EUKARYOTES share two major messenger RNA (mRNA) decay pathways that are both carried out by exonucleolytic digestion. mRNA degradation is initiated by gradual shortening of the poly(A) tail, followed by Xrn1-mediated 5′ to 3′ decay and RNA exosome-mediated 3′ to 5′ decay (Parker 2012). Because Xrn1 can only degrade RNAs with a 5′-monophosphate (Stevens and Poole 1995; Jinek et al. 2011), removal of the 5′ cap structure by Dcp2 is required in the 5′ to 3′ decay pathway. Importantly, deleting either DCP2 or XRN1 results in stabilization of many yeast mRNAs (Larimer et al. 1992; Dunckley and Parker 1999; He et al. 2003). The stabilization of mRNAs in dcp2 mutants indicates that yeast Dcp2 is the major decapping enzyme, and the 5′ to 3′ pathway is the major mRNA decay pathway. Other enzymes capable of decapping mRNAs have been described both in yeast and other organisms (Jiao et al. 2010; Song et al. 2010; Li et al. 2011; Fujimura and Esteban 2012; Zhou et al. 2015; Grudzien-Nogalska et al. 2016; Doamekpor et al. 2020), but their role in bulk cytoplasmic mRNA degradation has not been fully defined. Consistent with its importance for mRNA decay, deletion of XRN1 causes a slow growth defect, while the phenotype of dcp2∆ is reported inconsistently among different studies. Some studies have reported that dcp2∆ is viable but slow-growing, while others reported that dcp2∆ is lethal (Dunckley and Parker 1999; Giaever et al. 2002; Geisler et al. 2012; He and Jacobson 2015). It has been speculated that this difference between studies is attributable to differences between the strains used (He and Jacobson 2015), but this has not been critically analyzed.

Previously, suppressor screens of budding yeast decapping mutants (dcp1 or dcp2 conditional mutants) have identified EDC1, EDC2, EDC3, SBP1, and DCP2 itself as high-copy suppressors (Dunckley and Parker 1999; Dunckley et al. 2001; Kshirsagar and Parker 2004; Segal et al. 2006). In each case, the improved growth caused by suppressors was correlated with improved decapping activity and mRNA degradation, suggesting that the essential function of the Dcp1-Dcp2 decapping enzyme is indeed mRNA decapping. Although these studies showed that the major function of Dcp1 and Dcp2 is mRNA decapping, they are limited to high-copy suppressor screens of conditional alleles in the decapping enzyme, which may not have revealed the full functions of Dcp2.

To further understand the function of Dcp2, we sought to identify suppressors of the growth defect of a decapping mutant by a complementary experimental evolution of a dcp2 null strain, which can be more powerful in identifying smaller effects and double mutants. Surprisingly, we identified genes that have no obvious connection to mRNA degradation. Among the genes we identified, we focused on the karyopherin KAP123 and the leucine tRNA tL(GAG)G that are recurrently mutated. We showed that a null mutation of each gene is sufficient to suppress the growth phenotype of dcp2∆, and that kap123∆ and tl(gag)g∆ have additive effects. We also show that previously reported viable dcp1∆ and dcp2∆ strains had undetected mutations in KAP123, suggesting that they were mistakenly reported as viable due to the suppressor mutations. Instead, our results suggest that dcp2∆ grows extremely slowly and cannot be continuously cultured under standard conditions. Interestingly, suppression of the growth defect of dcp2∆ is not caused by improved cytoplasmic mRNA decay. Absence of Dcp2 causes a global disturbance of the transcriptome including not only mRNA, but also noncoding RNA, and the suppressor mutations we identified do not restore the transcriptome to normal. However, we do detect a widespread but modest amplitude effect in partially restoring RNA homeostasis. Whether these modest effects on transcripts are a cause or effect of improved growth, or a mixture of both, is not clear. These results indicate that the extremely poor growth of a strain lacking the decapping enzyme can be overcome by several independent mechanisms that have modest effects on the transcriptome compared to the global disruption of the transcriptome caused by dcp2 mutations.

Materials and MethodsStrains, plasmids, and oligonucleotides

The DCP2/dcp2∆ heterozygous diploid in the BY4743 (S288C) background was obtained from Open Biosystems, Huntsville, AL and all other strains (Supplemental Material, Table S1) used are derived from it through standard genetic procedures. Plasmids were generated by standard procedures and are listed in Table S2. Oligonucleotides (Sigma-Aldrich, St. Louis, MO) used in this study are listed in Table S3.

Yeast growth conditions

Yeast was grown either in standard yeast extract peptone (YEP) media containing 2% dextrose or galactose or in synthetic complete media lacking amino acids (Sunrise Science) as required. G418 (0.67 mg/ml), hygromycin B (325 U/ml), or clonNAT (100 µg/ml) was added to YEP plus dextrose media to select for knockouts. Cells were incubated at 30° unless otherwise indicated. The dcp2-7 cultures were incubated for 60 or 90 min at 37° to inactivate the decapping enzyme for the GAL mRNA stability and transcriptome sequencing experiments, respectively.

To induce sporulation, diploid cells were grown in nutrient-depleted media for 4–5 days. Sporulated cells were resuspended in water with Glusulase (Perkin Elmer). This reaction was incubated at 30° for 30 min. Ascus digestion was terminated using water. Haploids were obtained either by tetrad dissection or by random spore isolation (Rockmill et al. 1991). The dcp2∆ spores from the starting haploid formed pinprick-size colonies after 2 weeks of incubation at room temperature.

Experimental evolution was initiated from four haploid dcp2∆ strains, each derived from a different tetrad. Duplicate 5 ml cultures of each of the four haploid dcp2∆ strains were inoculated in YEP containing 2% dextrose, G418 (167 mg/liter), and ampicillin (50 mg/liter). Cultures were grown at 30° until the OD600 of the culture reached > 8.5. Then, 10 µl of this culture (containing on the order of 106 yeast cells) were transferred into 5 ml of fresh media of the same type. This culturing and 500-fold dilution was repeated 30 times. A 500-fold (or 28.97) dilution represents ∼9 doublings. Thus, after 30 cycles of culture and dilution the cultures had gone through ∼270 generations.

For the growth assay on solid media, exponentially growing cells were serially diluted (fivefold) and spotted on the indicated media. For the growth assay in liquid media, exponentially growing cells were diluted to OD600 of 0.1, and transferred to a 96-well plate. Cells were incubated at 30° in a BioTek′s Synergy Mx Microplate Reader. OD600 was measured every 10 min for ∼15 hr. Collected data were processed through Gen5 (BioTek).

Microscopy

To examine cell morphology, exponentially growing cells were analyzed on an Olympus BX60 microscope.

Whole-genome sequencing analysis

Total genomic DNA was isolated from exponentially growing cells using a phenol-chloroform extraction method and further purified with the use of a DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA) and a MasterPure Yeast DNA Purification Kit (Lucigen). PE150 libraries of the evolved strains were prepared and sequenced by Novogen.

To identify mutations, sequencing reads were trimmed with Trim Sequences (http://hannonlab.cshl.edu/fastx_toolkit/), quality checked with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and mapped with Bowtie2 (Langmead et al. 2009) to Saccharomyces cerevisiae reference genome R64-1-1 (www.ensembl.org). The overall alignment rate was ∼91–99%. Before calling variants, BAM data sets for the individual dcp2Δ strain and heterozygous diploid DCP2/dcp2Δ strain were merged using MergeSamFiles (http://broadinstitute.github.io/picard/). Data sets were further processed for left realignment through BamLeftAlign (https://arxiv.org/abs/1207.3907). To call all the variants, we used FreeBayes (https://arxiv.org/abs/1207.3907) for detection and SnpEff 4.3 (Cingolani et al. 2012) for annotation. Integrated Genome Viewer (https://software.broadinstitute.org/software/igv/download) was used to inspect candidate SNPs. True mutations were differentiated from sequencing errors and preexisting SNPs by being supported by the consensus of the reads in the evolved isolate(s), but not by the reads from the other evolved isolates or the heterozygous diploid DCP2/dcp2Δ strain that we had previously sequenced. The vast majority of preexisting SNPs that we identified in the DCP2/dcp2∆ starting diploid have previously been described in the genome sequences of BY4741 (http://sgd-archive.yeastgenome.org/sequence/strains/BY4741/) and/or BY4742 (http://sgd-archive.yeastgenome.org/sequence/strains/BY4742/). BY4741 and BY4742 are the parents of the diploid BY4743 strain, which in turn is the parent of our DCP2/dcp2∆ starting diploid.

MiModD Deletion Calling (https://sourceforge.net/projects/mimodd/) was used to search for deletions, which identified the ura3∆, his3∆, met15∆, lys2∆, and leu2∆ deletions of BY4743, but no other deletions. MiModD Coverage Statistics (https://sourceforge.net/projects/mimodd/) was used to measure coverage depth by chromosome, to search for aneuploidy.

Protein analysis

Total protein was extracted in IP50 buffer [50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 2 mM MgCl2, 0.1% Triton X-100] with 0.007% β-mercaptoethanol and 0.00174% PMSF, and complete protease EDTA-free mini tablet (Roche) by bead beating and analyzed by Western blot. Blots were probed with anti-Kap123 at 1:5000 (Patel and Rexach 2008; Floch et al. 2015) and anti-Pgk1 at 1:10,000 (Invitrogen, Carlsbad, CA), and developed using Amersham ECL Prime (GE Healthcare). Images were acquired and analyzed using an ImageQuant LAS 4000 biomolecular imager (GE Healthcare) and ImageQuant TL image analysis software.

RNA analysis using Northern blotting

For analyzing the steady-state RNA level, cells exponentially growing at 30° were harvested. For analyzing RNA stability, dcp2-7 mutants were grown in YEP containing 2% galactose at 21° and transferred into a 37° incubator for 1 hr to inactivate the decapping enzyme. Cells were washed with YEP, and dextrose (40% stock solution) was added to a final concentration of 2% to repress transcription of the GAL genes. Although cells were incubated at 37°, samples were collected at the indicated time points and immediately frozen.

For RNA preparation, the harvested cell pellet was lysed by vortexing with glass beads. RNA was purified through two rounds of phenol/chloroform/LET (LiCl-EDTA-Tris HCl, pH8.0) and one additional chloroform extraction, and ethanol precipitated.

Total RNA was analyzed through Northern blotting. Briefly, 10 μg of total RNA was analyzed by electrophoresis on denaturing gels, either 1.3% agarose/formaldehyde gels for mRNA analysis or 6% polyacrylamide (19:1) 8M urea gel for transfer RNA (tRNA) analysis, as indicated. RNA was transferred to a nylon membrane and probed with 32P 5′ end labeled oligonucleotides. For the Northern blots on mutant tRNAs, we prevented differences in detection efficiency by using probes that did not overlap with the mutations. Blots were imaged by phosphorimaging on a Typhoon FLA 7000 (GE Healthcare), and quantitated using ImageQuant software.

Finding kap123 mutations in published RNA-seq data

To determine whether previously published RNA-seq experiments inadvertently used kap123 mutant strains and to identify the mutations, we downloaded raw RNA-seq reads from the European Bioinformatics Institute (https://www.ebi.ac.uk/ena). Reads were trimmed with Trim Galore!, quality checked with FastQC, and then aligned with TopHat2 to the R64-1-1 reference genome. Aligned reads were analyzed in Integrated Genome Viewer. This identified the kap123-Y687X in three data sets from a dcp2∆ W303 strain (SRR4163304, SRR4163305, SRR4163306). For the dcp1∆ data sets (SRR4163301, SRR4163302), the TopHat alignment suggested a small deletion, but failed to precisely identify it. Aligning the same data sets with Bowtie2 in very sensitive local mode did precisely identify the deletion as a 21-bp deletion mediated by a GCGGAACC repeat in the wild-type gene. We found no mutations in the dcp1∆ or dcp2∆ strains for any of the other genes that are mutated in our evolved isolates. Similarly, analyzing the dcp2∆ RNA-seq data from reference (Geisler et al. 2012) (SRR364981), we identified the same tl(gag)g mutation as in our evolved isolates 4-1 and 4-2 and a novel kap123 mutation. We did not find kap123 mutations in wild-type controls (SRR4163289, SRR4163290, SRR4163291), xrn1∆ (SRR4163307, SRR4163308, SRR4163309), dcp2-7 (SRR2045250, SRR2045251, our RNA-seq data), dhh1∆ (SRR6362787), pat1∆ (SRR6362781), lsm1∆ (SRR6362784), dcp2-N245 (SRR6362793), dcp2-N245-E153Q (SRR6362796), dcp2-N245-E198Q (SRR6362799), scd6∆ (SRR7162931), caf1∆ (SRR7174202), or dhh1∆ (SRR3493892, SRR4418659) (Radhakrishnan et al. 2016; Wery et al. 2016; Celik et al. 2017; Jungfleisch et al. 2017; He et al. 2018; Webster et al. 2018; Zeidan et al. 2018). This suggests that only very severe decapping defects select for kap123 suppressors.

Data availability

The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article. RNA-seq data are available at the Sequence Read Archive under project number PRJNA626686. Supplemental data are available at https://doi.org/10.6084/m9.figshare.12985820. All yeast strains and plasmids used are available upon request.

ResultsDCP2 is required for normal growth of yeast

Although DCP2 is annotated as an essential gene in the Saccharomyces Genome Database (yeastgenome.org), other studies reported that DCP2 is not an essential gene (Dunckley and Parker 1999; Giaever et al. 2002; Geisler et al. 2012; He and Jacobson 2015). The genome-wide effort to identify essential genes was based on sporulating a heterozygous diploid DCP2/dcp2∆ strain and attempting to recover haploid dcp2∆ strains. We obtained this same commercially available heterozygous diploid DCP2/dcp2∆ strain and repeated sporulation and tetrad dissection (Figure 1A). We expected that this would produce viable wild-type and inviable dcp2∆ progeny in a 1:1 ratio. However, upon prolonged growth we isolated viable dcp2∆ (34%) along with wild-type (51%) and inviable dcp2∆ progeny (14%) (Figure 1B). Although we were able to recover viable dcp2∆ progeny, these spores formed much smaller colonies even after prolonged incubation.

Figure 1

Isolation of viable dcp2∆ cells with severe growth and morphological defects. (A) Diagram of tetrad analysis of heterozygous diploid DCP2/dcp2∆ strain. (B) Tetrad dissection results in wild-type and dcp2∆ colonies. If DCP2 is essential as annotated, 50% of the spores would be expected to be inviable. Instead 34% of the spores analyzed were dcp2∆ and viable. (C) dcp2∆ colonies resulting from tetrad dissection grow slowly. Serially diluted wild-type and viable dcp2∆ colonies from B were spotted on YPD solid media and grown at 30° for the indicated times. (D) dcp2∆ cells resulting from tetrad dissection have morphological defects. Cells were grown at 30° until OD600 reaches 0.3–0.4. Samples were diluted in YPD and examined by light microscopy. Bar represents 10 µm.

To further examine the growth and morphology of the recovered dcp2∆ strains, they were serially diluted, spotted on YPD, and cultured at 30°. Although viable, the dcp2∆ strains grow extremely slowly compared to wild type (Figure 1C). Examination through light microscopy revealed irregular and heterogeneous cell morphology, with many elongated cells in clumps (Figure 1D). Additionally, multiple vacuole-like organelles of different sizes accumulated in these cells. Taken together, this suggests that DCP2 is required for normal growth and morphology of budding yeast (see Discussion).

Experimental evolution of dcp2∆ strains results in improved growth and morphology

To understand the function of DCP2 that affects cell growth, we decided to identify suppressors of the growth defect of dcp2∆. We used an experimental evolution approach that allows cells to accumulate mutations and enriches for suppressors that are advantageous for fitness in the absence of DCP2. For this, we used four haploid progeny, each derived from a different tetrad (meiosis). Each haploid dcp2∆ strain was used to start duplicate liquid cultures. Once these cultures reached saturation, we diluted them into new media for several iterations (Figure 2A). Throughout the experimental evolution process, growth of all dcp2∆ populations was examined both by spotting serially diluted cultures on solid media (data not shown), and by measuring OD600 of cells growing in liquid media (Figure S1). During the course of the experimental evolution, we observed growth improvement at the 90th generation, and further growth improvement was observed at the 180th generation (Figure S1). However, in most cases, the growth improvement from 180 to 270 generations was minimal (Figure S1). Thus, we stopped the experimental evolution process after ∼270 generations and further analyzed these dcp2∆ populations (evolved dcp2∆). All eight evolved dcp2∆ populations grew better than their parental nonevolved dcp2∆ strain, although not as well as the wild-type strain (Figure 2B). The doubling time of the eight evolved dcp2∆ populations is 1.5- to 2-fold longer than that for the wild-type strain, but much shorter than the doubling time of four nonevolved dcp2∆ strains, which could not be calculated in the 16-hr period of the experiment because of the extremely slow growth. Similar to the growth improvement, the morphological defects in nonevolved dcp2∆ strains are partially restored in evolved dcp2∆ populations (Figure 2C and Figure S2). Evolved dcp2∆ cells had a more homogenous morphology, were less elongated, and less clumped compared to nonevolved dcp2∆ cells. These results suggest that the experimental evolution of dcp2∆ successfully selects suppressor mutations that confer growth improvement on dcp2∆ strains.

Figure 2

Experimental evolution of dcp2∆ strains results in growth and morphological improvement. (A) Diagram of experimental evolution. Four dcp2∆ strains (middle) isolated from distinct tetrads (left) were subject to serial passage. Two replicate samples of dcp2∆ strains were transferred to new media in iterations until the growth rate increases (right). (B) The dcp2∆ growth defect is partially improved in evolved dcp2∆ populations (blue) compared to nonevolved dcp2∆ strains (green). Cells were grown at 30° and OD600 was measured every 10 min for ∼15 hr. Shown is the average OD600 from replicate cultures and their standard deviations, plotted on a log scale. n = 8 for DCP2, n = 4 for each nonevolved dcp2∆, and n = 2 for each evolved dcp2∆. (C) Morphological defects are partially restored in evolved dcp2∆ populations. A representative microscopic image of an evolved dcp2∆ population is shown. Bar represents 10 µm.

Whole-genome sequence analysis identifies suppressors of dcp2∆ growth defects

To identify suppressor mutations that confer growth improvement to dcp2∆ we performed whole-genome sequence (WGS) analysis on evolved dcp2∆ strains. We suspected that the evolved populations were genetically heterogeneous, which complicates the analysis and interpretation of WGS. Thus, for each evolved dcp2∆ population, we picked a single colony to generate eight genetically homogeneous evolved dcp2∆ isolates. As we observed in the evolved dcp2∆ populations, all eight evolved dcp2∆ isolates grew better than their nonevolved dcp2∆ counterparts (Figure 3A). We then sequenced the genomes of the eight evolved dcp2∆ isolates as well as the starting diploid DCP2/dcp2∆ strain (average genome coverage 112-fold), and identified mutations that were present in the evolved isolates, but not in the starting diploid (Figure 3B and Figure S3A). Each evolved dcp2∆ isolate contains nonsynonymous mutations in two to six genes that are not present in the heterozygous diploid DCP2/dcp2∆ strain. All of them were point mutations, including substitution and deletion/insertion of a small number of bases. We did not detect any larger deletions or aneuploidy (Figure S3B), which is often seen after the deletion of an essential gene (Liu et al. 2015).

Figure 3

Whole-genome sequencing identifies multiple null mutations in KAP123 and tL(GAG)G. (A) The growth defect is partially improved in evolved dcp2∆ isolates. A single colony was isolated from each evolved population (blue) and haploid starting strain (green). Each of these genetically homogeneous strains was serially diluted, spotted on YPD solid media, and grown at 30°. Shown is the growth at day 2. (B) Whole-genome sequences were determined for the eight evolved dcp2∆ isolates and compared to the DCP2/dcp2∆ starting diploid. Nonsynonymous mutations that are not present in the starting diploid are listed. (C) Six of the evolved isolates have null mutations in KAP123 that generate a premature stop codon and they do not express Kap123. A representative Western blot analyzing the expression level of Kap123 (top) from the indicated strains is shown. Pgk1 (bottom) is used as loading control. (D) Three of the evolved isolates have null mutations in tL(GAG)G and do not express mature tL(GAG)G tRNA. A representative Northern blot of tL(GAG)G tRNA (top and middle) from the indicated strains is shown. The top panel is probed with an oligonucleotide complementary to mature tL(GAG)G, while the middle panel is probed with an oligonucleotide complementary to 5′ extended precursors of tL(GAG)G. SCR1 is used for loading control (bottom).

Strikingly, all eight evolved dcp2∆ isolates had a mutation in the KAP123 gene, which encodes one of the 14 karyopherins that mediate nucleocytoplasmic trafficking (Chook and Suel 2011; Aitchison and Rout 2012). In total, six different kap123 alleles were identified from the evolved dcp2∆ isolates. Isolates 2-1 and 2-2 are derived from the same haploid spore and both contained the kap123-E821X nonsense mutation. We conclude that this mutation arose very early, before the duplicate cultures were started. Similarly, each pair of evolved isolates shared at least one mutation, which must have arisen early, but also differed from its sister isolate by additional mutations (see Discussion).

Four kap123 mutant alleles have either a nonsense mutation or a frameshift mutation that generates a premature stop codon, and thus are likely loss-of-function mutations. Two of the mutations are missense mutations, A550V and R1068S, that both affect conserved residues that are structurally important (Figure S4A). Western blot analysis showed that the Kap123 protein was not detectable from the six evolved dcp2∆ isolates harboring nonsense or frameshift mutation, implying destabilization of mRNA, protein, or both. In contrast, Kap123-A550V and Kap123-R1068S were expressed (Figure 3C).

In addition to KAP123, we identified multiple alleles of tL(GAG)G and WHI2 in our evolved isolates. WHI2 encodes a protein involved in stress response and the TOR pathway in yeast (Kaida et al. 2002; Chen et al. 2018). One of the whi2 alleles introduces an early stop codon (whi2-Q20X) and thus is likely a loss-of-function allele. The tL(GAG)G gene encodes leucine tRNA with a GAG anticodon, and both of the mutations are predicted to disrupt tRNA folding (Figure S4B). Northern blot analysis indicated that the mutant tRNA was not expressed (Figure 3D). In addition, pre-tRNA with 5′ extensions appeared more abundant in the mutant, suggesting that the structural perturbations in tL(GAG)G interfere with 5′ end processing by RNase P. The nonevolved dcp2∆_4 strain did not produce the mature tRNA (Figure 3D). In addition, the two evolved dcp2∆ strains derived from the dcp2∆_4 had the same allele, implying that this mutation had already arisen in their common ancestor strain, nonevolved dcp2∆_4 (Figure S12A).

Overall, these data suggest that each of the evolved dcp2∆ isolates contains loss-of-function mutations in KAP123, tL(GAG)G, and/or WHI2, as well as other mutations of unclear significance, and that some of these mutations arose very early, whereas others arose later.

Null mutations of KAP123, tL(GAG)G, or WHI2 are sufficient to restore growth of dcp2∆

For genes that were mutated in multiple evolved dcp2∆ isolates, we tested whether a null mutation of each gene is individually sufficient to suppress dcp2∆ growth defects. Because our WGS indicated that dcp2∆ strains quickly accumulated suppressors, we were careful to minimize selection for undesired spontaneous suppressors that would complicate the analysis of the desired potential suppressors [kap123∆, tl(gag)g∆, and whi2∆]. We therefore started with the heterozygous DCP2/dcp2∆ diploid strain that we had sequenced, and that contained wild-type KAP123, WHI2, and tL(GAG)G genes. We then knocked out one of the alleles of KAP123, WHI2, or tL(GAG)G to generate DCP2/dcp2∆ KAP123/kap123∆, DCP2/dcp2∆ WHI2/whi2∆, and DCP2/dcp2∆ tL(GAG)G/tl(gag)g∆ diploids. Each of these three double heterozygous strains was then transformed with a plasmid that carried functional DCP2 and URA3 genes, and haploid progeny were generated and genotyped. Finally, the strains were grown on media lacking uracil (selecting for the DCP2 URA3 plasmid) or media containing 5FOA (counterselecting against the DCP2