In eukaryotes, Dom34 upregulates translation by securing levels of activatable ribosomal subunits. We found that in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, Dom34 interacts genetically with Pmt1, a major isoform of protein O-mannosyltransferase. In C. albicans, lack of Dom34 exacerbated defective phenotypes of pmt1 mutants, while they were ameliorated by Dom34 overproduction that enhanced Pmt1 protein but not PMT1 transcript levels. Translational effects of Dom34 required the 5′-UTR of the PMT1 transcript, which bound recombinant Dom34 directly at a CA/AC-rich sequence and regulated in vitro translation. Polysomal profiling revealed that Dom34 stimulates general translation moderately, but that it is especially required for translation of transcripts encoding Pmt isoforms 1,4 and 6. Because defective protein N- or O-glycosylation upregulates transcription of PMT genes, it appears that Dom34-mediated specific translational upregulation of the PMT transcripts optimizes cellular responses to glycostress. Its translational function as an RNA binding protein acting at the 5′-UTR of specific transcripts adds another facet to the known ribosome-releasing functions of Dom34 at the 3′-UTR of transcripts.
In eukaryotes, secretory proteins can get O-mannosylated at serine or threonine residues by protein mannosyltransferases (Pmt proteins). This modification occurs during or shortly after translation, during transit across the secretory pore complex into the ER lumen. O-mannosylation initiates the typical type of fungal O-chains, which mature in the Golgi. In mammalian cells O-mannosylation is a rare but important process, while the bulk of O-chains is formed post-translationally in the Golgi [1,2]. Seven and five Pmt isoforms forming Pmt1,2 and 4 subfamilies have been described in the yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans, respectively [1–3]. Pmt isoforms are largely specific for their protein substrates and the lack of the Pmt2 isoform in C. albicans or at least two isoforms in S. cerevisiae prevents growth [3,4]. In C. albicans, each Pmt isoform affects one or another aspect of fungal growth, morphogenesis and virulence [3,5]. Impaired O-mannosylation in pmt1 mutants or upon Pmt1 inhibition of a wild-type strain leads to transcriptional upregulation of PMT2 and PMT4 genes, while inhibition of N-glycosylation by tunicamycin upregulates PMT1 expression [6–8]. In both glycostress conditions, the increased levels of underglycosylated proteins also trigger the unfolded protein response (UPR), e. g. by increasing levels of the Kar2 chaperon and matured HAC1 transcript [6,7]. UPR induction is known to lower overall translation in yeast cells, although translation of some transcripts is increased [9]. In S. cerevisiae, the Dom34 protein is involved in the “no-go decay” (NGD) process, which is one of at least three mechanisms responding to the quality of translated mRNA. NGD relieves stalled translational complexes arising e. g. by secondary structures or rare codons through dissociation of ribosomal subunits and cleavage of mRNA [10,11]. Dom34 also rescues ribosomes that accumulate at the 3′-UTR of transcripts [12,13]. To promote subunit dissociation and peptidyl-tRNA drop-off from stalled ribosomes, Dom34 co-operates with its associated GTPase Hbs1 [10,11]. Very likely, this function of Dom34 is possible because of its high homology to the translational termination factor eRF1, as well as the structural similarities of the Dom34-Hbs1 and eRF1: eRF3 complexes that can occupy the ribosomal A-site. S. cerevisiae strains carrying single mutations for Dom34 or Hbs1 grow normally, while their combination with mutations impairing components of 40S (but not 60S) ribosomal subunits [14], mutations delaying translation by phosphorylation of eIF2 [15] and yet undefined mutations [16], leads to severe impairment of growth. The scarcity of 40S subunits due to “stuck” 80S ribosomes and the resultant impaired translational initiation has been suggested as the mechanism causing slow growth of dom34 hbs1 double mutants [14]. The homology of Dom34 to eRF1 only regards its central and C-terminal domains, while its N-terminal domain adopts a Sm-fold that is characteristic of RNA degradation or recognition domains [17,18]. However, recent results indicated that Dom34 is not the endonuclease that degrades mRNA in stalled ribosomes [19]. Besides NGD, other mechanisms including separation of free 80S ribosomes [14,20] and nonfunctional rRNA decay (NRD) [21,22] depend on Dom34 to maintain a sufficient supply of ribosomes for translation. Based on the previous discovery of a S. cerevisiae mutant with defective protein O-mannosylation [23] we report here a novel function of Dom34 in translational upregulation of the PMT1 transcript in C. albicans. By promoting the translational initiation of the PMT1 transcript, which under glycostress is strongly increased [6–8], Dom34 contributes to optimize the overall output of Pmt1 activity that helps to recover from damage to its glycostructures. Its mode of action as an RNA binding protein for specific transcripts differs from the previously described general roles and mechanisms of Dom34 in promoting translation in eukaryotes. By this action, Dom34 functionally links two essential processes in eukaryotic cells, translation and O-mannosylation.
Previously, a S. cerevisiae mutant (M577) defective in O-mannosylation of a heterologous protein (hIGF-1) and some homologous secretory proteins had been identified [23]. The mannosylation defect was recessive and segregated 2: 2 in crosses to a wild-type strain suggesting that it was caused by mutation of a single gene. Because M577 did not show easily scorable phenotypes, pmt1 or pmt2 mutations were introduced to explore synthetic phenotypes with mutations affecting O-mannosylation. This approach was led by the finding that pmt1 pmt2 double mutants, but not the single mutants, are resistant to the K1 killer toxin [24]. In agreement, we found that Pmt+- strains YE449 and mutant M577, as well as the pmt1 and pmt2 single mutants were toxin-sensitive (blue/dark appearance of colonies on indicator plates), while the pmt1 pmt2 double mutant was completely resistant to the toxin (white appearance of colonies on indicator plates) (Fig 1Aa). Importantly, similar to the pmt1 pmt2 double mutant, the pmt1 derivative of mutant M577 but not of the parental strain YE449 was completely toxin-resistant. Because the pmt2 derivative of M577 retained sensitivity, the results indicated that the unknown mutation in M577 generates a synthetic protein-O-mannosylation phenotype in combination with a pmt1 but not a pmt2 mutation. To identify the mutation in M577, its pmt1 derivative was transformed with a genomic bank in vector YEp13 and 80,000 transformants were screened for re-appearance of sensitivity to K1 killer toxin. Among 78 initial transformant isolates, 17 carried the complementing activity on a genomic insertion within the bank plasmid. Two genomic loci occurred repeatedly in overlapping inserts (Fig 1B): 5 plasmids contained a region close to the centromere of chromosome XIV and 4 plasmids contained a region close to the centromere of chromosome IX. In both cases, the overlapping clones had a single gene in common: the chromosome XIV clones contained DOM34, while the chromosome IX-clones contained YIL001w. To clarify if the K1 killer-resistance phenotype in strain M577 pmt1 was caused by mutation of DOM34, we introduced pmt1, dom34 and yil001w mutations singly or in combination into the genetic background of the parental strain YE449. At variance with an initial report [16] but in agreement with a subsequent report [15] we found that dom34 single mutants did not show significant growth defects. Importantly, we detected that the pmt1 dom34 mutant but not the pmt1 yil001w mutant was killer-resistant, resembling the M577 pmt1 strain (Fig 1Ab). Transformation with either PMT1-, DOM34- or YIL001w-overexpressing vectors restored killer-sensitivity of the pmt1 dom34 mutant (Fig 1Ac). These results suggested that in the parental strain M577, the DOM34 gene is mutated, while YIL001w may represent an extragenic suppressor of the pmt1 and dom34 mutations. In support of this conclusion we found that a diploid constructed from haploids M577 pmt1 and W21 (YE449 pmt1 dom34) was unable to sporulate, as expected for a homozygous dom34 diploid [15]. Furthermore, sequencing of the DOM34 ORF in mutant M577 revealed that it is mutated by insertion of a single T residue following position 366 generating a UAA stop codon leading to a truncated protein of 122 residues. The above results had suggested that in S. cerevisiae, the activities of specific elements involved in translation (Dom34) and protein-O-mannosylation (Pmt1) are functionally linked. We subsequently found that this cooperation not only prevents resistance (to killer toxin K1) but also enhances resistance to hygromycin B (HygB). HygB is an aminoglycoside antibiotic known to block translation [25], which is particularly active to block growth of glycosylation mutants [26]. dom34 pmt1 double mutants were significantly more sensitive than the pmt1 single mutant, although the dom34 single mutant did not show any sensitivity phenotype (Fig 1C). To generalize Dom34-Pmt1 functional interactions and because S. cerevisiae contains a DOM34 homolog (YCL001W-B) with unknown activity, we also studied the single DOM34 gene in the human fungal pathogen C. albicans that contains a family of well-studied Pmt proteins [3]. A homologue of S. cerevisiae DOM34, ORF19. 2419 in the C. albicans genome encodes a protein with 36%, 39% and 40% sequence identity to Dom34 proteins of S. cerevisiae, S. pombe and human, respectively. Sequence similarities are observed in the 3 domains of these proteins including a Sm-fold in domain 1 and a sequence in domain 3, which were both suggested to bind RNA (S1 Fig [13,15]). Compared to its homologues, the CaDom34 protein lacks a potential NLS sequence (position 173–177 in ScDom34p). Strains were constructed that lack both alleles of CaDOM34 in the wild-type background (SK47), in the pmt1 background (SK24) and in the pmt5 background (JH8-5-11) (disruption scheme in S2 Fig). Phenotypes were determined using mutant strains, in which URA3 was reconstituted at its authentic locus [27]. Similar to S. cerevisiae, a homozygous dom34 single mutation did not generate significant growth or morphogenetic defects in C. albicans; furthermore, this mutant was found not to be supersensitive to numerous tested antibiotics or inhibitors including HygB. On the other hand, the HygB-supersensitive phenotype of the pmt1 mutant was significantly increased by an additional dom34 mutation (Fig 1C). Thus, evidence in both yeast species supported a functional link between Dom34 and Pmt1 proteins to generate HygB resistance. However, for S. cerevisiae it cannot be excluded that the DOM34 paralog YCL001W-B contributes to this phenotype. Therefore, and because of its importance as a human pathogen, we focused subsequent analyses on the C. albicans DOM34 gene. The genetic interaction of dom34 and pmt1 mutations in C. albicans prompted experiments to study effects of DOM34 overexpression in this fungus. The DOM34 transcript level was determined by qPCR and showed an equal amount in homozygous pmt4, pmt5, pmt6 and heterozygous pmt2/PMT2 mutants as in the wild-type strain CAF2-1 but surprisingly, a 2–3 fold lower level in the pmt1 mutant (Fig 2A; S3 Fig). A transformant of the pmt1 mutant carrying plasmid pSK2 (MET3p-DOM34FLAG) that was grown in SD medium contained about sixfold higher DOM34 transcript levels than the untransformed strain and about threefold higher levels than the wild-type strain. Thus, using pSK2, a moderate overexpression of DOM34 was achieved in C. albicans. Overexpression of DOM34FLAG was able to rescue several known pmt1 mutant phenotypes [3]. The inability of pmt1 mutant colonies to form hyphae was partially suppressed by DOM34 overexpression, which was observed in > 80% of the colonies (Fig 2B). Furthermore, the sensitivity of pmt1 mutants to HygB and to high temperature (42°C) was also partially suppressed (Fig 2Ca). Interestingly, however, suppression was not achieved using a derivative of pSK2 carrying a point mutation in the DOM34 ORF that encodes the E21A variant Dom34 protein (Fig 2Cb). It has been suggested that this residue is important for RNase activity of Dom34 proteins [18]. We asked next if the suppression of pmt1 mutant phenotypes by DOM34 overexpression depended on the Pmt isoforms remaining in this strain. For this purpose, we constructed pmt1 pmt5 and pmt1 pmt6 double mutants, which both showed the supersensitive pmt1 mutant phenotype (Fig 2Cc and 2Cd), while single pmt5 or pmt6 mutants are not supersensitive to HygB [3]. Interestingly, double mutant transformants carrying the DOM34 overexpression plasmid pSK2 did not show any recovery of pmt mutant phenotypes (Fig 2Cc and 2Cd). This result indicates that Pmt5 and Pmt6 isoforms are required for the observed rescue by Dom34 overproduction. The relevance of the Pmt2 and Pmt4 isoforms could not be tested in this manner, because PMT2 is an essential gene and pmt1 pmt4 double mutants are not viable [3]. These experiments also revealed that unexpectedly, a C. albicans strain lacking all members of the Pmt1 subfamily (Pmt1 and Pmt5) is fully viable, although in S. cerevisiae heteromeric Pmt1-Pmt2 or Pmt1-Pmt5 complexes have been described to be essential for growth [28]. Conceivably, Dom34 could have suppressed Pmt1 deficiencies by several mechanisms, especially by increasing transcription/transcript levels of several PMT genes including PMT5 and/or PMT6, as suggested by the above experiments (Fig 2C). To explore this notion, we determined transcript levels in the DOM34 overexpression strain and found that none of the PMT transcript levels was increased (S4A Fig). In addition, the absence of Dom34 did not decrease PMT transcript levels, while PMT2/PMT4 transcript levels were increased in the pmt1 dom34 double mutant, as described for the pmt1 single mutant [6] (S4B Fig). Thus, increases and decreases of Dom34 levels were not related to PMT transcript levels. To explore effects on Pmt protein levels a C. albicans strain was constructed, in which one PMT1 allele was fused to sequences encoding the hemagglutinin (HA) -epitope (Fig 3), which was subsequently transformed with the DOM34 overexpression vector pSK2 or the corresponding empty vector. This strain produced considerably higher Pmt1HA protein levels as compared to a transformant carrying an “empty” control vector (Fig 3A). Scanning of band intensities revealed that DOM34 overexpression increased the mean Pmt1-HA/actin ratio 1. 78 fold (p = 0. 025). The 5′-UTR of the PMT1 transcript encompasses 190 or 218 nt [29,30] and contains an intriguing CA/AC-rich sequence, which is ordered into three overlapping 11-mer ACAACCACAAC repeats between nt -157 to -179 (Fig 3B, top). To examine if this sequence is involved in overproduction of the Pmt1 protein by DOM34 overexpression we generated genomic fusions containing two different lengths of the PMT1 upstream region joined to the RLUC reporter gene. One fusion did not contain most 5′-UTR sequences (pPdC3-HIS), while the second contained one full and one half of the 11-mer repeat (pPdC2-HIS) (Fig 3B top). These strains were transformed with the DOM34 overexpression plasmid pSK2 or a control plasmid and luciferase activity of the double transformants was determined. The results revealed that the construct containing 5′-UTR sequences including the 11-mer repeat was stimulated significantly by DOM34 overexpression, while no activation occurred for the construct lacking UTR sequences (Fig 3B bottom). As stated above, the C. albicans Dom34 protein lacks a consensus NLS sequence, suggesting that its primary action takes place outside of the nucleus. Furthermore, differential centrifugation of cell extracts identified HA-tagged Dom34HA to a large extent in the soluble fraction (cytoplasm) (S5 Fig). Collectively, the results suggest that Dom34 overproduction stimulates translation of Pmt proteins. The site of the Dom34 stimulatory activity appears to lie in a specific sequence within the 5′-UTR of target transcripts, as exemplified by the Dom34-mediated regulation of the PMT1-RLUC fusion. We next carried out polysome analyses to establish the role of Dom34 in translation of PMT1 and the ACT1 housekeeping transcripts. For this purpose, cellular lysates of the control strain CAF2-1 and the dom34 mutant JH47-2 were separated by sucrose gradient centrifugation to establish polysomal profiles (Fig 4A). Profile comparisons of both strains revealed that pre-polysomal fractions containing 40S, 60S and 80S rRNA were more pronounced in the dom34 mutant than in the control strain; furthermore, polysomal peaks were lower in the mutant and decreased at >2n polysomes, whereas in the control strain, the 3n peak was even greater than the 2n peak. These results indicate that in the dom34 mutant, translational efficiency is generally but moderately reduced. To examine the efficiency of PMT1 translation in control and dom34 mutant cells, the fractions of the polysomal gradient were examined for the presence of the PMT1 transcript by RT-qPCR, using a spiked-in control RNA as a reference. The result indicates clearly that the PMT1 transcript in the dom34 mutant is found predominantly in the pre-polysomal fraction, while in the control strain, significantly higher amounts reside in the polysomal fractions (Fig 4B). To establish if lower translational efficiency in the mutant concerns only the PMT1 transcript, we also examined the transcript profile for the PMT4 and PMT6 transcripts, which also were enriched in the polysomal fraction in the presence of Dom34 (S6A Fig) indicating that Dom34 positively regulates the translation of several PMT transcripts. In comparison, translation of the transcript for the housekeeping gene ACT1 appeared less affected by Dom34. These effects were quantitated the Kolmogorov-Smirnov test [31 (Fig 4C). By this algorithm, the calculated Dom34-dependent enrichment of transcripts in the polysomal fraction showed a statistically significant increase for the PMT1 transcript (D = 0. 6, p-value = 0. 031), the PMT4 transcript (D = 0. 5, p = 0. 042) and for the PMT6 transcript (D = 0. 5, p = 0. 066); in contrast, a lower and insignificant enrichment was calculated for the ACT1 transcript (D = 0. 3, p = 0. 675) (Fig 4C). Collectively, the results suggest that Dom34 generally enhances but is not absolutely required for translation in C. albicans. The degree of Dom34-mediated translational enhancement differs between transcripts and may particularly affect specific groups of transcripts including transcripts for different Pmt isoforms. Interestingly, the 5′-UTR of all PMT transcripts (but not the 5′-UTR of the ACT1 transcript) contains at least one CAAC motif, which in the above-described ACAACCACAAC repeat region of the PMT1 5′-UTR occurs eight times (S6B Fig). Conceivably, the positive action of Dom34 on translation of PMT transcripts is mediated by this sequence. Recombinant Dom34 (S7 Fig) was added to a rabbit reticulocyte in vitro translation system, using RNA carrying the coding region for click beetle green luciferase (CBGluc), either containing or not containing the PMT1 5′-UTR (Fig 5Aa). As expected, protein products of identical molecular masses of 60 kDa were obtained for both proteins (Fig 5Ab). Additional experiments indicated that the presence of the 5′-UTR augmented CBGluc biosynthesis, while added Dom34 reduced production, if the 5′-UTR was present, but not in its absence (Fig 5Ba). These effects were quantitated by measuring CBGluc luminescence of the samples, which demonstrated that in the presence of Dom34, for RNA containing the 5′-UTR, enzyme activity was decreased by about 40% in three independent measurements (Fig 5Bb and 5Bc). Next, using translational assays containing varying amounts of Dom34 protein, it was shown that already at 0. 1 μM, Dom34 reduces translation efficiency significantly, while it fully inhibits translation at 0. 25 μM. At higher concentrations of Dom34, CBGluc production increases and again to diminishes at 2. 5 μM. These results demonstrate clearly that Dom34 is able to strongly influence translational activity via the PMT1 5′-UTR sequence. Use of a heterologous in vitro system may explain the concentration-dependent, negative rather than the expected positive action of Dom34 on translation. To clarify the mechanisms how Dom34 influences translation we carried out electrophoretic mobility shift assays (EMSA) with recombinant Dom34 of the complete PMT1 5’-UTR sequence. The 5′-UTR was obtained by run off in vitro transcription using T7 polymerase using pRG01 as the template, which was cut by BglII at the 3′-end of the UTR. The 3′-[32PpCp] end-labelled RNA was incubated with increasing amounts of Dom34, separated by native PAGE (5% acrylamide) and examined by autoradiography. The 5′-UTR RNA migrated very slowly compared to the control 6S RNA from E. coli which is not much shorter and has a well-known compact secondary structure [32] (Fig 6A). This suggests a more bulky secondary structure for the PMT1 5’-UTR. At concentrations above 0. 3 μM, the binding of Dom34 to the UTR became visible resulting in two retarded complexes (I and II). The second complex appeared at high Dom34 concentrations suggesting that multiple proteins are bound to one RNA molecule. At 2. 5 μM of Dom34, no free RNA remained suggesting that all of the UTR was bound or degraded; in contrast, little binding of Dom34 (or Dom34E21A) to the 6S control RNA was observed (S8A and S8B Fig). Interestingly, starting already at very low concentrations of Dom34 (150 nM) some smaller-size degradation fragments of the UTR were observed (asterisks). Because the degradation products did not appear as sharp bands during native gel electrophoresis, we analyzed the same samples by denaturing gel electrophoresis (Fig 6B, right panel). Autoradiography clearly showed that already at 0. 1 μM Dom34 the 5′-UTR is partially degraded and that the degradation products have distinct lengths of 1 to 40 nt and around 100 to 120 nt suggesting endonucleolytic cleavage rather than 3’ or 5’ exonucleolytic degradation. Exonuclease-mediated degradation from the 3′- end would have removed the 3′-labeling, which would have decreased amounts of full-length UTR RNA, while degradation from its 5′-end would have generated a smear of cleavage products. However, the results of denaturing gel electrophoresis indicate that the slight decrease of full-length UTR occurring at Dom34 concentrations > 0. 6 μM is solely due to endonucleolytic fragmentation at specific sites. Under denaturing separation conditions protein-UTR interactions are disturbed, revealing the presence of full-length UTR that is not seen under native conditions. This result also provides an indirect proof for UTR binding by Dom34 at higher concentrations. The 5′-UTR structure predicted by the RNAfold program (http: //rna. tbi. univie. ac. at/cgi-bin/RNAfold. cgi) indicated that it contains several single-stranded regions including the presumed Dom34-binding CA/AC-rich sequence, as well as two double-stranded regions that compact the structure (S9A and S9B Fig). In the absence of Dom34, cleavage with single strand-specific RNase U2 confirmed that the CA/AC-region is unpaired, while in the presence of Dom34, this region was protected from RNase digestion, consistent with Dom34 binding in this region (S9C Fig). Furthermore, besides cutting the 5′-UTR at position 80–100 from 5′-end, binding of Dom34 made paired regions more accessible to RNase attack, thus indicating that significant rearrangement of the 5′-UTR had occurred during Dom34 binding. Specific sequences in Dom34 homologs have been suggested to be important for the translational functions of Dom34 [17–20,33]. A conserved glutamic acid residue in domain 1 (E21 in CaDom34) has been suggested to be important for RNase activity [18] and the importance of E21 for CaDom34 function was already shown above by the demonstration that the E21A variant is unable to suppress pmt1 phenotypes (Fig 2C). To test effects of the E21A mutation on binding or cleavage of the PMT1 UTR the Dom34-E21A variant was recombinantly produced in E. coli. Binding of Dom34-E21A to purified 5’-UTR was observed at very low protein concentrations (> 0. 1 μM Dom34); estimation of an apparent KD of about 100 nM deduced from these results demonstrates the very high affinity of Dom34-E21A to the UTR RNA, which is greater than the affinity observed for the wild-type Dom34 protein (Fig 6C). Importantly, minimal UTR cleavage is seen at concentrations > 0. 6 μM of Dom34-E21A. The denaturing gel confirms this result, because only at 1 μM Dom34-E21A minimal degradation products become visible, while the amount of full-length RNA is not changed significantly (Fig 6B, left panel). We conclude that Dom34 is an RNA binding protein, which favours certain RNA targets including the PMT1 5′-UTR. Furthermore, Dom34 has the capacity to endonucleolytically cleave bound target RNA and requires its E21 residue for this function. The above experiments had indicated that Dom34 stimulates translation (Fig 4) and that a specific sequence containing three overlapping 11-mer repeats within the 5′-UTR of the PMT1 is involved in this function (Fig 3). To explore if Dom34 directly interacts with this sequence we tested interaction of a corresponding RNA oligomer containing the repeat sequence with Dom34 protein in EMSA. These experiments showed a single retarded complex (complex I) with wild-type Dom34, which in the presence of lauryl sarcosinate (asterisks) split into two retarded complexes (I, II) (Fig 7). In contrast, BSA as a control protein did not bind the RNA oligonucleotide and the labelled RNA oligomer could be competed out using a 100-fold excess of unlabeled oligomer. The E21A Dom34 variant showed even enhanced binding compared to native Dom34, supporting its binding behavior to the full-length 5′-UTR (Fig 6). Furthermore, the Dom34N317A variant containing a mutation of a key residue in a sequence with high homology to RNA binding proteins [15] also bound to the 5′-UTR RNA oligomer as native Dom34. All Dom34 versions also led to a slight partial degradation of the labelled RNA oligonucleotide (smear emanating from free oligonucleotide); however, because a similar pattern was observed using the BSA control protein, it appears that no additional RNase activity is associated with Dom34.
By NGD, NRD and 80S release mechanisms, Dom34 and co-regulatory molecules including Hbs1 maintain sufficient numbers of ribosomes and thereby assure efficient translation in eukaryotes [10,14]. However, recent in vivo results have indicated that Dom34 affects ribosome occupancy at only 11% of all genes and it is yet unknown, why stalled ribosomes on some transcripts are resolved by Dom34, while other transcripts with similar structural impediments are not affected [13]. A restricted rather than a general function of Dom34 in translation was also suggested by the finding that S. cerevisiae dom34 mutants do not show a general growth phenotype in all genetic backgrounds [15,16]. Thus, the target specificity of Dom34 for specific transcripts remains to be clarified. Results presented here indicate for the first time that Dom34 can serve as an RNA binding protein that could enhance translation of specific transcripts. These conclusions were obtained using the yeast C. albicans as experimental organism, which contains a single allele for a Dom34 protein with high similarity to orthologs in other organisms, while S. cerevisiae, because of its ancient genome duplication [34], contains in addition to DOM34 a paralog (YCL001W-B) of unknown function. Our results indicate on the one hand a general function of Dom34 on translation, since polysome gradients in the dom34 mutant showed an increase of monosomes and decrease of polysomes, as compared to a wild-type strain. Furthermore, the abundance of the housekeeping ACT1 transcript encoding actin was shifted slightly to the monosomal fraction in the dom34 mutant suggesting reduced translation. However, the general translational effects of Dom34 appear to be moderate, since growth or morphogenesis of C. albicans was not affected in unstressed conditions. In contrast, in pmt1 mutants with defective O-mannosylation that lack a major isoform of Pmt proteins [3], the contribution of Dom34 to growth phenotypes was clearly apparent. Protein-O-mannosylation is essential for fungal growth and its absence triggers the UPR response, because of the accumulation of underglycosylated, wrongly folded proteins in the ER lumen [6,7]. The HAC1 transcript has recently been identified as a specific target of Dom34, which releases ribosomes stuck at the 3′-UTR [13]. One possible scenario explaining the genetic interaction of mutations in DOM34 and PMT1 is that Dom34, by its ribosome releasing function, assures efficient translation of the mature HAC1 transcript. By this action, UPR responses could stimulate growth of pmt1 mutant cells. Aside from this general activity for maintenance of ribosome levels, our results suggest a specific stimulatory function of Dom34 on the translation of certain transcripts. As shown by the polysome profiling experiments, the translation of the PMT1, PMT4 and PMT6 transcripts were more strongly affected by the presence of Dom34 than that of the ACT1 transcript. Furthermore, Dom34 binding to the 5′-UTR of the PMT1 transcript and activation of a reporter gene by this sequence supported a direct positive role of Dom34 on the translational initiation of the PMT1 transcript. This action is consistent with lowered Pmt1-mediated O-mannosylation of a heterologous protein (hIGF-1) during its massive overproduction in a dom34 single mutant of S. cerevisiae [23]. In this overproduction condition, Dom34-mediated improvement of Pmt1 activity appears to be needed to obtain full O-mannosylation of target proteins. On the other hand, DOM34 overexpression only rescued pmt1 mutant phenotypes, if PMT5 and PMT6 genes were present, suggesting that the posttranscriptional stimulatory action of Dom34 is not exclusive for the PMT1 transcript but applies to transcripts of several PMT genes, which was indeed confirmed for the PMT4 and PMT6 transcripts. Dom34 activity may be especially needed for translation of PMT2 and PMT4 transcripts, which in a compensatory response are upregulated in pmt1 mutants [7]. On the other hand, stalled ribosomes were not found previously in a dom34 mutant at any of the seven S. cerevisiae PMT transcripts [13], indicating that ribosome release by Dom34 is not a specific translational activation mechanism for PMT transcripts. The function of Dom34 as a relatively specific translational enhancer complements other posttranscriptional mechanisms in C. albicans that recently have been discovered to be essential for the biology and virulence of this fungus [35,36]. The ability of Dom34 to act as a RNA binding protein adds a new facet to the mode of action of Dom34 in eukaryotes. RNA binding was specific, because it did not occur with a control RNA, it was outcompeted efficiently and it occurred at low Dom34 concentrations. A stretch of residues in domain 3 of Dom34 had previously been shown to share high homology to RNA binding proteins [15,33]. However, mutation of a central residue in this sequence, N317 to N317A, did not affect binding of Dom34N317A to the 5′-UTR oligonucleotide suggesting that this Dom34 sequence may be important for binding to the 3′-UTR but not to the 5′-UTR of transcripts. The predicted structure of the 5′-UTR, which was supported by limited RNase digestion, was found to contain several single-stranded regions, one of which comprised a CA/AC-rich region with three ACAACCACAAC repeats. Binding of Dom34 protected the 5′-UTR at this region from RNase digestion and Dom34 bound to a corresponding oligonucleotide, thus identifying the CA/AC-rich region as the Dom34 binding site. Interestingly, 5′-UTR regions of all five transcripts for C. albicans Pmt isoform (but not the 5′-UTR of the ACT1 control transcript) contained at least one CAAC repeat (eight in the PMT1 5′-UTR), which may constitute the minimum requirement for Dom34 binding. Dom34 did not show a major general RNase activity in our experiments, in agreement with Passos et al. [19], although distinct levels of specific degradation products of the bound 5′-UTR were detected. A Dom34 mutant, in which the conserved glutamic acid residue (E21 in CaDom34) important for in vitro RNase activity [18] was altered (E21A), strongly bound to the 5′-UTR but did not cause its endonucleolytic degradation, suggesting that E21 is relevant for its RNase activity. In agreement, rescue of pmt1 phenotypes did not occur with overexpression of the DOM34E21A allele, thus confirming the importance of the E21 residue for the function of Dom34. Dom34 binding to the CA/AC-rich sequence caused cleavage of the 5′-UTR by its RNase activity at a distant site, within predicted double-stranded regions causing a major structural alteration of the 5′-UTR. The mechanism, by which Dom34-mediated binding and cleavage of 5′-UTR sequences is able to stimulate translation of specific transcripts remains to be established. Possibly, the actions of Dom34 on the 5′-UTR structure could improve the accessibility of the AUG codon to ribosomal factors and subunits. Whatever the mechanism may be, the results suggest that in addition to its general action at the 3′-UTR to release stalled ribosomes, Dom34 may act at the 5′-UTR of specific transcripts including PMT transcripts to stimulate translational initiation in glycostress conditions.
S. cerevisiae strains are listed in S1 Table. To disrupt PMT genes in strain YE449, a 3. 3 kb XbaI-SacI fragment of pDIS2, carrying pmt1Δ: : URA3, or/and a 2. 4 kb HindIII-BamHI fragment of pBDis [24], carrying pmt2Δ: : LEU2, were used for transformation, selecting prototrophs [37]. To disrupt DOM34 in strain YE449, a fragment generated by PCR on plasmid pUG6 [38] was used for transformation, selecting G418-resistance (PCR primers ScDOM34 disrupt for /rev) Oligonucleotides are listed in S2 Table. Likewise, to disrupt YIL001w, a PCR fragment generated by primers ScYIL001w disrupt for/rev was used. Correct integration of the disruption cassettes was verified by Southern blottings. C. albicans strains are listed in S1 Table. For disruption of CaDOM34 its 5′- and 3′-regions flanking the ORF were inserted into pSFU1 to frame the SAP2p-FLP and URA3 markers [39]. The DOM34 5′-region was amplified by genomic PCR on DNA of strain SC5314 using primers FPD34/RPD34 and the 3′-region was amplified using primers FDD34/RDD34. The resulting DOM34 5′-flanking region, as a SacII-NotI fragment, and the 3′-flanking region, as a XhoI-ApaI fragment, were inserted into the respective sites of pSFU1 to generate pJB28. JB28 was cut with ApaI and SacII and the large fragment was used for transformation of strain CAI4 or of pmt1 mutant CAP1-3121 [40]. Correct integration in the resulting strains SK47 and SK24, respectively, was verified by diagnostic colony PCR and confirmed by Southern blottings, using the 5′-region flanking the DOM34 ORF as the probe (S2 Fig). Removal of the URA3 cassette and disruption of the second allele was carried out as described [41]. Following disruption of both CaDOM34 alleles and eviction of the disruption cassette, URA3 was reconstituted at its authentic locus by transformation with a genomic fragment [3] to generate strains JH47-1/2 and JH24-4/5. The PMT5 gene was disrupted in the dom34 background (strain SK47) or in the pmt1 background (strain CAP1-3121) as previously described [3] to generate strains JH5-3-1 and P15-274, respectively. To generate a PMT1 gene encoding a C-terminally hemagglutinin (HA) epitope-marked Pmt1 protein PCR fragments were used containing the sat1 selectable marker and flanked by regions of homology to PMT1. For tagging pSAT1-3HA was used as template, which was constructed by replacing the URA3 gene in p3HA-URA [3] situated between PstI and BglII sites with the ACT1p-sat1-ACT1t-cassette of pFC1 [42] on a PstI to BamHI fragment. PCR was done using primers CaPMT1del-for/-rev to generate a tagging fragment for Pmt1, which was chromosomally integrated by transformation of strain CAI4 selecting for nourseothricin resistance (sat1); resulting strain CIS23. For C-terminal HA-tagging of Dom34, an insertion fragment was generated by PCR using p3HA-URA as template and primers Dom34-HA-for/rev. The insertion fragment was transformed in strain CAI4 selecting for uridine prototrophy; resulting strains JHCa1-1 (-2). Correct integration of tagging cassettes was verified by diagnostic PCR of transformants. S. cerevisiae and C. albicans strains were grown in complex YPD and synthetic SD media [37]. For hyphal induction of C. albicans the strains were grown for 3–4 days at 37°C on Spider-medium [43] or on 2% agar containing 5% horse serum. To compare killer sensitivities of several S. cerevisiae strains, YPD agar containing methylene blue and buffered to pH 4. 5 was autoclaved, cooled to 50°C and 17 μl of a saturated culture of the killer K1- secreting strain RC130 was added before pouring plates [44]. Strains to be tested were pre-grown on YPD medium and replica printed onto these plates and grown at 18°C for 4–7 days. Sensitive strains appeared blue at this time, while resistant strains remained white. To complement killer K1-resistance of strain M577 pmt1 by genomic clones, we transformed it with a genomic S. cerevisiae bank in YEp13 [45] and obtained 80 000 transformants on SD minimal medium. Colonies of transformants were replica-printed onto SD/methylene blue/pH 4. 5-medium containing killer K1 strain RC180. Blue colonies were picked, their plasmid was isolated and retransformed into M577 pmt1. Among 78 initial transformants, 17 transformants were identified, whose plasmids restored Killer-sensitivity upon retransformation. Insert ends in these plasmids were sequenced using primers YEp13-Bamflank-A/-B, flanking the BamHI insertion site of YEp13. The 7 kb BamHI-XhoI fragment of pDM3 carrying a genomic ScPMT1 fragment [46] was subcloned into YCplac111, to generate pSW20. Derivatives of YEp13 containing a 5. 59 kb genomic insert carrying ScDOM34 (pSW577/20) or containing a 4. 55 kb genomic insert carrying YIL001w were used in some complementation experiments. Expression vectors encoding HA-tagged proteins were constructed by PCR amplification of ORFs and introducing them into YCpIF17 [47]. DOM34 was amplified using primers ScDom34N/C, the 1. 2 kb product was digested with EcoRI and PstI and introduced into YCpIF17, resulting in plasmid pSW22. The GAL1p-HA-DOM34 fragment of pSW22 was excised with XhoI and XbaI and inserted into YCplac111 (SalI, XbaI) to generate pSW25. Likewise, YIL001w was amplified using primers YIL001wN/C, inserted into YCpIF17 to generate pSW21 and transferred into YCplac111 to generate pSW24. To construct a CaDOM34 expression vector its coding region was PCR amplified on gDNA using primers p1-DOM-FLAG and p2-DOM-FLAG. We then inserted the resulting PstI-SphI fragment downstream of the MET3 promoter into a derivative of pFLAG-Met3 [48], which had been modified by adding the CaARS2 replicator on an AatII fragment [49]. The resulting plasmid pSK2 was modified further by oligonucleotide-directed mutagenesis to encode the Dom34-E21A variant; for this purpose, pSK2 was used as template with primers Dom34Mut21for/rev in the QuikChange protocol (Stratagene) to generate pSK2mut. Using anti-FLAG antibody the FLAG-tagged Dom34 protein could be identified as a 45 kDa protein in immunoblottings of cellular extracts, although several cross-reacting proteins prevented immunocytological analyses. The C. albicans DOM34 ORF was inserted downstream of the T7 promoter, between the NdeI and XhoI restriction sites of expression vector pET22b (Invitrogen). The resulting vector pET22b-Dom34 encoded a Dom34 protein containing six histidine residues at its C-terminal end. The single CTG sequence encoding non-standard S288 in C. albicans was then altered to standard serine-encoding TCG by oligonucleotide-directed mutagenesis, using primers Dom34-Leu (mut) for/rev according to the QuikChange protocol (Agilent). The resulting vector pET22b-Dom34+ was modified further similarly to encode variants potentially important for the function of Dom34. For the E21A variant and the N317A variant, oligonucleotides Dom34Mut21fw/rev and Dom34Mut317fw/rev were used for mutagenesis. All expression vectors were verified by sequencing and transformed in E. coli strain Rosetta (Novagen). Transformant cultures were grown to OD600 = 0. 6 in LB medium containing 100 μg/ml ampicillin and 50 μg/ml chloramphenicol, 100 mM IPTG was added to induce the T7 promoter and cultures were incubated further at room temperature. Cell pellets were resuspended in buffer (50 mM TrisHCl/pH 7. 9; 500 mM NaCl; 10% glycerol) containing protease inhibitors and disrupted by ultrasonication, followed by centrifugation (10,000 x g for 20 min). To assure the solubility of recombinant proteins, 0. 5% lauryl sarcosinate was added to the buffer in some experiments. Soluble Dom34-His6 proteins in the supernatant were purified by affinity chromatography using Histrap Crude-Agarose columns (GE) using 5 mM, 50 mM and 250 mM imidazole for elution. Elution fractions were collected using a ÄKTAprime collector (GE Healthcare) and analyzed by SDS-PAGE (4–20% acrylamide) followed by Coomassie Blue staining or by immunoblotting using an HRP-coupled anti-His tag antibody (Qiagen) (S7 Fig). About 2 mg of Dom34 variants with at least 95% purity were obtained. Relative transcript levels (RTL) of specific C. albicans genes were determined by quantitative reverse transcription PCR (qPCR) using the ACT1 transcript as reference, as described [7,8]. Oligonucleotides designated RT (S2 Table) were used for this purpose. For the in vitro generation of transcripts, plasmid pUC18-T7CBG was constructed, which contains the promoter of bacteriophage T7 upstream of the coding region for click beetle green luciferase (CBGluc). For this purpose, primers CBG-Stu/Bam for/rev were used to amplify the coding region for CBGluc from plasmid CBGluc-pMK-RQ [50], which was inserted into pUC18T7 [51]. The resulting plasmid was modified further by insertion of the PMT1 5′-UTR sequence (-218 to -1 [29]) that was amplified using primers PMT1_5′UTR/-rev BglII_long from gDNA (strain CAF2-1); the resulting plasmid was named pRG01. Plasmids were linearized by BamHI (pUC18-T7-CBG) or BglII (pRG01) downstream of the CBGluc coding region and were used as template for in vitro run-off transcription using T7 RNA polymerase (Ambion SP6 in vitro transcription kit) according to Gildehaus et al. [51]). The resulting transcripts were used for in vitro translation in a rabbit reticulocyte lysate kit (Promega L4960) that incorporates biotinylated lysine in the protein products (Promega Transcend Detection System). Divergent from the manufacturer' s protocol the reaction was reduced to a total volume of 20 μl with 0. 75 μl Transcend™ Biotin-Lysyl-tRNA. For each reaction 4. 9 fmol RNA (244 nM) and variable amounts of Dom34 were preincubated for 10 min at RT and the reaction was started by adding a premix of reticulocyte-lysate, amino acids and RNasin. After 100 min at 30°C, 1 μl was separated by SDS-PAGE (10% acrylamide), followed by reaction with horseradish peroxidase-coupled streptavidin, as described by the manufacturer. Furthermore, CBGluc luminescence was measured by adding 45 μl of water to 5 μl of the translation mix, followed by addition of 50 μl Chroma-Glo substrate (Promega). Measurements were done in wells of 96-microtiter plates using TriStar LB 941 luminometer (Berthold Technologies) for 1 sec, as described [50]. Labelled RNA encompassing the 5′-UTR was obtained by multiple rounds of run-off transcription of pRG01 linearized by BglII and T7 RNA polymerase [48]. pUC18-T7-6S linearized by StuI served to generate 6S control RNA [51]. After purification on agarose-gels followed by glass wool-elution, the radioactive labeling at the 3′-end was performed by T4-RNA ligase-catalyzed addition of 32P-pCp, as described [52]. If necessary, the labeled RNA was purified in a second step by electrophoresis on a 5% denaturing polyacrylamide gel. RNA-Dom34 complex formation was assayed by incubating 50–100 cps 32P-labeled RNA together with variable amounts of Dom34 for 10 min at 30°C, in 10 mM Tris-HCl/pH 8. 0,100 mM NaCl. Complexes were then challenged by adding of heparin (final concentration 50 ng/μl) for 10 min and separated on native 5% polyacrylamide gels or denaturing 10% polyacrylamide gels. For EMSA of the PMT1-5′UTR RNA oligonucleotide, it was biotinylated using the Pierce RNA 3′ End Biotinylation Kit (Thermo Scientific) according to the instructions of the manufacturer. Protein-RNA binding assays were done using the LightShift Chemoluminescent RNA EMSA Optimization and Control Kit (Thermo Scientific). In a total volume of 20 μl, binding assays contained 20 nM biotinylated RNA oligonucleotide and 400 nM purified Dom34-His6 (or its E21A or N317A variants), which were supplemented in part by 2 μM unlabelled oligonucleotide. Following incubation at room temperature for 20 min, 5 μl of 5x REMSA loading buffer was added and the assay components were separated by native PAGE (6% acrylamide) and electroblotted onto a nylon membrane; the biotinylated RNA was fixed on the membrane by a short UV treatment and was detected using the Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Scientific). Cells of wild-type strain CAF2-1 and of dom34Δ mutant JH47-2 cells were grown exponentially in YPD media to OD600 0. 4–0. 6. Preparation of cells and polysome gradients were performed as described by Garre et al. [53] with some modifications. A portion of the culture (80 ml) was recovered and chilled for 5 min on ice in the presence of 0. 1 mg/ml cycloheximide (CHX). Cells were harvested by centrifugation at 6000 x g for 4 min at 4°C and resuspended in lysis buffer (20 mM Tris-HCl, pH 8,140 mM KCl, 5 mM MgCl2,0. 5 mM dithiothreitol, 1% Triton X-100,0. 1 mg/ml CHX, and 0. 5 mg/ml heparin). After washing, cells were resuspended in 700 μl of lysis buffer, a 0. 3 ml volume of glass beads was added, and cells were disrupted by shaking on a Vortex Genie 2 (setting 8) using 6 cycles for 40 s at 6. 5 ms-1. Between cycles cells were placed on ice for 5 min. Lysates were cleared by centrifuging twice for 5 min, first at 5,000 rpm, and then the supernatant was recovered and was centrifuged at 8,000 rpm. Finally, glycerol was added to the supernatant at a final concentration of 5%, before storing extracts at -70°C. Samples of 10–20 A260 units were loaded onto 10–50% sucrose gradients and were separated by ultracentrifugation for 2 h and 40 min at 35,000 rpm in a Beckman SW41 rotor at 4°C. Then, gradients were fractionated using isotonic pumping of 60% sucrose from the bottom, followed by a recording of the polysomal profiles by online UV detection at 260 nm (Density Gradient Fractionation System, Teledyne Isco, Lincoln, NE). To analyze the RNA of the polysomal fractions, RNA from 200 μl of each fraction was extracted using GeneJet RNA extraction kit (STREK, Biotools). To each sample 1 μg of in vitro transcribed RNA (HiScribe™ T7 High Yield RNA Synthesis Kit, NEB) was added and used as spiked-in mRNA for normalization of the transcripts. After reverse transcription of the purified RNA (Maxima First Strand cDNA synthesis kit, Thermo Scientific) quantitative PCR (RT-qPCR) was performed using gene specific primer pairs to quantify mRNAs of PMT1, PMT4, PMT6 and ACT1. For each fraction three technical replicates were assayed on a Mx3000P LightCycler (Stratagene), with 10 μl of cDNA, 4 μl EvaGreen QPCR-mix II (Bio-Budget) and 3 μl each of forward and reverse oligonucleotide primers (400 pmol/μl) in each reaction. The polymerase was activated at 95°C for 10 min, annealing was performed at 60°C for 15 s, extension at 72°C for 30 s and the denaturation step was performed at 95°C for 30 s in a total of 50 cycles. For the statistical assessment of the difference between the transcript distributions in the reference strain CAF2-1 and the dom34 mutant strain, the Kolmogorov-Smirnov test (KS-test) was performed [31]. The KS-test was computed using the ks. test CRAN package in the R statistical software environment.
Fungi respond to damages of their glycostructures in their cell wall by transcriptional upregulation of genes that specify compensatory activities. Upon block of protein N-glycosylation, the human fungal pathogen Candida albicans increases transcription of PMT1 encoding a major isoform of protein O-mannosyltransferase. Here we demonstrate that the Dom34 protein aids in glycostress responses by upregulating the translation of several PMT isoform transcripts. Dom34 has previously been implicated in mechanisms to secure high levels of ribosomal subunits that promote translation in general, e. g. by no-go decay at the 3′-UTR of transcripts. By binding to the 5′-UTR and activating translational initiation of PMT transcripts we add a novel mode of action and suggest a preferred class of targets for the translational activities of the Dom34 protein. The combination of transcriptional and Dom34-mediated translational upregulation of PMT genes optimizes effective recovery and survival of fungal cells upon glycostress.
medicine and health sciences
pathology and laboratory medicine
nucleases
enzymes
pathogens
messenger rna
microbiology
dna-binding proteins
enzymology
nucleotides
fungi
model organisms
untranslated regions
molecular biology techniques
fungal pathogens
research and analysis methods
saccharomyces
mycology
proteins
medical microbiology
ribonucleases
microbial pathogens
denaturation
gene expression
molecular biology
yeast
biochemistry
rna
candida
hydrolases
nucleic acids
phenotypes
oligonucleotides
protein translation
genetics
biology and life sciences
yeast and fungal models
saccharomyces cerevisiae
rna denaturation
organisms
candida albicans
Dom34 Links Translation to Protein O-mannosylation