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 five 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 Saccharomyces cerevisiae functional and structural 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 and decreases splicing in vivo of reporter genes harboring intron substitutions that limit the rate of exon ligation. When splicing is assayed in vitro, whole-cell extracts lacking Fyv6 accumulate first-step products and exhibit a defect in exon ligation. Moreover, loss of Fyv6 causes a change in 3' splice site (SS) selection in both a reporter gene and the endogenous SUS1 transcript in vivo. Together, these data suggest that Fyv6 is a component of the yeast spliceosome that influences 3' SS usage and the potential homolog of human FAM192A.

Keywords: FAM192A; Fyv6; RNA splicing; spliceosome; yeast.

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. The three putative Fyv6 α helices identified by Zhan and coworkers are annotated next to the corresponding EM density in panel B. 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 first and second 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 d 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.

Accumulation of splicing intermediates occurs in the absence of Fyv6 in in vitro splicing assays. (A) Products of the first and second steps after incubation of a radioactively labeled RP51A pre-mRNA (lane 1) in WCE from cup1Δ strains (yAAH0434 and yAAH3353, Supplemental Table 1) for 45 min (lanes 2,3). Quantitation of (B) lariat intermediates or (C) Second-step splicing efficiency from three replicates. Statistical significance was determined by unpaired Welch's two-tailed t-test (P = 0.006415 and 0.01788 for fraction of lariat intermediate and second-step efficiency, respectively). The fraction of the lariat intermediate represents the fraction of that species relative to the substrate and all splicing products while the second-step efficiency represents the fraction of mRNA relative to the sum of first- and second-step products (lariat intermediate plus mRNA). Details for calculations can be found in the Materials and Methods.

FIGURE 5.

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 the two experimental groups were compared with an unpaired Welch's two-tailed t-test (P = 0.04262).

FIGURE 6.

Loss of Fyv6 results in the use of an alternative 3′ SS in SUS1. (A) Representative gel image of RT-PCR of SUS1 in strains with (WT) and without (fyv6Δ) Fyv6 in a upf1Δ background. (+RT reactions contain reverse transcriptase; −RT control reactions do not contain reverse transcriptase). (B) Quantification of band intensities of each isoform as a fraction of the total SUS1 product in a lane. The bars indicate the average of three experiments with standard deviation. (C) Portion of the Sanger sequencing chromatogram of the SUS1 splice variant identified as an RT-PCR product in the fyv6Δ strain. The bar above the nucleotides indicates those from intron 1 included in the splice variant due to the use of an alternative 3′ SS. (D) Diagram of the SUS1 gene structure with the BS adenosine and the two, alternative 3′ SS of intron 1 indicated. The numbering of the nucleotides begins at the first nucleotide of intron 1. The newly identified 3′ SS is at position 60.

Katherine Senn

Karli Lipinski