Widespread splicing changes due to loss of Fyv6.

A) Pre-mRNA splicing reaction steps and products. B) Growth of WT (cup1Δ), fyv6Δ, upf1ti and fyv6tiupf1ti yeast at 16, 23, 30, and 37°C. Images were taken on the indicated days. White lines indicate where images were cropped, as all dilutions for all strains were on the same plate, but not necessarily directly adjacent to one another. C) Number of alternative splicing events observed in a fyv6tiupf1ti strain relative to a strain containing FYV6 after a temperature shift to 16, 30, and 37°C. D) Venn diagram of shared alternative 3′ SS splicing events at 16 (blue), 30 (green), and 37°C (red). E) Box plots of the log10 of the ratios of fyv6ti upf1ti to WT (upf1ti) Fraction of Annotated Splicing (FAnS) values for RPGs (red) and non-RPGs (black) for 3′ SS splicing events. FAnS values represent the ratio of the alternative splicing event (here, selection of an alternate 3′ SS) to the annotated, canonical splice isoform.

Loss of Fyv6 activates BP proximal, non-consensus 3ʹ SS.

A) Plot of the log10 of Fraction of Annotated Splicing (FAnS) values for differences between alternative 3ʹ SS usage in fyv6Δ upf1Δ and upf1Δ strains. High FAnS values indicate increased usage of the alternate 3ʹ SS relative to the annotated site. Points are colored based on whether they are upstream (pink) or downstream (blue) to the canonical 3ʹ SS. B) Violin plots of the ratios of fyv6Δupf1Δ to the upf1Δ FAnS values based on the distances between the alternative and canonical 3ʹ SS (Δ3ʹ SS = 3ʹ SSAlt. - 3ʹ SScanonical). FAnS ratios >0 are indicative of the site being upregulated in fyv6Δ. C) Violin plots of the ratios of fyv6Δupf1Δ to the upf1Δ FAnS values based on the distances between the alternative 3ʹ SS and the annotated BP. FAnS ratios >0 are indicative of the site being upregulated in fyv6Δ. D) Diagram of ACT1-CUP1 reporter showing BP-3ʹ SS distances. E) Representative primer extension analysis of RNA products generated from splicing of the ACT1-CUP1 reporter in the presence (WT) or absence of Fyv6 (fyv6Δ). Bands for fully spliced mRNA and lariat intermediate are indicated. U6 snRNA was detected as a loading control. The * indicates an unknown product present in every lane. F) Quantification of the primer extension results from N=3 replicates represented by the ratio of band intensities for mRNA/(mRNA+lariat intermediate). Bars represent the average ratio of the replicates ±SD. Means between WT and fyv6Δ groups for each reporter were compared with an unpaired Welch’s two-tailed t-test. Significance is indicated: n.s. no significance; ** p<0.01; *** p<0.001. G) Sequence logos of alternative 3ʹ SS with FAnS > 0 sorted by either upstream or downstream of the canonical 3ʹ SS compared against the canonical 3ʹ SS for genes with FAnS > 0. H) The log10 of the FAnS ratio for alternative 3ʹ SS sorted by the sequences of the 3ʹ SS.

Cryo-EM structure of the yeast P complex spliceosome at 2.3 Å resolution.

A) A composite density map for P complex showing focused refinements of the Prp22, NTC, U2 snRNP, U5 Sm ring, and Cwc22 N-terminal domain regions. B) Overall model for the P complex spliceosome. C) Cryo-EM density for state I of P complex (above, low-pass filtered) and for the active site and 3ʹ SS (below, sharpened). D,E) As for C) but for states II (D) and III (E).

Fyv6 and its interactors in the P complex cryo-EM structure.

A) Cryo-EM density segmented around Fyv6. B) Structure of Prp22. C) Structure of the NTC within P complex. D) IP6 site and cryo-EM density in NTC. E) Interaction of the hook domain of Fyv6 with the Prp22 RecA2 domain. F) Interaction of Fyv6 with Slu7 and the region of the intron between the BP and 3ʹ SS. G) Interaction of Fyv6 with Syf1 and an analogous view of the interaction of Syf1 with Yju2 in C complex (Wilkinson et al., 2021).

Structure-based analysis of Fyv6 domain function.

A) Diagram of Fyv6 protein structure, protein interactors, and protein truncations. NLS= nuclear localization signal B) Spot dilution assay of strains with Fyv6 truncations on –Trp DO plates at different temperatures. Plates were imaged after the number of days indicated. C) Representative gel image of SUS1 RT-PCR in strains with an empty vector or expressing the indicated Fyv6 variant. (+RT reactions contain reverse transcriptase; -RT control reactions do not contain reverse transcriptase.) D) Diagram of Syf1 protein structure and truncations. E) Spot dilution assay of strains with Syf1 truncations on –Trp DO plates at different temperatures. Plates were imaged after the number of days indicated. F) Representative gel image of SUS1 RT-PCR in strains with an empty vector or expressing the indicated Syf1 variant. G) Temperature growth assay of strains with Syf1 truncations in a fyv6Δ background on YPD plates. Plates were imaged after the number of days indicated. H) Representative gel image of RT-PCR of SUS1 in strains with plasmids containing WT Syf1 or a Syf1 truncation in a fyv6Δ background.

Identification of novel suppressors of fyv6ti temperature sensitivity.

A) Workflow for the suppressor screen. B) P complex spliceosome structure with fyv6ti suppressor mutations labelled. (See Supplemental Table S5) C) Close-up view of Cef1 and Prp8 suppressor mutations in P complex. D) Spot dilution temperature growth assay with Cef1 mutants on YPD plates. Plates were imaged after the number of days indicated. E) Close-up view of Prp8 mutations in P complex. F) Spot dilution temperature growth assay with Prp8 mutants on YPD plates. Plates were imaged after the number of days indicated.

Genetic interactions between Prp22 and Fyv6.

A) Location of the Prp22 I1133R fyv6Δ suppressor in the P complex structure. B) Spot dilution growth assay with Prp22 mutants in WT or fyv6Δ backgrounds grown on –Trp DO or –Trp+5-FOA plates. Plates were imaged after 3 days at 30°C. C) Spot dilution growth assay with strains containing Prp22 mutants on YPD plates at different temperatures. Plates were imaged after the number of days indicated. D) Representative gel of SUS1 RT-PCR products in strains with Prp22 mutants in the presence or absence of Fyv6. E) Quantitation of band intensities for each SUS1 splice isoform as a fraction of total product in the lane. Bars indicate average ± SD of N=3 replicates. F) ACT1-CUP1 reporter construct with the location of the 3ʹ SS substitution noted. G) Copper tolerance for each ACT1-CUP1 reporter with each Prp22 allele in strains with (▪) or without (○) Fyv6 present. Shown is a representative, single replicate of N=3.

Model for the role of Fyv6 during the 2nd step.

Fyv6 interacts with Syf1 and Prp22 and promotes usage of BP distal 3ʹ SS. When Fyv6 is absent, BP proximal 3ʹ SS are favored. It is possible that lack of Fyv6 also results in the presence of Yju2 during exon ligation and relaxing of the requirement for Prp22 at this step.