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. Author manuscript; available in PMC: 2015 Aug 21.
Published in final edited form as: Cell Rep. 2014 Aug 7;8(4):966–973. doi: 10.1016/j.celrep.2014.07.004

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

Anindita Basak 1, Charles C Query 1,2
PMCID: PMC4425566  NIHMSID: NIHMS613783  PMID: 25127136

SUMMARY

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

Keywords: pseudouridine, filamentation, snRNA, RNA modification

INTRODUCTION

Pre-mRNA splicing generates mature mRNAs from nascent transcripts and is catalyzed by the spliceosome, a dynamic macromolecular machine consisting of snRNAs and auxiliary proteins. Five snRNPs (U1, U2, U4, U5 and U6) constitute the ribonucleoprotein subunits of the spliceosome, the assembly of which requires extensive compositional and conformational rearrangements (reviewed in Hoskins and Moore, 2012). U1 snRNP binds the 5' splice site, and U2 snRNP binds the branch site to form a pre-spliceosome (reviewed in Wahl et al., 2009). U4/U6.U5 tri-snRNP then joins; after conformational rearrangements, U1 and U4 snRNPs are released and the remaining U2/U6.U5 snRNAs form the core of the catalytic spliceosome, with phosphates on U6 snRNA providing coordination of catalytic magnesium metal ions (Fica et al., 2013).

Spliceosomal snRNAs are extensively modified, most so in vertebrates and predominantly subject to pseudouridylation and 2'-O-methylation (Reddy and Busch, 1988). Of all modified nucleotides in RNA, pseudouridine (Ψ, the C5 glycoside isomer of uridine; Fig. S1A) is the most abundant and is clustered in functionally important regions (Ge and Yu, 2013). Although pseudouridines are proposed to rigidify local structures (Davis, 1995; Newby and Greenbaum, 2002) or alter protein–RNA interactions (Yu et al., 1998), their physiologic effects are mostly unknown.

Pseudouridylation is catalyzed by either H/ACA RNPs or protein-only pseudouridine synthases. This modification was thought constitutive until recently, when additional pseudouridines on U2 snRNA were found upon heat shock and nutrient-starvation (Wu et al., 2011).

In response to environmental stresses, many yeast species shift from yeast form to filamentous or pseudo-hyphal form (Cullen and Sprague, 2012 and references therein), a morphogenic switch characterized by cell elongation, unipolar budding, altered cell cycle, and enhanced cell-cell adhesion, resulting in filamentous extensions (hyphae) into the growth media. Four signaling pathways, Ras2/cAMP-PKA, SNF1, TOR, and MAPK, are implicated in filamentation control (reviewed in Cullen and Sprague, 2012).

In a prior Synthetic Genetic Array (SGA) screen for factors that altered splicing efficiency, we identified a pseudouridine synthase (unpublished); in searching for potential substrates, we found an unrelated, novel pseudouridine on U6 snRNA. U6-Ψ28 is induced upon activation of filamentation but not by other standard stress conditions. Likewise, the pseudouridylase responsible, Pus1p, but not other pseudouridylases, is up-regulated upon filamentation induction. U6 snRNA mutations as well as designer snoRNA that modifies Ψ28 activate pseudo-hyphal growth, arguing that Ψ28 is a potent component of the filamentation program. Lastly, we observed changes in splicing efficiency of suboptimal introns upon induction of filamentation, dependent upon Pus1p. Here, we report the first pseudouridine modification on yeast U6 snRNA, the interplay between three seemingly discrete phenomena – pseudouridylation, pseudo-hyphal growth, and splicing – and physiologic effects of a single pseudouridine modification.

RESULTS

A novel pseudouridine residue on U6 snRNA

We examined all S. cerevisiae spliceosomal snRNAs for previously undetected pseudouridine modifications, using CMC modification and primer extension in which pseudouridines retain a chemical adduct that blocks elongation by reverse transcriptase (Bakin and Ofengand, 1993). When yeast were grown under conditions typical for SGA analysis (solid media), we detected a non-canonical pseudouridine on U6 snRNA (Fig. 1A & S1B, lane 2) at position 28 between the U6 5' stem and U2/U6 helix III (Fig. 1B). This site was not reported under previously examined stress conditions (heat shock or nutrient deprivation) that induce pseudouridines in U2 snRNA (Wu et al., 2011). Consistent with this, we did not detect U6-Ψ28 in log-phase cultures or in cultures exhausted of nutrition (OD 1.5 for our strain in SC) (Fig. 1A, lanes 4 and 6). Thus, we detect a novel, condition-dependent, pseudouridine on U6 snRNA.

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

Figure 1

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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.

U6-Ψ28 is induced upon filamentation

In consideration of environmental conditions that solid media might mimic, we tested the filamentation response. Over-expression of WHI2, a gene required for cell cycle arrest upon nutrient deficiency, induces filamentation (Radcliffe et al., 1997) (Fig. 1C). RNA analysis from cells over-expressing WHI2 revealed U6-Ψ28 induction in conditions where U6-Ψ28 is otherwise not observed (liquid media) (Fig. 1D, cf. lanes 2 and 4).

Fusel alcohols, such as butanol, also induce a filamentation response (Lorenz et al., 2000). As a second test of a filamentation–pseudouridylation link, we treated 46ΔCUP (Fig. 1E&F, cf. lanes 2 and 4) and haploid and diploid strains of Σ1278b, customarily used for filamentation studies (Fig. S1C,D&E), with butanol; in all cases, we observed U6-Ψ28 induction in addition to characteristic elongated pseudo-hyphal cells.

General environmental stress response is studied by subjecting S. cerevisiae to various stresses: heat shock, DNA damage (MMS), oxidative damage (H2O2), heavy metal toxicity (CdCl2), and osmotic shock (sorbitol) (Gasch et al., 2000; Momose and Iwahashi, 2001). To test whether U6-Ψ28 induction is typical of stress responses, we analyzed RNA from yeast cells at 5, 30, and 120 min after subjection to each stress. U6-Ψ28 was not readily detected under any of these conditions as compared to solid media (Fig. S2A, cf. lanes 1–24 to 26; Fig. S2B, cf. lanes 1–16 to 18), whereas positive controls for each condition were substantially up-regulated (Fig. S2C). Thus, U6-Ψ28 is not generally induced as part of stress response pathways, and we conclude that U6 snRNA pseudouridylation is a specific part of the filamentation response.

Pus1p catalyzes U6 pseudouridylation and is up-regulated by filamentous growth

To identify the enzyme responsible for U6-Ψ28, we analyzed RNA from strains deleted for each non-essential pseudouridine synthase or that contained the cbf5-D95A catalytically-inactive mutant (Zebarjadian et al., 1999). Using conditions that induce U6-Ψ28 in wild-type cells, we did not detect pseudouridylation of U6-U28 in pus1Δ cells, whereas U6-Ψ28 was detected in all other pseudouridylase mutant strains (Fig. 2A, cf. lanes 3–4 to 1–2 and 5–18).

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

Figure 2

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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.

In addition, expression of PUS1, but not other pseudouridylases, increased ~10-fold under filamentation-like growth (Fig. 2C, cf. bars i and iii), suggesting that PUS1 expression correlates with the filamentation program.

To confirm that Pus1p catalyzes U6-Ψ28, we used conditions in which U6-U28 is not ordinarily modified and over-expressed PUS1 from a galactose-inducible plasmid (Fig. 2C, iii). Under standard growth (liquid media), PUS1 over-expression resulted in U6-Ψ28, in contrast to the control (Fig. 2B, cf. lanes 2 and 4). Under filamentation-like growth, because endogenous PUS1 was already physiologically up-regulated, the galactose-inducible PUS1 plasmid resulted in only an additional two-fold increase in PUS1 mRNA with a concomitant small increase in U6-Ψ28 (Fig. 2B, cf. lanes 6 and 8; Fig. 2C, iii–iv).

To test whether U6-Ψ28 modification during filamentation is due to Pus1p, we over-expressed WHI2 (Fig. S3A) in wild-type or pus1Δ cells. WHI2 over-expression led to a 3-fold over-expression of PUS1 mRNA (Fig. 2E) and to U6-Ψ28 induction in wild-type PUS1 but not in pus1Δ cells (Fig. 2D, cf. lanes 4 and 8). In contrast to PUS1, neither CBF5 nor any other pseudouridine synthase was strongly (>1.5 fold up-regulated) by WHI2 over-expression (Fig. 2E). The effect of filamentous growth was specific for U6-Ψ28, as the two pseudouridines induced on U2 snRNA (Wu et al., 2011) were not affected by the WHI2 pathway, nor did we detect any additional pseudouridines on U2 or U6 snRNAs (Fig. S3B, cf. lanes 1–8). We conclude that U6-Ψ28 is induced as part of filamentous growth in yeast through up-regulation of PUS1.

To investigate whether U6-Ψ28 affects splicing, we used an ACT1-CUP1 reporter (Lesser and Guthrie, 1993), the splicing efficiency of which determines resistance to copper in the growth media. Over-expression of either WHI2 (Fig. S3D, lane 2) or PUS1 (lane 5) inhibited growth of cells carrying branch site A-to-C (BS-C) and U257C reporters that are suboptimal for splicing (Fig. S3C), whereas wild-type ACT1-CUP1 pre-mRNA was unaffected. In pus1Δ cells, inhibition of the BS-C and U257C reporters upon WHI2 over expression was relieved, indicating that effects of Whi2p on splicing required a functional Pus1p enzyme (Fig. S3D, cf. lane 4 to lanes 2 and 5). These data, validated from primer extensions on in vivo total mRNA levels (Fig. S3E), indicate that pseudouridylation at U6-U28 alters spliceosome function.

Mutations in U6 snRNA result in robust U6-U28 pseudouridylation and a concomitant filamentous response

The U6 region that contains U6-Ψ28 carries three consecutive uridines, at positions 27–29. To confirm the location of the observed pseudouridine and to test effects of mutants in this region, we made various U6 mutations (U27A, -C, -G, and U28A, -C, -G) and tested their consequences through CMC-based primer-extension assays under conditions that induce U6-Ψ28. As expected, U6-U28C abolished Ψ28 (Fig. 3A, lane 4). Unexpectedly, mutations of the neighboring nucleotide to purine (U6-U27G and -U27A) as well as some mutations distant from U6-U28 (U6-U36C and -G50U) resulted in robust pseudouridylation at position U28 (Fig. 3A, lanes 6–8, and 9–12, respectively), providing additional tools to investigate the consequences of U6-Ψ28 without overexpression of any factor.

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

Figure 3

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(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.

To further investigate the pseudouridylation profile in this region of U6, we over-expressed Pus1p in strains carrying each U6-U27 and -U28 mutation. In addition to the modification of U6-Ψ28 due to U6-U27A and -G (Fig. S4A, cf. lanes 26–28, and 22–24), U6-U28A and -G resulted in modification of both U6-Ψ27 and U6-Ψ29 (Fig. S4A, cf. lanes 10–12, and 14–16). PUS1 over-expression resulted in a filamentation phenotype in U6-U27G and -A mutants. Compared to U28, U29 modification (due to U6-U28A and -G) resulted in weaker filamentation, but was stronger than U6-U28C (Fig. S4A). None of these modifications were detectable in the absence of PUS1 (Fig. S4B). Thus, in the absence of U6-U28, a secondary neighboring uridine becomes subject to pseudouridylation, albeit to a lesser extent, indicating that increased expression of Pus1p results in a tendency to modify the U6-U27–29 region.

All U6 snRNA mutations that resulted in strong pseudouridylation at position 28 (U6-U27G, -U27A, -U36C and -G50U; see Fig. 1B) also exhibited a pseudo-hyphal growth phenotype. Yeast cells bearing any of these U6 snRNA mutations had an elongated morphology, ranging from ~9–12% of examined cells (Fig. 3B, i, ii, vii, and viii). Such a pseudo-hyphal phenotype was rarely observed in wild-type yeast cells (Fig. 3B, v) or in cells harboring the non-modifiable mutation U6-U28C (Fig. 3B, vi). Pseudo-hyphal morphology was dependent upon the presence of U6-Ψ28 and not the U6 mutations, as PUS1 was required for this effect (Fig. 3B, iii and iv). In contrast, both the non-modifiable U6-U28C and pus1Δ reduced the pseudo-hyphal phenotype of WHI2 over-expression, whereas U6-U27A & -G resulted in stronger pseudo-hyphal phenotype (Fig. 3C, i–vi). In addition, the pseudohyphal response to butanol was blunted in both pus1Δ and U6-U28C mutants (Fig. S1F). We conclude that U6-Ψ28 pseudouridylation, per se, both can activate and is needed for an optimal filamentation growth program.

Designer H/ACA snoRNA targeting U6 snRNA activates pseudo-hyphal growth

To verify independently the effects of U6-Ψ28 on cell morphology, we expressed a guide snoRNA [‘snR-Ψ28’, based on snR81 (Chen et al., 2010)] in both wild-type and pus1Δ strains to target pseudouridylation of U6-U28 by Cbf5p (Fig. 3D). In both cases, we observed an induction of U6-Ψ28 by snR-Ψ28 but not by snR81 (Fig. 3D, lanes 4 and 8) as well as mild filamenting properties (~5% of cells) and cells with morphological abnormalities not sufficiently pronounced to be called as true pseudo-hyphal phenotype (“rotund” cells) ranging up to 40% of the total population (Fig. 3E, ii and iv), confirming that U6-Ψ28 causes morphologic changes. It is unclear if the snoRNA resulted in the same extent of modification as did the U6-U27G/A mutant, and this may explain phenotypic differences. It is also possible that binding of a snoRNP to U6 RNA results in altered dynamics of U6 structures or localization.

DISCUSSION

We describe a novel pseudouridine residue on spliceosomal U6 snRNA induced during filamentation-like environmental stress. U6-Ψ28 is neither present in log-phase growth nor significantly induced under general environmental stress response (heat shock, DNA damage, oxidative damage, heavy metal toxicity and osmotic shock). We identified Pus1p as the Ψ-synthase for the novel U6-Ψ28, and we show that mutants in U6 snRNA that are strongly pseudouridylated at position 28 activate a pseudo-hyphal growth program and alter the splicing of sensitive splicing reporters (dependent upon PUS1); conversely, inability to pseudouridylate U6-U28 inhibits pseudo-hyphal growth. Thus, Pus1p and U6-Ψ28 pseudouridylation are novel components of the filamentation response (Fig. 4).

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

Figure 4

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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.

Pus1p substrate specificity

The sequence and structural features that present a Pus1p substrate are unclear (Czudnochowski et al., 2013 and references therein). Pus1p modifies positions 27, 28 in the anticodon stem of many tRNAs, positions 34, 36 in certain tRNA introns, position 1 in tRNAArg, and position 44 in U2 snRNA. Yeast Pus1p can also pseudouridylate tRNA positions 26, 65 and 67 in vivo, whereas human and mouse tRNAs lack Ψ at these positions (Sibert and Patton, 2012 and references therein). The site in U2 snRNA modified by Pus1p, U2-Ψ44, does not contain sequence elements in common with the tRNA substrate sites (Massenet et al., 1999), although this region is now known to form a stem in the branch-recognition stemloop (BSL) in one U2 conformation (Perriman and Ares, 2010), and this may present a substrate structure more analogous to tRNA than previously thought.

Unlike the features that favor Ψ27 on tRNAs, U6-Ψ28 is present in a short presumed single-stranded region adjacent to a stem loop (Fig. 1B). Multiple structural rearrangements occur during U6 biogenesis and its transition into a catalytic component of the spliceosome (Vidaver et al., 1999; Dunn and Rader, 2010); it is unclear which of these conformations may be the substrate for Pus1p. The hyper-pseudouridylation that occurs with certain U6 mutations far removed from Ψ28 (Fig. 3A and Fig. 1B) suggests that relative stabilization of one conformation of U6 snRNA may allow for increased presentation of U6-U28 to Pus1p for modification.

The relation of U6-Ψ28 to the filamentation response

Consistent with the induction of U6-Ψ28, we found PUS1 expression increased ~10-fold under filamentation-like growth (Fig. 2C). Although PUS1 was not significantly up-regulated in a microarray dataset assessing feedback controls on the filamentation pathway (Chen and Fink, 2006), this filamentation was induced by conditions different from those used in our experiments and in different yeast strains. Our experiments with WHI2, a gene described previously to induce filamentation (Radcliffe et al., 1997), show that WHI2 up-regulates PUS1 expression (Fig. 2E) and induces U6-Ψ28 (Fig. 1D). WHI2 overexpression phenocopies PUS1 overexpression in its splicing effects (Fig. S3D); thus, it is likely that WHI2 expression indirectly activates the transcription of Pus1p. As candidates linking Whi2p to Pus1p in an activation cascade, we investigated Msn2p/Msn4p, documented transcription factors regulated by WHI2 and PSR1 phosphatase (Kaida et al., 2002), but our experiments did not support a role of Msn2p/4p in activation of PUS1 by Whi2p (data not shown). The filamentation response is an exceptionally complex process, and it is likely that other transcriptional activators may be part of the WHI2-PUS1 pathway. We propose a model for Whi2p activation of the filamentous growth program through Pus1p (Fig. 4). Similar to the general stress response wherein Whi2p and its interacting partner, Psr1-phosphatase, dephosphorylates Msn2p, a filamentation-specific transcription factor (TF) is dephosphorylated and, thus, activated by Whi2p-Psr1p. This activated TF binds to a STRE-like (stress response element) sequence within the PUS1 promoter to up-regulate it. Such Pus1p up-regulation is experimentally observed by over-expressing Whi2p (Fig. 2E). Indeed, we identified a STRE-like element ~500 bp upstream of the promoter of PUS1 (100% identity to the conserved ‘AGGGG’ sequence reported for MSN2), a potential binding site for a filamentation-specific TF. A consequent Pus1p up-regulation results in filamentation-specific pseudouridylation at U6-U28. U6-Ψ28 alters spliceosome function (Fig. S3) and likely activates the filamentous growth program through altered splicing of target genes.

How may the splicing of genes in vivo be affected through pseudouridylation? Cwc2p, an NTC (NineTeen Complex) component, directly binds to the U6-ISL with its RRM domain and to the vicinity of U6-Ψ28 with its Zn finger and connector element; this bipartite RNA-binding is critical to catalytic activation of the spliceosome (Schmitzová et al., 2012). We speculate that pseudouridylation at U6-U28 may create local RNA distortion that affects, and thus may regulate, Cwc2p binding. Although this model remains to be validated by future experiments, it is an attractive possibility to explain altered splicing efficiency of sensitive introns.

What mRNAs may be downstream in-vivo targets of this filamentation-specific pseudouridylation? We propose that mRNAs containing non-consensus splice or branch sites may be affected by U6-Ψ28. It is possible that they, in turn, modulate splicing of additional pre-mRNAs; more likely, however, is that U6-Ψ28 alters the splicing of one or more gene products that are limiting for initiation of the filamentation program, or that it inhibits the splicing of a negative regulator. Our results suggest that pseudouridylation may have a significant effect on gene expression and cell physiology by regulating splicing of a specific subset of genes; in this case, pseudouridylation of a catalytic core component of the spliceosome mediates activation of the filamentation program.

EXPERIMENTAL PROCEDURES

Growth conditions, cell culture and RNA isolation

S. cerevisiae were grown on solid medium+agarose (2%) [synthetic complete (SC), CSM, or auxotrophic media depending on plasmids, if any] at 30°C for 3 days. Cells were scraped off the media-surface, washed with chilled water to remove agarose traces, and either quick-frozen on dry ice and stored at −80°C or broken by bead beating. Total RNA was isolated with TRIzol (Invitrogen).

Primer extension based pseudouridylation assays

Pseudouridylation assays were as described (Bakin and Ofengand, 1993). Briefly, 20 µg total RNA was treated with 1-cyclohexyl-3(2-morpholinoethyl) carbodiimide metho-ptoluenesulphonate (CMC) at 37°C for 20 min in BEU buffer (50 mM bicine, pH 8.3, 4 mM EDTA, 7 M urea), subsequently incubated at 37°C for 2 hours in sodium bicarbonate buffer (50 mM NaHCO3, pH 10.5), and subjected to primer extension (Wu et al., 2011), using a 5' 32P-labeled oligo complementary to nts 51–70 of S. cerevisiae U6 snRNA. Products were visualized by phosphorimaging.

Construction of a designer snoRNA targeting U6-U28

Based on H/ACA-snoRNA snR81, we constructed a U6-U28-specific guide snoRNA (‘snR-Ψ28’). The 5' pseudouridylation pocket of snR81 was altered to fit the new target site (U6-U28); specifically nts 9–15 were altered to ‘attgacc’ and nts 65–71 to ‘atgtcca’.

Supplementary Material

01
02

HIGHLIGHTS.

  • U6 snRNA is pseudouridylated during filamentous growth.

  • Pus1p catalyzes this modification.

  • Constitutive U6 pseudouridylation stimulates filamentous growth.

  • Inability to pseudouridylate U6 inhibits filamentous growth.

ACKNOWLEDGMENTS

We are grateful to Jon Warner, Ian Willis, Tom Meier, and Query lab members for helpful discussions and critical readings of the manuscript. We thank Ian Willis for providing over-expression plasmids, Anuj Kumar for yeast Σ1278b strains, and Yi-Tao Yu and Audrey Gasch for helpful suggestions. This work was supported by NIH grant GM57829 to C.C.Q. and by a Cancer Center Support (core) grant from the NCI to AECOM. C.C.Q. is a scholar of the Irma T. Hirschl Trust.

Footnotes

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REFERENCES

  1. Bakin A, Ofengand J. Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique. Biochemistry. 1993;32:9754–9762. doi: 10.1021/bi00088a030. [DOI] [PubMed] [Google Scholar]
  2. Chen C, Zhao XL, Kierzek R, Yu YT. A Flexible RNA Backbone within the Polypyrimidine Tract Is Required for U2AF65 Binding and Pre-mRNA Splicing In Vivo. Mol. Cell. Biol. 2010;30:4108–4119. doi: 10.1128/MCB.00531-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chen H, Fink GR. Feedback control of morphogenesis in fungi by aromatic alcohols. Genes Dev. 2006;20:1150–1161. doi: 10.1101/gad.1411806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cullen PJ, Sprague GF., Jr The regulation of filamentous growth in yeast. Genetics. 2012;190:23–49. doi: 10.1534/genetics.111.127456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Czudnochowski N, Wang AL, Finer-Moore J, Stroud RM. In human pseudouridine synthase 1 (hPus1), a C-terminal helical insert blocks tRNA from binding in the same orientation as in the Pus1 bacterial homologue TruA, consistent with their different target selectivities. J Mol Biol. 2013;425:3875–3887. doi: 10.1016/j.jmb.2013.05.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Davis DR. Stabilization of RNA stacking by pseudouridine. Nucleic Acids Res. 1995;23:5020–5026. doi: 10.1093/nar/23.24.5020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dunn EA, Rader SD. Secondary structure of U6 small nuclear RNA: implications for spliceosome assembly. Biochem. Soc. Trans. 2010;38:1099–1104. doi: 10.1042/BST0381099. [DOI] [PubMed] [Google Scholar]
  8. Fica SM, Tuttle N, Novak T, Li NS, Lu J, Koodathingal P, Dai Q, Staley JP, Piccirilli JA. RNA catalyses nuclear pre-mRNA splicing. Nature. 2013;503:229–234. doi: 10.1038/nature12734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, Storz G, Botstein D, Brown PO. Genomic expression programs in the response of yeast cells to environmental changes. Mol. Biol. Cell. 2000;11:4241–4257. doi: 10.1091/mbc.11.12.4241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ge JH, Yu YT. RNA pseudouridylation: new insights into an old modification. Trends in Biochemical Sciences. 2013;38:210–218. doi: 10.1016/j.tibs.2013.01.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hoskins AA, Moore MJ. The spliceosome: a flexible, reversible macromolecular machine. Trends in Biochemical Sciences. 2012;37:179–188. doi: 10.1016/j.tibs.2012.02.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaida D, Yashiroda H, Toh-e A, Kikuchi Y. Yeast Whi2 and Psr1-phosphatase form a complex and regulate STRE-mediated gene expression. Genes Cells. 2002;7:543–552. doi: 10.1046/j.1365-2443.2002.00538.x. [DOI] [PubMed] [Google Scholar]
  13. Lesser CF, Guthrie C. Mutational analysis of pre-mRNA splicing in Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1. Genetics. 1993;133:851–863. doi: 10.1093/genetics/133.4.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lorenz MC, Cutler NS, Heitman J. Characterization of alcohol-induced filamentous growth in Saccharomyces cerevisiae. Mol. Biol. Cell. 2000;11:183–199. doi: 10.1091/mbc.11.1.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Massenet S, Motorin Y, Lafontaine DLJ, Hurt EC, Grosjean H, Branlant C. Pseudouridine mapping in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (snRNAs) reveals that pseudouridine synthase Pus1p exhibits a dual substrate specificity for U2 snRNA and tRNA. Mol. Cell. Biol. 1999;19:2142–2154. doi: 10.1128/mcb.19.3.2142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Momose Y, Iwahashi H. Bioassay of cadmium using a DNA microarray: genome-wide expression patterns of Saccharomyces cerevisiae response to cadmium. Environ. Toxicol. Chem. 2001;20:2353–2360. doi: 10.1897/1551-5028(2001)020<2353:bocuad>2.0.co;2. [DOI] [PubMed] [Google Scholar]
  17. Newby MI, Greenbaum NL. Sculpting of the spliceosomal branch site recognition motif by a conserved pseudouridine. Nat. Struct. Biol. 2002;9:958–965. doi: 10.1038/nsb873. [DOI] [PubMed] [Google Scholar]
  18. Perriman R, Ares M. Invariant U2 snRNA Nucleotides Form a Stem Loop to Recognize the Intron Early in Splicing. Mol. Cell. 2010;38:416–427. doi: 10.1016/j.molcel.2010.02.036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Radcliffe PA, Binley KM, Trevethick J, Hall M, Sudbery PE. Filamentous growth of the budding yeast Saccharomyces cerevisiae induced by overexpression of the WHi2 gene. Microbiology. 1997;143(Pt 6):1867–1876. doi: 10.1099/00221287-143-6-1867. [DOI] [PubMed] [Google Scholar]
  20. Reddy R, Busch H. Small nuclear RNAs: RNA sequences, structure, and modifications. In: Birnsteil ML, editor. Structure and function of major and minor small nuclear ribonucleoprotein particles. Heidelberg: Springer-Verlag Press; 1988. pp. 1–37. [Google Scholar]
  21. Schmitzová J, Rasche N, Dybkov O, Kramer K, Fabrizio P, Urlaub H, Lührmann R, Pena V. Crystal structure of Cwc2 reveals a novel architecture of a multipartite RNA-binding protein. EMBO J. 2012;31:2222–2234. doi: 10.1038/emboj.2012.58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sibert BS, Patton JR. Pseudouridine synthase 1: a site-specific synthase without strict sequence recognition requirements. Nucleic Acids Res. 2012;40:2107–2118. doi: 10.1093/nar/gkr1017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Vidaver RM, Fortner DM, Loos-Austin LS, Brow DA. Multiple functions of Saccharomyces cerevisiae splicing protein Prp24 in U6 RNA structural rearrangements. Genetics. 1999;153:1205–1218. doi: 10.1093/genetics/153.3.1205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wahl MC, Will CL, Lührmann R. The Spliceosome: Design Principles of a Dynamic RNP Machine. Cell. 2009;136:701–718. doi: 10.1016/j.cell.2009.02.009. [DOI] [PubMed] [Google Scholar]
  25. Wu G, Xiao M, Yang C, Yu YT. U2 snRNA is inducibly pseudouridylated at novel sites by Pus7p and snR81 RNP. EMBO J. 2011;30:79–89. doi: 10.1038/emboj.2010.316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yu YT, Shu MD, Steitz JA. Modifications of U2 snRNA are required for snRNP assembly and pre-mRNA splicing. EMBO J. 1998;17:5783–5795. doi: 10.1093/emboj/17.19.5783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Zebarjadian Y, King T, Fournier MJ, Clarke L, Carbon J. Point mutations in yeast CBF5 can abolish in vivo pseudouridylation of rRNA. Mol. Cell. Biol. 1999;19:7461–7472. doi: 10.1128/mcb.19.11.7461. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

01
NIHMS613783-supplement-01.pdf (11.4MB, pdf)
02
NIHMS613783-supplement-02.pdf (852.8KB, pdf)

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