Post‐transcriptional regulation of transcript abundance by a conserved member of the tristetraprolin family in Candida albicans

In Candida albicans, deep sequencing of zfs1Δ/Δ mutants revealed significant increases (>1.5-fold) in 156 transcripts.

“In the human pathogen Candida albicans, deep sequencing of mutants lacking the orthologous protein, Zfs1, revealed significant increases (> 1.5-fold) in 156 transcripts.”

• Also stated in: § Introduction · plain

  “Using mRNA-Seq analysis, we identified 156 transcripts that were significantly increased by 1.5-fold or more in zfs1Δ/Δ mutant strains compared with WT.”

No changes were found in expression of orthologs of transcripts previously shown to be regulated by Cth1/2 in S. cerevisiae (ACO1, SDH4, HEM15, HEM13 and HMX1) in C. albicans zfs1Δ/Δ mutants using RT-PCR, in either iron-rich or iron-depleted conditions.

“We found no changes in expression of these transcripts when we compared WT and zfs1Δ/Δ mutants in either iron-rich or iron-depleted conditions (data not shown).”

Of the 56 up-regulated transcripts, 44 (79%) contained at least a single 7-mer binding site with sequence UAUUUAU.

“Of the 56 up-regulated transcripts, 44 (79%) contained at least a single 7-mer binding site with this sequence”

• Also stated in: § Zfs1 target conservation · plain

  “Of these transcripts, 44 contained at least one 7-mer potential binding site.”

• AssumptionRests on an assumption: The presence of a predicted TTP family member binding site (7-mer UAUUUAU) in a transcript's 3′UTR indicates that the transcript is a direct target of Zfs1.

There was no difference in hyphal formation in zfs1Δ/Δ mutants on liquid or solid medium.

“we failed to detect a difference in hyphal formation in zfs1Δ/Δ mutants on liquid or solid medium (data not shown).”

None of the down-regulated transcripts contained a 7-mer potential binding site, with the exception of the deleted gene itself.

“In contrast, none of the down-regulated transcripts contained a 7-mer potential binding site (Table 2), with the exception of the deleted gene itself.”

• AssumptionRests on an assumption: The presence of a predicted TTP family member binding site (7-mer UAUUUAU) in a transcript's 3′UTR indicates that the transcript is a direct target of Zfs1.

zfs1Δ/Δ mutants had slight increases in cell growth during stationary phase.

“slight increases in cell growth during stationary phase”

• Also stated in: § Phenotype of the zfs1Δ/Δ mutants in C. albicans · plain

  “the loss of ZFS1 resulted in a slight but significant increase in cell density during stationary phase (Fig. 1A).”

Another 100 transcripts were increased between 1.5- and 2-fold (P < 0.05) in zfs1Δ/Δ mutants.

“In addition, another 100 transcripts were increased between 1.5- and 2-fold (P < 0.05)”

zfs1Δ/Δ mutants were viable.

“Phenotypic analysis confirmed that zfs1Δ/Δ mutants were viable”

No differences were found in the growth of zfs1Δ/Δ mutants on lactate, galactose, glycerol, sucrose or sorbitol compared with the WT strain.

“However, we found no differences in the growth of zfs1Δ/Δ mutants on lactate, galactose, glycerol, sucrose or sorbitol, compared with the WT strain.”

No significant changes were found in any of the histidine pathway transcripts, with the exception of the HIS3 mRNA itself.

“We found no significant changes in any of the histidine pathway transcripts, with the exception of the HIS3 mRNA itself (Fig. 3).”

No overlaps were found, with the possible exceptions of the three transcripts involved in the TCA cycle, between the lists of putative targets in the three organisms.

“However, we did not find any overlaps, with the possible exceptions of the three transcripts involved in the TCA cycle, between the lists of putative targets in the three organisms.”

C. albicans Zfs1 bound to the ideal mammalian TTP binding site with high affinity.

“C. albicans Zfs1 bound to the ideal mammalian TTP binding site with high affinity”

• Also stated in: § Characteristics of binding of Zfs1 to an ARE RNA oligonucleotide · plain

  “purified full-length Zfs1:MBP fusion protein bound with high affinity (Kd of 1.8 nM at 24°C) to the FL-ARE13 probe”

zfs1Δ/Δ mutants had minor alterations in biofilm architecture, specifically a slight, reproducible enhancement of the basal biofilm layer on polystyrene plates.

“with minor alterations in biofilm architecture”

• Also stated in: § Phenotype of the zfs1Δ/Δ mutants in C. albicans · plain

  “We found a slight, but reproducible, enhancement of the basal biofilm layer on polystyrene plates, consistent with the enhanced cell density we observed during stationary phase (Fig. 1B – G).”

Predicted Zfs1 binding sites in these mRNAs often disappeared with increasing evolutionary distance from the parental species.

“while the predicted Zfs1 binding sites in these mRNAs often ‘disappeared’ with increasing evolutionary distance from the parental species.”

• Also stated in: § Zfs1 target conservation · plain

  “the numbers of 7-mer binding sites generally decreased with increasing evolutionary distance from C. albicans (Fig. 5A – C).”

The zfs1Δ/Δ mutant was resistant to 3-amino-1,2,4-triazole (3-AT) when grown in minimal medium.

“The zfs1Δ/Δ mutant was resistant to 3-amino-1,2,4-triazole”

• Also stated in: § Characterization of Zfs1 targets · plain

  “We found that the zfs1Δ/Δ mutants were resistant to increasing doses of 3-AT when grown in minimal medium (Fig. 4)”

No changes were observed in transcripts involved in iron homeostasis or cell-cell adhesion in C. albicans zfs1Δ/Δ mutants, with the exception of FRE10.

“we did not observe changes in any transcripts in C. albicans that were shown to be involved in either of these processes, with the exception of FRE10, encoding a major cell-surface ferric reductase under low-iron conditions.”

There were no differences in exponential growth between zfs1Δ/Δ mutants and WT.

“Although there were no differences in exponential growth”

Sequence analysis of the two independent zfs1Δ/Δ strains and their isogenic WT control strain showed no differences in the numbers of ATTT repeat elements within the HIS3 3′UTR.

“Sequence analysis of the two independent zfs1Δ/Δ strains used here and their isogenic WT control strain showed no differences in the numbers of ATTT repeat elements within the HIS3 3′UTR.”

Down-regulated transcripts were not significantly overrepresented in any specific GO category.

“However, we did not find that the down-regulated transcripts were significantly overrepresented in any specific category.”

46 additional transcripts were down-regulated between 1.5 and 2-fold (P < 0.05) in zfs1Δ/Δ mutants.

“and 46 additional transcripts were down-regulated between 1.5 and 2-fold (P < 0.05)”

Sequences of the proteins encoded by the putative Zfs1 targets were highly conserved among other species within the fungal CTG clade.

“Sequences of the proteins encoded by the putative Zfs1 targets were highly conserved among other species within the fungal CTG clade”

• Also stated in: § Introduction · plain

  “The sequences of the proteins encoded by the putative Zfs1 targets were highly conserved among other members of the CTG clade.”

Of the 156 up-regulated transcripts, 113 (72%) contained at least one predicted TTP family member binding site in their 3′UTR.

“Of these, 113 (72%) contained at least one predicted TTP family member binding site in their 3′UTR”

• Also stated in: § Transcript changes in the zfs1Δ/Δ mutants · plain

  “a total of 156 transcripts were up-regulated in the zfs1Δ/Δ cells, with 113 (72%) containing at least one optimal potential TTP family member binding site.”

• AssumptionRests on an assumption: The presence of a predicted TTP family member binding site (7-mer UAUUUAU) in a transcript's 3′UTR indicates that the transcript is a direct target of Zfs1.

zfs1Δ/Δ mutants exhibited no apparent defects in cell-cell interactions.

“They exhibited no apparent defects in cell–cell interactions.”

• Also stated in: § Phenotype of the zfs1Δ/Δ mutants in C. albicans · hedged

  “the zfs1Δ/Δ mutants remained completely in suspension, suggesting that there was no defect in cell–cell adhesion”

Zfs1 was associated with target transcripts after co-immunoprecipitation.

“Zfs1 was associated with target transcripts after co-immunoprecipitation.”

• Also stated in: § RIP of Zfs1 targets · plain

  “10 of 11 of the putative target transcripts tested were enriched in the immunoprecipitated RNA from the Zfs1:Myc strain as compared with the control strain lacking the Myc tag (Fig. 8).”

The frequency of 7-mer-containing transcripts in C. albicans (17%) is slightly lower than in S. pombe (22%).

“This is slightly lower than the frequency seen in S. pombe, which contains 22% (Wells et al., 2012).”

• cites Wells et al., 2012Not checked yet

  Not checked in the free run.

Approximately 17% of C. albicans transcripts contained at least a single 7-mer.

“We found that approximately 17% of C. albicans transcripts contained at least a single 7-mer.”

No differences in vacuole morphology were observed in zfs1Δ/Δ mutants compared with the WT strain.

“We observed no differences in vacuole morphology in zfs1Δ/Δ mutants as compared with the WT strain (data not shown).”

GO analysis found significant enrichment (6 of 56) of transcripts involved in the TCA cycle in the mRNA-Seq dataset, compared with transcripts included in the GO dataset (16 of 6525) (P = 4.9E−07).

“We found that there was a significant enrichment (6 of 56) of transcripts involved in the tricarboxylic acid (TCA) cycle in our mRNA-Seq dataset, as compared with the transcripts included in the GO dataset (16 of 6525) (P = 4.9E−07) (Table 3).”

Of the 56 down-regulated transcripts, only 3 (5%) contained at least one predicted TTP family member binding site.

“compared with only 3 of 56 (5%) down-regulated transcripts.”

• Also stated in: § Transcript changes in the zfs1Δ/Δ mutants · plain

  “only 3 of the 56 down-regulated transcripts (not including the deleted transcript) contained potential binding sites of this type.”

• AssumptionRests on an assumption: The presence of a predicted TTP family member binding site (7-mer UAUUUAU) in a transcript's 3′UTR indicates that the transcript is a direct target of Zfs1.

Recombinant Zfs1 could bind to a synthetic RNA 'target' containing the optimal mammalian binding sequence with low nanomolar affinity, similar to that seen with a human TTP binding domain.

“Finally, we showed that recombinant Zfs1 could bind to a synthetic RNA ‘target’ containing the optimal mammalian binding sequence with low nanomolar affinity, similar to that seen with a human TTP binding domain.”

Orthologs in several species sometimes had no binding sites whatsoever.

“In fact, the orthologs in several species sometimes had no binding sites whatsoever (Fig. 5A – C).”

Orthologs of Zfs1 were found in 13 species of the CTG clade.

“We found orthologs of Zfs1 in 13 species”

In one case, C. albicans orf19.1340, encoding a putative aldose reductase, there was an increase from one 7-mer in C. albicans to eight overlapping 7-mers in L. elongisporus.

“However, in one case, C. albicans orf19.1340, encoding a putative aldose reductase, there was an increase from one 7-mer in C. albicans to eight overlapping 7-mers in L. elongisporus.”

Increases in steady state levels of mRNAs in zfs1Δ/Δ mutants occurred in many transcripts that contained potential Zfs1 binding sites.

“These changes occurred in many transcripts that contained potential Zfs1 binding sites”

The zfs1Δ/Δ mutants in C. albicans did not appear to have defects in cell-cell adhesion, as evidenced by the lack of flocculation both in normal growth conditions and after calcium treatment.

“However, unlike the situation in S. pombe, the zfs1Δ/Δ mutants in C. albicans did not appear to have defects in cell–cell adhesion, as evidenced by the lack of flocculation both in normal growth conditions and after calcium treatment.”

10 of 11 of the putative target transcripts tested were enriched in the immunoprecipitated RNA from the Zfs1:Myc strain as compared with the control strain lacking the Myc tag.

“Following immunoprecipitation of Zfs1:Myc with the Myc antibody, we found that 10 of 11 of the putative target transcripts tested were enriched in the immunoprecipitated RNA from the Zfs1:Myc strain as compared with the control strain lacking the Myc tag (Fig. 8).”

The proteins encoded by the putative target mRNAs were highly conserved among other CTG clade species.

“As expected, the proteins encoded by the putative target mRNAs were highly conserved among other CTG clade species”

The top target identified by mRNA-Seq analysis was the HIS3 mRNA.

“The ‘top’ target identified by our mRNA-Seq analysis was the HIS3 mRNA.”

Each of the 13 CTG clade species appeared to express only a single member of the Zfs1 protein family.

“and each species appeared to express only a single member of this protein family.”

The full-length Zfs1 protein of C. albicans can bind to the optimal mammalian target sequence UUAUUUAUU with very nearly identical affinity to a purified synthetic TZF domain peptide from human TTP, using the same fluorescence anisotropy assay.

“We were able to demonstrate directly that the full-length Zfs1 protein of C. albicans can bind to the optimal mammalian target sequence, UUAUUUAUU, with very nearly identical affinity to that seen with a purified synthetic TZF domain peptide from human TTP, using the same fluorescence anisotropy assay.”

Several proposed Zfs1 target transcripts in C. albicans encoded proteins previously identified as playing a role in cell adhesion and pathogenicity.

“Several of the proposed Zfs1 target transcripts in C. albicans encoded proteins previously identified as playing a role in cell adhesion and pathogenicity.”

6-12 7-mer binding sites were found within the HIS3 3′UTR sequences from the 4 different genomic sequences representing 4 different C. albicans sequences currently available in GenBank.

“We found 6–12 7-mer binding sites within the HIS3 3′UTR sequences from the 4 different genomic sequences representing 4 different C. albicans sequences currently available in GenBank (AVAX01000022, AACQ01000253, AVAZ01000065, AVAW01000080).”

Attempts to assay transcripts from two different alleles of widely different microsatellite lengths in the same C. albicans cells have not demonstrated obvious differences in steady state transcript levels.

“To date, our attempts to assay the transcripts from two different alleles of widely different microsatellite lengths in the same C. albicans cells have not demonstrated obvious differences in steady state transcript levels”

212 transcripts had steady state levels changed by more than 1.5-fold with P < 0.05 in zfs1Δ/Δ mutants.

“Altogether, we found 212 transcripts whose steady state levels were changed by more than 1.5-fold with P < 0.05.”

The presence or absence of binding sites in Zfs1 targets correlated well with the overall relatedness of the Zfs1 TZF domain sequences.

“The presence or absence of binding sites in Zfs1 targets correlated well with the overall relatedness of the Zfs1 TZF domain sequences (Figs 5 and 6B).”

Essentially no binding of the purified Zfs1:MBP fusion protein to a control polyU probe was observed.

“We observed essentially no binding of the purified Zfs1:MBP fusion protein to a control polyU probe (5′-FL-UUUUUUUUUUUUU-3′) (Fig. 7A).”

The zfs1Δ/Δ mutants lacked an obvious growth or morphological phenotype.

“Because the zfs1Δ/Δ mutants lacked an obvious growth or morphological phenotype, we could investigate the biochemical functions of Zfs1 as a potential mRNA-binding and destabilizing protein in the absence of major phenotypic changes.”

There was no significant enrichment of either orf19.558 or orf19.5525, both transcripts that were increased in zfs1Δ/Δ mutants but that lacked obvious binding sites.

“There was no significant enrichment of either orf19.558 or orf19.5525, both transcripts that were increased in zfs1Δ/Δ mutants but that lacked obvious binding sites (Fig. 8).”

Increases in steady state levels of mRNAs in zfs1Δ/Δ mutants occurred in the absence of a gross phenotype in the deletion mutants.

“and also occurred in the absence of a gross phenotype in the deletion mutants, which might alter gene expression independently.”

No differences in growth were found between WT and zfs1Δ/Δ strains under hypoxic conditions.

“We also found no differences in growth between WT and zfs1Δ/Δ strains under hypoxic conditions (data not shown).”

The potential Zfs1 targets observed in the present study are not obviously part of an iron-related regulon.

“and the potential Zfs1 targets observed in the present study are not obviously part of an iron-related regulon.”

The predicted 3′UTRs of the putative target mRNAs were much less well conserved among other CTG clade species.

“but the predicted 3′UTRs were much less well conserved.”

The mRNA-Seq analysis was able to quantitate results from 5585 transcripts.

“Because our mRNA-Seq analysis was able to quantitate results from 5585 transcripts”

No differences were found in the growth of zfs1Δ/Δ mutants under conditions that cause oxidative stress, such as hydrogen peroxide.

“In addition, there were no differences in the growth of the zfs1Δ/Δ mutants under conditions that cause oxidative stress, such as hydrogen peroxide (data not shown).”

Zfs1 protein-coding sequences are present in at least 13 members of the fungal CTG clade, in addition to C. albicans, that have reasonably complete genomic sequences in GenBank.

“Zfs1 protein-coding sequences are present in at least 13 members of the fungal CTG clade, in addition to C. albicans, that have reasonably complete genomic sequences in GenBank.”

The TZF domain sequences of various CTG clade species differ from human TTP.

“This is despite many differences in the sequences of the TZF domain from the various CTG clade species compared with human TTP.”

The zfs1Δ/Δ mutation resulted in major expression changes in approximately 4.6% of the C. albicans transcriptome.

“this indicates that the zfs1Δ/Δ mutation resulted in major expression changes in approximately 4.6% of the C. albicans transcriptome.”

Increased expression of HIS3 mRNA and presumably protein in the zfs1Δ/Δ mutant could overcome the growth inhibitory effect of 3-AT under minimal medium conditions.

“perhaps because of increased expression of the potential target transcript encoded by HIS3.”

• Also stated in: § Characterization of Zfs1 targets · hedged

  “suggesting that increased expression of HIS3 mRNA and presumably protein in the mutant could overcome the growth inhibitory effect of 3-AT under these conditions.”

The increase in HIS3 mRNA observed in zfs1Δ/Δ mutants most likely reflects direct regulation of HIS3 mRNA turnover rather than an overall perturbation of the pathway.

“Taken together, the increase in HIS3 mRNA observed in the zfs1Δ/Δ mutants most likely reflects direct regulation of HIS3 mRNA turnover rather than an overall perturbation of the pathway.”

In Schizosaccharomyces pombe, the single TTP family protein Zfs1 regulates cell-cell interactions.

“the single protein expressed in Schizosaccharomyces pombe, Zfs1, regulates cell–cell interactions.”

• Also stated in: § Introduction · hedged

  “These data suggested that a major physiological function of Zfs1 in S. pombe is to regulate the levels of cell surface proteins involved in cell–cell interactions.”

• cites Wells et al., 2012Not checked yet

  Not checked in the free run.

Three up-regulated transcripts involved in the TCA cycle, KGD1, SDH2 and CIT1, had been previously proposed to be Cth1/Cth2 targets.

“we identified three up-regulated transcripts involved in the TCA cycle, KGD1, SDH2 and CIT1, that had been previously proposed to be Cth1/Cth2 targets (Puig et al., 2005).”

• cites Puig et al., 2005Not checked yet

  Not checked in the free run.

ZFS1 expression is regulated in C. albicans during the yeast to hyphae switch.

“However, ZFS1 expression is regulated in C. albicans during the yeast to hyphae switch, a step important for biofilm formation (Nantel et al., 2002).”

• cites Nantel et al., 2002Not checked yet

  Not checked in the free run.

Taken together with data from S. pombe and S. cerevisiae, the data in C. albicans support the concept that the biochemical functions of TTP family members in these species-their ability to bind AREs in mRNA with high affinity and promote mRNA decay-are highly conserved in these diverse species.

“Taken together with the data from S. pombe and S. cerevisiae, the data in C. albicans support the concept that the biochemical functions of the TTP family members in these species, i.e. their ability to bind AREs in mRNA with high affinity and promote mRNA decay, are highly conserved in these diverse species.”

These findings supported the averaging of read counts from all seven zfs1Δ/Δ samples.

“These findings supported the averaging of read counts from all seven zfs1Δ/Δ samples.”

Even within members of the CTG clade, and previously in members of the Schizosaccharomyces genus, TTP family member binding sites within the 3′UTR are often not conserved within orthologous transcripts.

“In fact, even within the members of the CTG clade, as seen here, and previously in members of the Schizosaccharomyces genus (Wells et al., 2012), the TTP family member binding sites within the 3′UTR are often not conserved within the orthologous transcripts.”

• cites Wells et al., 2012Not checked yet

  Not checked in the free run.

In S. cerevisiae and S. pombe, TTP family member proteins can bind ARE sequences in mRNA and promote the decay of those mRNAs, similar to their mammalian counterparts.

“In both S. cerevisiae and S. pombe, the TTP family member proteins seem to act like their mammalian counterparts, in that they can bind to ARE sequences in mRNA and promote the decay of those mRNAs.”

• cites Cuthbertson et al., 2008Not checked yet

  Not checked in the free run.

• cites Vergara et al., 2011Not checked yet

  Not checked in the free run.

The HIS3 gene has polymorphic alleles that encode varying numbers of repeats of the microsatellite DNA sequence (ATTT)n located within the 3′UTR of the transcript.

“The HIS3 gene is known to have polymorphic alleles that encode varying numbers of repeats of the microsatellite DNA sequence (ATTT)n that are located within the 3′UTR of the transcript (Botterel et al., 2001).”

• cites Botterel et al., 2001Not checked yet

  Not checked in the free run.

The down-regulated transcripts are likely to be secondary effects, perhaps related to one or more transcription factors that were up-regulated in zfs1Δ/Δ cells.

“According to this formulation, the down-regulated transcripts, which were fewer in number and rarely contained potential Zfs1 binding sites, are likely to be secondary effects, perhaps related to one or more of the transcription factors that were up-regulated in the zfs1Δ/Δ cells.”

• AssumptionThe authors state this as an assumption: Increases in steady state levels of mRNAs in zfs1Δ/Δ mutants indicate that those mRNAs are likely to be direct targets of Zfs1.

When raw reads greater than 0 from the zfs1Δ/Δ samples were compared with each other, the minimum correlation coefficient between any two samples was 0.96.

“When the raw reads greater than 0 from the zfs1Δ/Δ samples were compared with each other, the minimum correlation coefficient between any two samples was 0.96.”

The increase in CIT1 expression does not result in an obvious growth advantage on alternative carbon sources, at least under the conditions tested.

“These data suggest that the increase in CIT1 expression does not result in an obvious growth advantage on alternative carbon sources, at least under our conditions.”

The three organisms C. albicans, S. pombe and S. cerevisiae have used their specific TTP family member proteins for their own specific purposes, which differ from one species to the next.

“At first glance, therefore, it appears that the three organisms have used their specific TTP family member proteins for their own specific purposes, which differ from one species to the next.”

The RNA binding module of TTP family proteins appears to have been maintained essentially intact during the more than one billion years since the common ancestor of fungi and mammals.

“Although a common ancestor of fungi and mammals is thought to have existed more than a billion years ago (Stajich et al., 2009), it is remarkable that this RNA binding module appears to have been maintained essentially intact during that evolutionary time.”

• cites Stajich et al., 2009Not checked yet

  Not checked in the free run.

The optimum binding site found in studies of human TTP, UUAUUUAUU, is also a high-affinity binding site for the full-length C. albicans protein.

“These data demonstrate that the optimum binding site found in studies of human TTP, UUAUUUAUU, is also a high-affinity binding site for the full-length C. albicans protein.”

When average reads from the two zfs1Δ/Δ strains were compared, only one relatively poorly expressed transcript (FDH1) was statistically different at 1.68-fold.

“In addition, when the average reads from the two zfs1Δ/Δ strains were compared, only one relatively poorly expressed transcript (FDH1) was statistically different at 1.68-fold.”