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Author(s): Sebastian M. Fica (corresponding author) [1]; Chris Oubridge [1]; Wojciech P. Galej [1]; Max E. Wilkinson [1]; Xiao-Chen Bai [1]; Andrew J. Newman [1]; Kiyoshi Nagai (corresponding author) [1] [...]

Precursor mRNA (pre-mRNA) splicing proceeds by two consecutive transesterification reactions via a lariatintron intermediate. Here we present the 3.8 cryo-electron microscopy structure of the spliceosome immediately after lariat formation. The 5-splice site is cleaved but remains close to the catalytic Mg[sup.2+] site in the U2/U6 small nuclear RNA (snRNA) triplex, and the 5-phosphate of the intron nucleotide G(+1) is linked to the branch adenosine 2OH. The 5-exon is held between the Prp8 amino-terminal and linker domains, and base-pairs with U5 snRNA loop 1. Non-WatsonCrick interactions between the branch helix and 5-splice site dock the branch adenosine into the active site, while intron nucleotides +3 to +6 base-pair with the U6 snRNA ACAGAGA sequence. Isy1 and the step-one factors Yju2 and Cwc25 stabilize docking of the branch helix. The intron downstream of the branch site emerges between the Prp8 reverse transcriptase and linker domains and extends towards the Prp16 helicase, suggesting a plausible mechanism of remodelling before exon ligation., Author(s): Wojciech P. Galej (corresponding author) [1]; Max E. Wilkinson [1]; Sebastian M. Fica [1]; Chris Oubridge [1]; Andrew J. Newman [1]; Kiyoshi Nagai (corresponding author) [1] The spliceosome is [...]

Pre-mRNA splicing is catalyzed in two steps: 5' splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (1 st and 2 nd step factors). We recently identified Fyv6 (FAM192A in humans) as a 2 nd step factor in S. cerevisiae ; however, we did not determine how widespread Fyv6's impact is on the transcriptome. To answer this question, we have used RNA-seq to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3' SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-EM structure of a yeast product complex spliceosome containing Fyv6 at 2.3 Å. The structure reveals that Fyv6 is the only 2 nd step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the 1 st step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv6Δ suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of consensus, BP distal 3' SS.