Biochemical and Genetic Evidence Supports Fyv6 as a Second-Step Splicing Factor in Saccharomyces cerevisiae

Abstract

Precursor mRNA (pre-mRNA) splicing is an essential process for gene expression in eukaryotes catalyzed by the spliceosome in two transesterification steps. The spliceosome is a large, highly dynamic complex composed of 5 small nuclear RNAs and dozens of proteins, some of which are needed throughout the splicing reaction while others only act during specific stages. The human protein FAM192A was recently proposed to be a splicing factor that functions during the second transesterification step, exon ligation, based on analysis of cryo-electron microscopy (cryo-EM) density. It was also proposed that Fyv6 might be the functional S. cerevisiae homolog of FAM192A; however, no biochemical or genetic data has been reported to support this hypothesis. Herein, we show that Fyv6 is a splicing factor and acts during exon ligation. Deletion of FYV6 results in genetic interactions with the essential splicing factors Prp8, Prp16, and Prp22; decreases splicing in vivo of reporter genes harboring intron substitutions that limit the rate of exon ligation; and changes 3’ splice site (SS) selection. Together, these data suggest that Fyv6 is a component of the spliceosome and the potential functional and structural homolog of human FAM192A.

Figure 1.. Sequence Alignment of Fyv6 with FAM192 and Unassigned EM Density in Yeast Spliceosome Structures.

A) Sequence based alignment of S. cerevisiae Fyv6 and human FAM192A using EMBOSS Needle (Needleman and Wunsch, 1970). B), C) Superposition of the atomic models for the spliceosome C* (panel B, 5MQ0) and P (panel C, 6BK8) complexes with the unassigned EM density shown in blue spacefill. Images were prepared using ChimeraX (Pettersen et al., 2021). D) Impact of fyv6Δ on yeast growth at various temperatures. Plates were imaged on the days shown.

Figure 2.. Genetic interactions between Fyv6 and Prp8, Prp16, or Prp22.

A) Diagram of how Prp8, Prp16, and Prp22 alleles impact the 1^st and 2^nd steps of splicing. B) Alleles of Prp8 were combined with fyv6Δ in Prp8 shuffle strains and grown on -Trp or -Trp+5-FOA plates. Yeast growth was imaged after 3 days at 30°C. C) Prp8^P986T/fyv6Δ strains were tested for suppression or exacerbation of temperature-dependent growth phenotypes. D, E) Alleles of Prp16 and Prp22 were combined with fyv6Δ and tested for suppression or exacerbation of temperature-dependent growth phenotypes. For panels C-E, yeast were plated on YPD and imaged after 3 (30°C), 4 (23°C and 37°C), or 10 (16°C) days.

Figure 3.. Impact of FYV6 deletion on yeast copper tolerance using the ACT1-CUP1 assay.

A) Schematic of the WT ACT1-CUP1 reporter along with intronic substitutions. B) Images of representative yeast growth on copper containing media shown after 48 (WT) or 72 h (fyv6Δ) for strains containing the indicated ACT1-CUP1 reporters. C) Maximum copper tolerances observed for each strain for N=3 replicates (dots). Bars represent the average values.

Figure 4.. Loss of Fyv6 Changes 3’ SS Selection in Yeast.

A) Schematic of the 3’ SS competition reporter (3’ SS comp) showing relative locations of the proximal and distal sites. B) Images of representative yeast growth on copper containing media shown after 48 (WT) or 72 h (fyv6Δ) for strains containing the 3’ SS comp ACT1_CUP1 reporter. C) Maximum copper tolerances observed for each strain for N=3 replicates (dots). Bars represent the average values. D) Representative primer extension analysis of mRNAs generated by yeast using the distal (mRNA_D) or proximal (mRNA_P) 3’ SS in the presence (WT) or absence of Fyv6 (fyv6Δ). U6 snRNA was analyzed as a loading control. E) Quantification of the primer extension results from N=3 replicates (dots) expressed as a ratio of mRNA_P/mRNA_D . Bars represent the average of the replicates ±SD. Means between two experimental groups were compared with an unpaired student’s t-test of equal variance (p = 0.01014).