extended in the human spliceosome, which potentially compensates for the degenerate appearance of the corresponding signal sequences in the pre-mRNA of higher eukaryotes. In addition, metazoan-specific proteins such as RBM22 or IBP160 (Aquarius) can be localized and likely assist in modulating the splicing activity by interacting with the pre-mRNA and proximal protein factors. Large-scale remodelling events of the remaining U2 snRNP components are furthermore found to convey their functionally essential dynamic trajectories onto the much smaller entities at the catalytic core of the C* complex. For example, the BSH that spatially occupies the catalytic centre in the post-step 1 C complex is repositioned accordingly in the pre-step 2 C* complex. Besides clarifying the molecular architecture of the spliceosome itself, the results presented in this work contribute towards a better understanding of the involved assembly pathways and the mechanism of catalysis. The substantial differences between yeast and human spliceosomes during catalytic activation and in the handling of pre-mRNA stabilization within catalytically activated spliceosomes may furthermore add to the evolutionary understanding of RNA splicing.