A pseudouridine residue in the spliceosome core is part of the filamentous growth program in yeast

Abstract

Although pseudouridine nucleobases are abundant in tRNAs, rRNAs, and small nuclear RNAs (snRNAs), they are not known to have physiologic roles in cell differentiation. We have identified a pseudouridine residue (Ψ28) on spliceosomal U6 snRNA that is induced during filamentous growth of Saccharomyces cerevisiae. Pus1p catalyzes this modification and is upregulated during filamentation. Several U6 snRNA mutants are strongly pseudouridylated at Ψ28. Remarkably, these U6 mutants activate pseudohyphal growth, dependent upon Pus1p, arguing that U6-Ψ28 per se can initiate at least part of the filamentous growth program. We confirmed this by using a designer small nucleolar RNA (snoRNA) targeting U6-U28 pseudouridylation. Conversely, mutants that block U6-U28 pseudouridylation inhibit pseudohyphal growth. U6-U28 pseudouridylation changes the splicing efficiency of suboptimal introns; thus, Pus1p-dependent pseudouridylation of U6 snRNA contributes to the filamentation growth program.

Figure 1. A novel pseudouridine residue on U6 snRNA is induced under conditions akin to filamentation response

A) CMC-based primer extension on S. cerevisiae (strain 46ΔCUP) U6 snRNA. Even-numbered lanes=CMC treated; odd-numbered lanes=untreated. RNA was collected from solid and liquid media. The position of U6-Ψ28 is indicated. 46ΔCUP can be maximally grown up to OD ~1.5; yeast were grown in synthetic complete (SC)+glucose media for 2.5–3 days to ensure nutrient deprivation. (B) Schematic representation of the first-step catalytic center showing S. cerevisiae U2 snRNA, U6 snRNA and the pre-mRNA. Canonical U2 snRNA pseudouridine residues (35, 42, 44) and U6-Ψ28 are indicated. (C) Microscopy of 46ΔCUP yeast cells over-expressing WHI2. ‘Control’ and ‘WHI2 O/E’ respectively denote an empty plasmid and a galactose-inducible URA3-marked WHI2-plasmid. (D) WHI2 over-expression induces U6-Ψ28 (lanes 3 and 4). (E) Microscopic observation of yeast morphological changes upon butanol induction. Cells were grown in YPD or YPD+1% vol/vol butanol (+glucose). (F) CMC-based primer extension on U6 snRNA. Yeast grown in YPD+glucose were either treated with 1% vol/vol butanol for 10 hours (lanes 3, 4) or untreated (lanes 1, 2). U6-Ψ28 is indicated with black arrows. See also Figures S1 & S2.

Figure 2. Pus1p catalyzes U6-Ψ28 and is induced by filamentous growth

A) Pseudouridylation on S. cerevisiae U6 snRNA, comparing wild-type yeast (strain BY4741) with deletions of non-essential pseudouridine synthase genes and the catalytically inactive Cbf5p (cbf5-D95A). Even-numbered lanes=CMC treated; odd-numbered lanes=untreated. U6-Ψ28 and two nucleotides 3' (U27 and A26) are marked. Cells grown in solid SC+glucose media. (B) ‘Control’ and ‘PUS1 O/E’ respectively denote an empty plasmid and a galactose-inducible PUS1-plasmid. Yeast (46ΔCUP) grown either in (SC-Ura) liquid media to OD 0.7 (lanes 1–4), or in (SC-Ura) solid media (lanes 5–8) were subjected to pseudouridylation assays. (C) Quantitative RT-PCR data of PUS1 levels (normalized to ACT1) from yeast cells grown under standard (liquid) or filamentation-like (solid) media. Bars represent the mean and error bars the Standard Error of the Mean. A cutoff of greater than 1.5 fold indicates significant increase (D) The effect of WHI2 over-expression (in wild-type and pus1Δ cells) on U6-Ψ28 induction tested with pseudouridylation assays in 46ΔCUP. ‘Control’ and ‘WHI2 O/E’, respectively, refer to an empty plasmid and a galactose-inducible WHI2-plasmid in liquid (SCUra) media. Even-numbered lanes=CMC treated; odd-numbered lanes=untreated. U6-Ψ28 is marked. (E) Quantitative RT-PCR of the levels of all pseudouridine synthases upon WHI2 over-expression. The fold changes in expression are normalized to endogenous actin. See also Figure S3 & Table S2.

Figure 3. Mutations in U6 snRNA result in robust pseudouridylation at U6-Ψ28

(A) CMC-based U6 snRNA primer extension assays with the primary modification site, U6-U28, mutated to U6-U28C (lanes 3, 4). Upon mutating U6-U27 to U6-U27G (lanes 5, 6) or U6-U27A (lanes 7, 8), U28 was strongly pseudouridylated. Other U6 snRNA mutations (U36C and G50U) resulting in robust U6-Ψ28 induction are shown (lanes 10, 12). RNA collected from solid media (SC+glucose) was CMC-treated or untreated as marked. (B) Microscopic observation of yeast cell morphologies in liquid SC+glucose media harboring different U6 snRNA mutations. Indicated are WT-U6 snRNA, U6-U28C that abolishes U6-Ψ28, and U6 snRNA mutations that result in robust U6-Ψ28 (U6-U27G, -U27A, -U36C and -G50U). In the absence of Pus1p, and hence U6-Ψ28, neither U6-U27G nor -U27A resulted in an elongated morphology. (C) Microscopic observation of yeast cells over-expressing control plasmid or WHI2 in combination with WT-U6 (Ci,ii), U6-U28C (Ciii), U6-U27A (Cv), U6-U27G (Cvi) and in pus1Δ (Civ). (D) CMC-based primer extension assays on U6 snRNA in liquid -His+glucose media using control snoRNA (snR81-WT) and a U6-U28 specific snoRNA-guide (‘snR-Ψ28’) in WT (lanes 1–4) and pus1Δ (lanes 5–8) yeast strains. (E) Yeast cell phenotypes harboring snR81-WT and snR-Ψ28 guides observed by microscopy. Panels i–ii and iii–iv show morphologies in WT and pus1Δ strains, respectively. All experiments were done in 46ΔCUP. See also Figure S4.

Figure 4. Model for WHI2 activation of the filamentous growth program via PUS1-mediated modification of U6 snRNA

The phosphorylated form of a filamentous-growth program-specific transcription factor (TF) is inactive. The Whi2p-Psr1p-phosphatase complex dephosphorylates and thereby activates the TF, which binds to STRE-like (Stress Response Elements) within the PUS1 promoter. ‘TSS’ denotes the PUS1 transcription start site. The thus up-regulated pseudouridine synthase PUS1 catalyzes the filamentation-specific Ψ28 (red) on U6 snRNA (green). This modification results in altered spliceosome activity and altered splicing of target genes, activating the filamentous growth program as a downstream cascade.