Functional analysis of the zinc finger modules of the Saccharomyces cerevisiae splicing factor Luc7.

Identification of splice sites is a critical step in pre-messenger RNA (pre-mRNA) splicing because the definition of the exon/intron boundaries controls what nucleotides are incorporated into mature mRNAs. The intron boundary with the upstream exon is initially identified through interactions with the U1 small nuclear ribonucleoprotein (snRNP). This involves both base-pairing between the U1 snRNA and the pre-mRNA as well as snRNP proteins interacting with the 5' splice site (5'ss)/snRNA duplex. In yeast, this duplex is buttressed by two conserved protein factors, Yhc1 and Luc7. Luc7 has three human paralogs (LUC7L, LUC7L2, and LUC7L3), which play roles in alternative splicing. What domains of these paralogs promote splicing at particular sites is not yet clear. Here, we humanized the zinc finger (ZnF) domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. Although we were unable to determine a function for the first ZnF domain, humanization of the second ZnF domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5'ss. In contrast, the corresponding ZnF domain of LUC7L3 could not support yeast viability. Further, humanization of Luc7 can suppress mutation of the ATPase Prp28, which is involved in U1 release and exchange for U6 at the 5'ss. Our work reveals a role for the second ZnF of Luc7 in splice site selection and suggests that different ZnF domains may have different ATPase requirements for release by Prp28.

Nonfunctional coq10 mutants maintain the ERMES complex and reveal true phenotypes associated with the loss of the coenzyme Q chaperone protein Coq10.

Coenzyme Q (CoQ) is a redox-active lipid molecule that acts as an electron carrier in the mitochondrial electron transport chain. In Saccharomyces cerevisiae, CoQ is synthesized in the mitochondrial matrix by a multisubunit protein-lipid complex termed the CoQ synthome, the spatial positioning of which is coordinated by the endoplasmic reticulum-mitochondria encounter structure (ERMES). The MDM12 gene encoding the cytosolic subunit of ERMES is coexpressed with COQ10, which encodes the putative CoQ chaperone Coq10, via a shared bidirectional promoter. Deletion of COQ10 results in respiratory deficiency, impaired CoQ biosynthesis, and reduced spatial coordination between ERMES and the CoQ synthome. While Coq10 protein content is maintained upon deletion of MDM12, we show that deletion of COQ10 by replacement with a HIS3 marker results in diminished Mdm12 protein content. Since deletion of individual ERMES subunits prevents ERMES formation, we asked whether some or all of the phenotypes associated with COQ10 deletion result from ERMES dysfunction. To identify the phenotypes resulting solely due to the loss of Coq10, we constructed strains expressing a functionally impaired (coq10-L96S) or truncated (coq10-R147∗) Coq10 isoform using CRISPR-Cas9. We show that both coq10 mutants preserve Mdm12 protein content and exhibit impaired respiratory capacity like the coq10Δ mutant, indicating that Coq10's function is vital for respiration regardless of ERMES integrity. Moreover, the maintenance of CoQ synthome stability and efficient CoQ biosynthesis observed for the coq10-R147∗ mutant suggests these deleterious phenotypes in the coq10Δ mutant result from ERMES disruption. Overall, this study clarifies the role of Coq10 in modulating CoQ biosynthesis.

Formin tails act as a switch, inhibiting or enhancing processive actin elongation.

Formins are large, multidomain proteins that nucleate new actin filaments and accelerate elongation through a processive interaction with the barbed ends of filaments. Their actin assembly activity is generally attributed to their eponymous formin homology (FH) 1 and 2 domains; however, evidence is mounting that regions outside of the FH1FH2 stretch also tune actin assembly. Here, we explore the underlying contributions of the tail domain, which spans the sequence between the FH2 domain and the C terminus of formins. Tails vary in length from ∼0 to >200 residues and contain a number of recognizable motifs. The most common and well-studied motif is the ∼15-residue-long diaphanous autoregulatory domain. This domain mediates all or nothing regulation of actin assembly through an intramolecular interaction with the diaphanous inhibitory domain in the N-terminal half of the protein. Multiple reports demonstrate that the tail can enhance both nucleation and processivity. In this study, we provide a high-resolution view of the alternative splicing encompassing the tail in the formin homology domain (Fhod) family of formins during development. While four distinct tails are predicted, we found significant levels of only two of these. We characterized the biochemical effects of the different tails. Surprisingly, the two highly expressed Fhod-tails inhibit processive elongation and diminish nucleation, while a third supports activity. These findings demonstrate a new mechanism of modulating actin assembly by formins and support a model in which splice variants are specialized to build distinct actin structures during development.

Nanoblot: an R-package for visualization of RNA isoforms from long-read RNA-sequencing data.

RT-PCR and northern blots have long been used to study RNA isoforms usage for single genes. Recent advancements in long-read sequencing have yielded unprecedented information about the usage and abundance of these RNA isoforms. However, visualization of long-read sequencing data remains challenging due to the high information density. To alleviate these issues, we have developed NanoBlot, an open-source R-package that generates northern blot and RT-PCR-like images from long-read sequencing data. NanoBlot requires aligned, positionally sorted and indexed BAM files. Plotting is based around ggplot2 and is easily customizable. Advantages of NanoBlot include a robust system for designing probes to visualize isoforms including excluding reads based on the presence or absence of a specified region, an elegant solution to representing isoforms with continuous variations in length, and the ability to overlay multiple genes in the same plot using different colors. We present examples of nanoblots compared to actual northern blot data. In addition to traditional gel-like images, the NanoBlot package can also output other visualizations such as violin plots and 3'-RACE-like plots focused on 3'-end isoforms visualization. The use of the NanoBlot package should provide a simple answer to some of the challenges of visualizing long-read RNA-sequencing data.

Splicing factor Prp18p promotes genome-wide fidelity of consensus 3'-splice sites.

The fidelity of splice site selection is critical for proper gene expression. In particular, proper recognition of 3'-splice site (3'SS) sequences by the spliceosome is challenging considering the low complexity of the 3'SS consensus sequence YAG. Here, we show that absence of the Prp18p splicing factor results in genome-wide activation of alternative 3'SS in S. cerevisiae, including highly unusual non-YAG sequences. Usage of these non-canonical 3'SS in the absence of Prp18p is enhanced by upstream poly(U) tracts and by their potential to interact with the first intronic nucleoside, allowing them to dock in the spliceosome active site instead of the normal 3'SS. The role of Prp18p in 3'SS fidelity is facilitated by interactions with Slu7p and Prp8p, but cannot be fulfilled by Slu7p, identifying a unique role for Prp18p in 3'SS fidelity. This fidelity function is synergized by the downstream proofreading activity of the Prp22p helicase, but is independent from another late splicing helicase, Prp43p. Our results show that spliceosomes exhibit remarkably relaxed 3'SS sequence usage in the absence of Prp18p and identify a network of spliceosomal interactions centered on Prp18p which are required to promote the fidelity of the recognition of consensus 3'SS sequences.

Splicing inactivation generates hybrid mRNA-snoRNA transcripts targeted by cytoplasmic RNA decay.

Many small nucleolar RNAs (snoRNA)s are processed from introns of host genes, but the importance of splicing for proper biogenesis and the fate of the snoRNAs is not well understood. Here, we show that inactivation of splicing factors or mutation of splicing signals leads to the accumulation of partially processed hybrid messenger RNA-snoRNA (hmsnoRNA) transcripts. hmsnoRNAs are processed to the mature 3' ends of the snoRNAs by the nuclear exosome and bound by small nucleolar ribonucleoproteins. hmsnoRNAs are unaffected by translation-coupled RNA quality-control pathways, but they are degraded by the major cytoplasmic exonuclease Xrn1p, due to their messenger RNA (mRNA)-like 5' extensions. These results show that completion of splicing is required to promote complete and accurate processing of intron-encoded snoRNAs and that splicing defects lead to degradation of hybrid mRNA-snoRNA species by cytoplasmic decay, underscoring the importance of splicing for the biogenesis of intron-encoded snoRNAs.

Stress-induced inhibition of mRNA export triggers RNase III-mediated decay of the BDF2 mRNA.

The expression of bromodomain-containing proteins that regulate chromatin structure and accessibility must be tightly controlled to ensure the appropriate regulation of gene expression. In the yeast S. cerevisiae, Bromodomain Factor 2 (BDF2) expression is extensively regulated post-transcriptionally during stress by RNase III-mediated decay (RMD), which is triggered by cleavage of the BDF2 mRNA in the nucleus by the RNase III homolog Rnt1p. Previous studies have shown that RMD-mediated down-regulation of BDF2 is hyperactivated in osmotic stress conditions, yet the mechanisms driving the enhanced nuclear cleavage of BDF2 RNA under these conditions remain unknown. Here, we show that RMD hyperactivation can be detected in multiple stress conditions that inhibit mRNA export, and that Rnt1p remains primarily localized in the nucleus during salt stress. We show that globally inhibiting mRNA nuclear export by anchoring away mRNA biogenesis or export factors out of the nucleus can recapitulate RMD hyperactivation in the absence of stress. RMD hyperactivation requires Rnt1p nuclear localization but does not depend on the BDF2 gene endogenous promoter, and its efficiency is affected by the structure of the stem-loop cleaved by Rnt1p. Because multiple stress conditions have been shown to mediate global inhibition of mRNA export, our results suggest that the hyperactivation of RMD is primarily the result of the increased nuclear retention of the BDF2 mRNA during stress.

Robust mapping of polyadenylated and non-polyadenylated RNA 3' ends at nucleotide resolution by 3'-end sequencing.

3'-end poly(A)+ sequencing is an efficient and economical method for global measurement of mRNA levels and alternative poly(A) site usage. A common method involves oligo(dT)19V reverse-transcription (RT)-based library preparation and high-throughput sequencing with a custom primer ending in (dT)19. While the majority of library products have the first sequenced nucleotide reflect the bona fide poly(A) site (pA), a substantial fraction of sequencing reads arise from various mis-priming events. These can result in incorrect pA site calls anywhere from several nucleotides downstream to several kilobases upstream from the bona fide pA site. While these mis-priming events can be mitigated by increasing annealing stringency (e.g. increasing temperature from 37 °C to 42 °C), they still persist at an appreciable level (∼10%) and computational methods must be used to prevent artifactual calls. Here we present a bioinformatics workflow for precise mapping of poly(A)+ 3' ends and handling of artifacts due to oligo(dT) mis-priming and sample polymorphisms. We test pA site calling with three different read mapping programs (STAR, BWA, and BBMap), and show that the way in which each handles terminal mismatches and soft clipping has a substantial impact on identifying correct pA sites, with BWA requiring the least post-processing to correct artifacts. We demonstrate the use of this pipeline for mapping pA sites in the model eukaryote S. cerevisiae, and further apply this technology to non-polyadenylated transcripts by employing in vitro polyadenylation prior to library prep (IVP-seq). As proof of principle, we show that a fraction of tRNAs harbor CCU 3' tails instead of the canonical CCA tail, and globally identify 3' ends of splicing intermediates arising from inefficiently spliced transcripts.

RNA polymerase I stability couples cellular growth to metal availability.

Zinc is an essential cofactor of all major eukaryotic RNA polymerases. How the activity of these enzymes is coordinated or regulated according to cellular zinc levels is largely unknown. Here we show that the stability of RNA polymerase I (RNAPI) is tightly coupled to zinc availability in vivo. In zinc deficiency, RNAPI is specifically degraded by proteolysis in the vacuole in a pathway dependent on the export in Xpo1p and deubiquitination of the RNAPI large subunit Rpa190p by Ubp2p and Ubp4p. RNAPII is unaffected, which allows for the expression of genes required in zinc deficiency. RNAPI export to the vacuole is required for survival during zinc starvation, suggesting that degradation of zinc-binding subunits might provide a last resort zinc reservoir. These results reveal a hierarchy of cellular transcriptional activities during zinc starvation and show that degradation of the most active cellular transcriptional machinery couples cellular growth and proliferation to zinc availability.

Protein Methylation and Translation: Role of Lysine Modification on the Function of Yeast Elongation Factor 1A.

To date, 12 protein lysine methyltransferases that modify translational elongation factors and ribosomal proteins (Efm1-7 and Rkm 1-5) have been identified in the yeast Saccharomyces cerevisiae. Of these 12, five (Efm1 and Efm4-7) appear to be specific to elongation factor 1A (EF1A), the protein responsible for bringing aminoacyl-tRNAs to the ribosome. In S. cerevisiae, the functional implications of lysine methylation in translation are mostly unknown. In this work, we assessed the physiological impact of disrupting EF1A methylation in a strain where four of the most conserved methylated lysine sites are mutated to arginine residues and in strains lacking either four or five of the Efm lysine methyltransferases specific to EF1A. We found that loss of EF1A methylation was not lethal but resulted in reduced growth rates, particularly under caffeine and rapamycin stress conditions, suggesting EF1A interacts with the TORC1 pathway, as well as altered sensitivities to ribosomal inhibitors. We also detected reduced cellular levels of the EF1A protein, which surprisingly was not reflected in its stability in vivo. We present evidence that these Efm methyltransferases appear to be largely devoted to the modification of EF1A, finding no evidence of the methylation of other substrates in the yeast cell. This work starts to illuminate why one protein can need five different methyltransferases for its functions and highlights the resilience of yeast to alterations in their posttranslational modifications.

Common genomic elements promote transcriptional and DNA replication roadblocks.

RNA polymerase II (Pol II) transcription termination by the Nrd1p-Nab3p-Sen1p (NNS) pathway is critical for the production of stable noncoding RNAs and the control of pervasive transcription in Saccharomyces cerevisiae To uncover determinants of NNS termination, we mapped the 3'-ends of NNS-terminated transcripts genome-wide. We found that nucleosomes and specific DNA-binding proteins, including the general regulatory factors (GRFs) Reb1p, Rap1p, and Abf1p, and Pol III transcription factors enhance the efficiency of NNS termination by physically blocking Pol II progression. The same DNA-bound factors that promote NNS termination were shown previously to define the 3'-ends of Okazaki fragments synthesized by Pol δ during DNA replication. Reduced binding of these factors results in defective NNS termination and Pol II readthrough. Furthermore, inactivating NNS enables Pol II elongation through these roadblocks, demonstrating that effective Pol II termination depends on a synergy between the NNS machinery and obstacles in chromatin. Consistent with this finding, loci exhibiting Pol II readthrough at GRF binding sites are depleted for upstream NNS signals. Overall, these results underscore how RNA termination signals influence the behavior of Pol II at chromatin obstacles, and establish that common genomic elements define boundaries for both DNA and RNA synthesis machineries.

Mutations of EXOSC3/Rrp40p associated with neurological diseases impact ribosomal RNA processing functions of the exosome in S. cerevisiae.

The RNA exosome is a conserved multiprotein complex that achieves a large number of processive and degradative functions in eukaryotic cells. Recently, mutations have been mapped to the gene encoding one of the subunits of the exosome, EXOSC3 (yeast Rrp40p), which results in pontocerebellar hypoplasia with motor neuron degeneration in human patients. However, the molecular impact of these mutations in the pathology of these diseases is not well understood. To investigate the molecular consequences of mutations in EXOSC3 that lead to neurological diseases, we analyzed the effect of three of the mutations that affect conserved residues of EXOSC3/Rrp40p (G31A, G191C, and W238R; G8A, G148C, and W195R, respectively, in human and yeast) in S. cerevisiae We show that the severity of the phenotypes of these mutations in yeast correlate with that of the disease in human patients, with the W195R mutant showing the strongest growth and RNA processing phenotypes. Furthermore, we show that these mutations affect more severely pre-ribosomal RNA processing functions of the exosome rather than other nuclear processing or surveillance functions. These results suggest that delayed or defective pre-rRNA processing might be the primary defect responsible for the pathologies detected in patients with mutations affecting EXOSC3 function in residues conserved throughout eukaryotes.

Widespread use of non-productive alternative splice sites in Saccharomyces cerevisiae.

Saccharomyces cerevisiae has been used as a model system to investigate the mechanisms of pre-mRNA splicing but only a few examples of alternative splice site usage have been described in this organism. Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many S. cerevisiae intron-containing genes exhibit usage of alternative splice sites, but many transcripts generated by splicing at these sites are non-functional because they introduce premature termination codons, leading to degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3' splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3'-splice site in GCR1. The use of non-productive alternative splice sites can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results show that alternative splicing is frequent in S. cerevisiae but masked by RNA degradation and that the use of alternative splice sites in this organism is mostly aimed at controlling transcript levels rather than increasing proteome diversity.

Widespread impact of nonsense-mediated mRNA decay on the yeast intronome.

Nonsense-mediated mRNA decay (NMD) eliminates transcripts carrying premature translation termination codons, but the role of NMD on yeast unspliced pre-mRNA degradation is controversial. Using tiling arrays, we show that many unspliced yeast pre-mRNAs accumulate in strains mutated for the NMD component Upf1p and the exonuclease Xrn1p. Intron identity and suboptimal splicing signals resulting in weak splicing were found to be important determinants in NMD targeting. In the absence of functional NMD, unspliced precursors accumulate in the cytoplasm, possibly in P-bodies. NMD can also complement RNase III-mediated nuclear degradation of unspliced RPS22B pre-mRNAs, degrades most unspliced precursors generated by a 5' splice site mutation in RPS10B, and limits RPS29B unspliced precursors accumulation during amino acid starvation. These results show that NMD has a wider impact than previously thought on the degradation of yeast-unspliced transcripts and plays an important role in discarding precursors of regulated or suboptimally spliced transcripts.

Translational roles of elongation factor 2 protein lysine methylation.

Methylation of various components of the translational machinery has been shown to globally affect protein synthesis. Little is currently known about the role of lysine methylation on elongation factors. Here we show that in Saccharomyces cerevisiae, the product of the EFM3/YJR129C gene is responsible for the trimethylation of lysine 509 on elongation factor 2. Deletion of EFM3 or of the previously described EFM2 increases sensitivity to antibiotics that target translation and decreases translational fidelity. Furthermore, the amino acid sequences of Efm3 and Efm2, as well as their respective methylation sites on EF2, are conserved in other eukaryotes. These results suggest the importance of lysine methylation modification of EF2 in fine tuning the translational apparatus.

Sequential RNA degradation pathways provide a fail-safe mechanism to limit the accumulation of unspliced transcripts in Saccharomyces cerevisiae.

The nuclear exosome and the nonsense-mediated mRNA decay (NMD) pathways have been implicated in the degradation of distinct unspliced transcripts in Saccharomyces cerevisiae. In this study we show that these two systems can act sequentially on specific unspliced pre-mRNAs to limit their accumulation. Using steady-state and decay analyses, we show that while specific unspliced transcripts rely mostly on NMD or on the nuclear exosome for their degradation, some unspliced RNAs are stabilized only when both the nuclear exosome and NMD are inactivated. We found that the mechanism of degradation of these unspliced pre-mRNAs is not influenced by promoter identity. However, the specificity in the pre-mRNAs degradation pathways can be manipulated by changing the rate of export or retention of these mRNAs. For instance, reducing the nuclear export of pre-mRNAs mostly degraded by NMD results in a higher fraction of unspliced transcripts degraded by the nuclear exosome. Reciprocally, inactivating the Mlp retention factors results in a higher fraction of unspliced transcripts degraded by NMD for precursors normally targeted by the nuclear exosome. Overall, these results demonstrate that a functional redundancy exists between nuclear and cytoplasmic degradation pathways for unspliced pre-mRNAs, and suggest that the degradation routes of these species are mainly determined by the efficiency of their nuclear export rates. The presence of these two sequential degradation pathways for unspliced pre-mRNAs underscores the importance of limiting their accumulation and might serve as a fail-safe mechanism to prevent the expression of these nonfunctional RNAs.

Quality control of MATa1 splicing and exon skipping by nuclear RNA degradation.

The MATa1 gene encodes a transcriptional repressor that is an important modulator of sex-specific gene expression in Saccharomyces cerevisiae. MATa1 contains two small introns, both of which need to be accurately excised for proper expression of a functional MATa1 product and to avoid production of aberrant forms of the repressor. Here, we show that unspliced and partially spliced forms of the MATa1 mRNA are degraded by the nuclear exonuclease Rat1p, the nuclear exosome and by the nuclear RNase III endonuclease Rnt1p to prevent undesired expression of non-functional a1 proteins. In addition, we show that mis-spliced forms of MATa1 in which the splicing machinery has skipped exon2 and generated exon1-exon3 products are degraded by the nuclear 5'-3' exonuclease Rat1p and by the nuclear exosome. This function for Rat1p and the nuclear exosome in the degradation of exon-skipped products is also observed for three other genes that contain two introns (DYN2, SUS1, YOS1), identifying a novel nuclear quality control pathway for aberrantly spliced RNAs that have skipped exons.

Cryptic transcription mediates repression of subtelomeric metal homeostasis genes.

Nonsense-mediated mRNA decay (NMD) prevents the accumulation of transcripts bearing premature termination codons. Here we show that Saccharomyces cerevisiae NMD mutants accumulate 5'-extended RNAs (CD-CUTs) of many subtelomeric genes. Using the subtelomeric ZRT1 and FIT3 genes activated in response to zinc and iron deficiency, respectively, we show that transcription of these CD-CUTs mediates repression at the bona fide promoters, by preventing premature binding of RNA polymerase II in conditions of metal repletion. Expression of the main ZRT1 CD-CUT is controlled by the histone deacetylase Rpd3p, showing that histone deacetylases can regulate expression of genes through modulation of the level of CD-CUTs. Analysis of binding of the transcriptional activator Zap1p and insertion of transcriptional terminators upstream from the Zap1p binding sites show that CD-CUT transcription or accumulation also interferes with binding of the transcriptional activator Zap1p. Consistent with this model, overexpressing Zap1p or using a constitutively active version of the Aft1p transcriptional activator rescues the induction defect of ZRT1 and FIT3 in NMD mutants. These results show that cryptic upstream sense transcription resulting in unstable transcripts degraded by NMD controls repression of a large number of genes located in subtelomeric regions, and in particular of many metal homeostasis genes.

Trafficking of mitochondrial double-stranded RNA from mitochondria to the cytosol.

In addition to mitochondrial DNA, mitochondrial double-stranded RNA (mtdsRNA) is exported from mitochondria. However, specific channels for RNA transport have not been demonstrated. Here, we begin to characterize channel candidates for mtdsRNA export from the mitochondrial matrix to the cytosol. Down-regulation of SUV3 resulted in the accumulation of mtdsRNAs in the matrix, whereas down-regulation of PNPase resulted in the export of mtdsRNAs to the cytosol. Targeting experiments show that PNPase functions in both the intermembrane space and matrix. Strand-specific sequencing of the double-stranded RNA confirms the mitochondrial origin. Inhibiting or down-regulating outer membrane proteins VDAC1/2 and BAK/BAX or inner membrane proteins PHB1/2 strongly attenuated the export of mtdsRNAs to the cytosol. The cytosolic mtdsRNAs subsequently localized to large granules containing the stress protein TIA-1 and activated the type 1 interferon stress response pathway. Abundant mtdsRNAs were detected in a subset of non-small-cell lung cancer cell lines that were glycolytic, indicating relevance in cancer biology. Thus, we propose that mtdsRNA is a new damage-associated molecular pattern that is exported from mitochondria in a regulated manner.

Spliceosomal mutations decouple 3' splice site fidelity from cellular fitness.

The fidelity of splice site selection is thought to be critical for proper gene expression and cellular fitness. In particular, proper recognition of 3'-splice site (3'SS) sequences by the spliceosome is a daunting task considering the low complexity of the 3'SS consensus sequence YAG. Here we show that inactivating the near-essential splicing factor Prp18p results in a global activation of alternative 3'SS, many of which harbor sequences that highly diverge from the YAG consensus, including some highly unusual non-AG 3'SS. We show that the role of Prp18p in 3'SS fidelity is promoted by physical interactions with the essential splicing factors Slu7p and Prp8p and synergized by the proofreading activity of the Prp22p helicase. Strikingly, structure-guided point mutations that disrupt Prp18p-Slu7p and Prp18p-Prp8p interactions mimic the loss of 3'SS fidelity without any impact on cellular growth, suggesting that accumulation of incorrectly spliced transcripts does not have a major deleterious effect on cellular viability. These results show that spliceosomes exhibit remarkably relaxed fidelity in the absence of Prp18p, and that new 3'SS sampling can be achieved genome-wide without a major negative impact on cellular fitness, a feature that could be used during evolution to explore new productive alternative splice sites.