Tethered Scd6 represses expression of a GFP reporter mRNA

Previously, it was shown that Scd6 interacts with mRNA decapping factors and can inhibit translation initiation in vitro [7, 22]; however, it was unknown whether Scd6 can reduce the abundance and repress the translation of specific mRNAs in vivo. Because native mRNA substrates of Scd6 were unknown, we employed a tethered-function assay to examine the consequences of Scd6 binding to reporter mRNA—an approach used successfully to demonstrate regulation of mRNA translation and turnover by the decapping activator/translational repressor Dhh1 [6]. A fusion of Scd6 to bacteriophage MS2 coat protein (CP), or MS2 CP alone, each tagged with three FLAG epitopes (henceforth, Scd6-MS2-F or MS2-F), was expressed in wild-type (WT) cells from mRNA containing the 5’ and 3’ untranslated regions (UTRs) of SCD6 driven by the native SCD6 promoter from a low copy plasmid. A GFP reporter mRNA, harboring the 5’UTR and 3’UTR of PGK1 mRNA with tandem MS2 RNA recognition elements inserted in the 3’ UTR, and driven by a galactose-inducible GAL1 UAS and PGK1 hybrid promoter [6], was expressed from a low copy plasmid in the same strains by culturing cells with galactose as carbon source. As a positive control, we expressed the Dhh1-MS2 fusion shown previously to evoke translational repression of the same GFP reporter [6]. Western blotting with anti-GFP antibodies and either Northern blotting or qRT-PCR analyses were conducted to measure steady-state expression of GFP protein and GFP reporter mRNA, respectively, and the ratio of these measurements yielded the translational efficiency (TE) of the reporter mRNA (Fig 1A).

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Fig 1. Tethered Scd6 destabilizes GFP mRNA in vivo.

(A) Schema of the MS2 CP tethering assay with Scd6-MS2-F fusion protein targeted to stem-loops in the 3’ UTR of the GFP reporter; with summary of assays used to measure GFP protein or mRNA abundance and translation efficiency (TE) of the reporter. (B) WT cells (BY4741) were co-transformed with an expression plasmid for MS2(2μ) (pJC398), Dhh1-MS2 (pJC236), Scd6-MS2-F (pQZ127) or MS2-F (pQZ130), along with GFP reporter plasmid (pJC429), cultured in synthetic complete medium without leucine or uracil (SC-L-U) with 2% galactose/2% raffinose replacing dextrose at 30°C for at least two cell divisions, and harvested at A₆₀₀ ~0.6–0.7. Total protein was extracted under denaturing conditions and subjected to Western blot analysis with antibodies against FLAG, GFP or Prt1 (as loading control). Typical results from three biological replicates are shown. (C) Relative GFP protein signal normalized to Prt1 protein signal by densitometry analysis of samples in (B). Average results (± S.E.M.s) from at least three biological replicates are shown. (D) Transformants from (A), and WT strain BY4741 harboring empty vector YCpLac111 and GFP reporter pJC429, were cultured as in (B). Total RNA was extracted from whole cell extracts (WCEs) and reporter mRNA abundance was quantified by RT-qPCR, relative to that of ACT1 mRNA. Average results (± S.E.M.s) from at least three biological replicates are shown. (E) Total RNA samples from (D) were subjected to Northern blot analysis and probed for GFP mRNA; blots were stripped and re-probed for PYK1 mRNA as a loading control. (F) Changes (Δ) in expression of GFP reporter protein or mRNA, or reporter TE, on tethering Scd6-MS2-F, as ratios of values in cells harboring Scd6-MS2-F versus MS2-F. Mean values (± S.E.M.s) were determined from at least three biological replicates. Calculations of S.E.M.s for mean ratios of GFP protein and mRNA expression shown in (F), and determination of P-values from significance testing of differences in mean values in (C-D) using an unpaired Student’s t-test, were conducted as described in the supporting file S1 Text “Analysis and Explanation of Supporting Data Files”. P-values are summarized as: **, P <0.01; *, P <0.05. P-values for these and all other statistical tests performed in this study can be found in the supplementary data files provided for the respective figures.

https://doi.org/10.1371/journal.pgen.1007806.g001

Tethering Scd6-MS2-F reduced expression of GFP protein and reporter mRNA by comparable amounts, ~2.5- to 3-fold, compared to tethering MS2-F alone (Fig 1B–1D, Scd6-MS2-F vs. MS2-F; P <0.0001 in C & D). These results are similar to those observed on tethering Dhh1-MS2 versus MS2 alone expressed from a high-copy plasmid (Fig 1B–1D, Dhh1-MS2 vs. MS2(2μM)), in agreement with previous findings [6]. Expression of GFP protein and mRNA in cells expressing MS2-F was essentially indistinguishable from that measured in transformants of the same strain harboring empty vector (S1A Fig, Fig 1C and 1D). Northern analysis confirmed the repression of GFP mRNA by both Scd6-MS2-F and Dhh1-MS2 in comparison to empty vector and MS2-alone controls (Fig 1E). These results are consistent with the possibility that tethering Scd6 increases the rate of GFP mRNA turnover to lower its steady-state abundance, with attendant reduction in GFP protein expression. Owing to comparable repression of reporter protein and mRNA, tethered Scd6-MS2-F evokes little change in the TE of the GFP reporter (Fig 1F).

To determine whether tethering Scd6-MS2 to GFP mRNA accelerates its degradation, we measured the half-life of GFP mRNA following a shift from galactose to glucose medium that should repress the GAL promoter and halt new synthesis of reporter mRNA. On tethering Scd6-MS2-F, the half-life of GFP mRNA was significantly reduced from 2.8 ± 0.06 min (tethering MS2-F alone) to 1.9 ± 0.15 min (P = 0.03; S1B Fig). A similar fold-reduction in half-life was observed on tethering Dhh1-MS2 vs MS2 alone (2.3 ± 0.09 min vs. 3.20 ± 0.02 min, respectively; P = 0.01; S1C Fig). We conclude that tethered Scd6-MS2-F reduces GFP mRNA abundance by accelerating its degradation.

Because it was shown previously that Npl3 and Sbp1 can also repress translation initiation in vitro dependent on interaction of their RGG domains with eIF4G in a manner similar to Scd6 [22], we examined MS2 fusions of these proteins, expressed from plasmid constructs containing the native promoters and 5’- and 3’-UTRs of NPL3 or SBP1, respectively, along with the corresponding MS2 control proteins. In contrast to results obtained for Scd6-MS2-F, neither Npl3-MS2-F nor Sbp1-MS2-F had any significant effect on GFP expression from the same reporter analyzed above (S2A, S2B, S2C and S2D Fig) despite being expressed at levels exceeding that of Scd6-MS2–F (S2E Fig). These results underscored the specificity of the effects of Scd6-MS2-F and led us to probe its mechanism of repression.

Tethered Scd6 evokes Dcp2-dependent reduction of reporter mRNA abundance and Dhh1-dependent translational repression

As Scd6 can bind to Dcp2, the catalytic component of the decapping enzyme [7], we asked whether the repression of reporter mRNA abundance evoked by tethered Scd6-MS2-F is attenuated in a dcp2Δ strain. The isogenic WT DCP2 strain employed for this experiment exhibited reductions in GFP mRNA and GFP protein expression conferred by Scd6-MS2-F versus MS-F alone (Fig 2A–2D, WT data) equal to, or greater than those observed in the different WT strain employed above (Fig 1B–1D). Tethering Scd6-MS2-F to the GFP reporter in dcp2Δ cells reduced the abundance of GFP mRNA (Fig 2C, dcp2Δ, Scd6-MS2-F vs. MS2-F; P = 0.03). Importantly, however, the reduction in reporter mRNA abundance conferred by Scd6-MS2-F was diminished in dcp2Δ versus WT cells, with the ratio of GFP mRNA in cells expressing Scd6-MS2-F versus MS2-F increasing from ~0.4 in WT to ~0.7 in dcp2Δ cells (Fig 2D, white bars, WT vs. dcp2Δ; P = 0.009). Expression of GFP protein in cells containing MS2-F alone was reduced somewhat in the dcp2Δ vs. WT strain; however, this reduction occurred independently of tethering as it was also observed in the corresponding strains containing empty vector (S3A and S3B Fig). Tethering Scd6-MS2-F in dcp2Δ cells conferred a strong reduction in GFP protein expression compared to MS2-F alone (Fig 2B, dcp2Δ, Scd6-MS2-F vs. MS2-F; P = 0.002), that was only slightly smaller in magnitude compared to that seen in WT cells (Fig 2D, black bars, WT vs. dcp2Δ). As a consequence of relatively greater repression of GFP protein versus GFP mRNA on tethering Scd6-MS2-F versus MS2-F alone in the dcp2Δ mutant (Fig 2D, WT vs. dcp2Δ, black vs. white bars), the TE of GFP reporter mRNA is diminished in dcp2Δ versus WT cells by ~33% (Fig 2E, WT vs. dcp2Δ; P = 0.005). These findings suggest that: (i) Dcp2 is required for strong repression of reporter mRNA abundance, and (ii) translational repression of the GFP mRNA is unveiled in cells lacking Dcp2, where mRNA turnover by tethered Scd6-MS2 is diminished. Similar conclusions were reached previously for tethered Dhh1-MS2 [6].

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Fig 2. Evidence that translational repression of the GFP reporter by tethered Scd6-MS2-F is coupled to mRNA turnover via Dcp2 and requires Dhh1 in vivo.

(A-C) Transformants of WT (HFY114), dcp2Δ (CFY1016), dhh1Δ (YQZ127), or dcp2Δdhh1Δ (QZY128) strains (isogenic in the W303 background) harboring expression plasmids for MS2-F (pQZ130] or Scd6-MS2-F (pQZ127) and GFP reporter pJC429, were analyzed for reporter protein (A-B) and mRNA (C) expression as in Fig 1B–1D. Mean values (± S.E.M.s) from three different biological replicates per group are shown. (D-E) Changes in expression of GFP reporter protein or mRNA (D), or in TE (E), on tethering Scd6-MS2-F versus MS2-F for the strains analyzed in panels A-C, reported as in Fig 1F. Mean values (± S.E.M.s) were determined from at least three biological replicates. Calculations of S.E.M.s for changes in mean ratios of GFP protein and mRNA expression shown in (D), and determination of P-values from significance testing of differences in mean values in (B-E) using an unpaired Student’s t-test, were conducted as described in the supporting file S1 Text “Analysis and Explanation of Supporting Data Files”. P-values are summarized as: **, P <0.01; *, P <0.05; n.s., not significant. In (E), the entries directly above the bars in cols. 2–3 refer to differences in mean values between the indicated mutants vs. WT (col. 1).

https://doi.org/10.1371/journal.pgen.1007806.g002

The concordance between effects on GFP reporter mRNA and protein expression conferred by tethered Scd6-SM-2 (Fig 2A–2E) and Dhh1-MS2 (Sweet et al., 2012) in WT and dcp2Δ cells raised the possibility that Dhh1 is required for translational repression conferred by tethered Scd6-MS2-F. To address this possibility, we examined the regulation of GFP reporter expression in dhh1Δ cells. In contrast to our findings for isogenic dcp2Δ cells, we observed greater repression of reporter mRNA abundance by tethered Scd6-MS2-F in dhh1Δ versus WT cells (Fig 2C, Scd6-MS2-F vs. MS2-F, WT vs. dhh1Δ), with the Scd6-MS2-F/MS2-F repression ratio for GFP mRNA declining ~2.4-fold from ~0.4 in WT to ~0.17 in dhh1Δ cells (Fig 2D, white bars, WT vs. dhh1Δ; P = 0.007). Thus, unlike Dcp2, Dhh1 is dispensable for repression of reporter mRNA abundance by tethered Scd6-MS2-F. One possible explanation for the enhanced repression of GFP mRNA in dhh1Δ cells (vs. WT) might be that Dhh1 impedes Dcp1/Dcp2-mediated decapping of the reporter in the presence of tethered Scd6-MS2-F. Repression of GFP protein by Scd6-MS2-F was also intact in the dhh1Δ strain (Fig 2B, Scd6-MS2-F vs. MS2-F; P = 0.001), with an Scd6-MS2-F/MS2-F repression ratio for GFP protein ~1.6-fold lower than that seen in WT cells (Fig 2D, black bars, WT vs. dhh1Δ; P = 0.03). However, owing to even greater Scd6-MS2-F-mediated repression of GFP mRNA compared to GFP protein, the TE of the GFP reporter was increased by ~1.4-fold in dhh1Δ vs. WT cells (Fig 2E; P = 0.009). This observation is consistent with the possibility that Dhh1 contributes to translational repression by tethered Scd6-MS2-F.

We reasoned that if Dcp2 and Dhh1 are respectively required for repression of mRNA abundance and translation by tethered Scd6-MS2-F, then eliminating both proteins in a dcp2Δ dhh1Δ double mutant should abrogate repression of both GFP protein and GFP mRNA by Scd6-MS2-F. Indeed, nearly identical expression levels of GFP protein and mRNA were observed in the dcp2Δ dhh1Δ mutant whether expressing Scd6-MS2-F or MS2–F (Fig 2B and 2C, dcp2Δ dhh1Δ, Scd6-MS2-F vs. MS2-F), yielding near-unity Scd6-MS2-F/MS2-F repression ratios in this strain for both reporter mRNA and protein expression (Fig 2D, dcp2Δ dhh1Δ, black and white bars), which are markedly increased from the corresponding repression ratios of 0.36 and 0.40 in WT cells (Fig 2D, dcp2Δ dhh1Δ vs. WT, white and black bars; P = 0.004 (Protein), P = 0.0009 (mRNA)). Importantly, comparing the reporter TEs in the double mutant to the dcp2Δ single mutant reveals a loss of translational repression in dcp2Δ dhh1Δ cells (Fig 2E, dcp2Δ dhh1Δ vs. dcp2Δ; P = 0.04), supporting a requirement for Dhh1 in translational repression of GFP mRNA abundance by tethered Scd6-MS2-F. That the residual repression of mRNA abundance seen in the dcp2Δ single mutant is eliminated by dhh1Δ in the double mutant (Fig 2D, white bars, dcp2Δ dhh1Δ vs. dcp2Δ; P = 0.02) might indicate that Dhh1 mediates a Dcp2-independent mechanism of mRNA degradation evoked by tethered Scd6-MS2-F in dcp2Δ cells, such as involvement of the exosome or a ribosome-associated endonuclease. In summary, analyses of dcp2Δ and dhh1Δ mutants indicate that efficient repression of reporter mRNA abundance is dependent on Dcp2, whereas Dhh1 participates in translational repression, by tethered Scd6-MS2-F.

Tethered Scd6-MS2-F alters the polysomal distribution of GFP mRNA

Having observed that the occurrence of translational repression of GFP mRNA is unmasked in dcp2Δ cells, owing to partial stabilization of reporter mRNA, we examined the effect of tethered Scd6-MS2-F on the size distribution of GFP mRNA in polysomes, 80S monosomes, ribosomal subunits, and free mRNPs. Using sedimentation through sucrose density gradients and qRT-PCR analysis of GFP and actin (ACT1) mRNA in the gradient fractions, we reproducibly observed a shift in the fractions with peak abundance of GFP mRNA from polysomes containing 3 to 6 ribosomes (fractions 6–8, 3- to 6-mers) in cells expressing MS2-F alone to those containing 80S monosomes and 2- to 3-mers (fractions 3–5) in cells expressing Scd6-MS2–F (Fig 3A and 3B (P-values for fractions 7 and 4 of 0.031 and 0.0005); S4A and S4B Fig). By contrast, the fractions containing the peak abundance of actin mRNA (fractions 8–10, 5- to 8-mers) did not differ reproducibly between cells expressing Scd6-MS2-F versus MS2–F (Fig 3A & 3C). Despite the shift in peak GFP mRNA abundance from 3- to 5-mers to 2-mers and monosomes, there was no reduction in the proportion of GFP mRNA found in the largest polysomes near the bottom of the gradient on tethering Scd6-MS2-F versus MS–F (Fig 3A and 3B, fractions 10–15). One way to explain these findings is to propose that tethering Scd6-MS2-F inhibits translation to a greater extent at the initiation versus elongation stage of protein synthesis for the fraction of GFP mRNA that shifts towards monosomes and free mRNP, while inhibiting elongation more than initiation on a minority fraction that is retained in heavier polysomes.

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Fig 3. Polysome size distribution of GFP reporter mRNA is altered on tethering Scd6-MS2-F.

(A-C) dcp2Δ transformants from Fig 2A harboring the GFP reporter and expressing Scd6-MS2-F or MS2-F were cultured as in Fig 1B and WCEs were separated by velocity sedimentation on sucrose density gradients and fractionated with continuous monitoring at A₂₅₄. A representative profile is shown in (A). The abundance of GFP mRNA (B) or ACT1 mRNA (C) was quantitated by RT-qPCR in total RNA extracted from the gradient fractions and plotted as the percentage of total RNA signal in the gradient summed across all fractions. Average results (and ±S.E.M.s) from three biological replicates are shown, which are presented in S4A and S4B Fig. An unpaired Student’s t-test showed that the proportions of mRNA in fractions 7 and 4, representing 4-mers and 1- to 2-mer polysomes, respectively, differed significantly between the Scd6-MS2-F vs. MS2-F transformants, with P-values summarized as: **, P <0.01; *, P <0.05.

https://doi.org/10.1371/journal.pgen.1007806.g003

Elimination of Ccr4 enhances repression of both GFP mRNA abundance and translational efficiency by tethered Scd6-MS2-F

Having implicated Dcp2 in the reduction of reporter mRNA abundance by tethered Scd6-MS2-F, and noting that mRNA degradation in yeast frequently proceeds via removal of the poly(A) tail followed by decapping [4], we asked next whether repression of GFP mRNA expression by Scd6-MS2-F requires Ccr4, the major cytoplasmic deadenylase in yeast [32]. Unexpectedly, the reduction in reporter mRNA abundance by Scd6-MS2-F was enhanced rather than diminished in cells lacking Ccr4. We observed a modest (~30%) reduction in GFP mRNA levels in the ccr4Δ mutant containing empty vector or MS2–F (Fig 4B, vector & MS2-F, white vs. grey bars), but tethering Scd6-MS2-F conferred ~3-fold lower GFP mRNA abundance in ccr4Δ vs. WT cells (Fig 4B, Scd6-MS2-F, white vs. grey bars). The resulting >5-fold reduction in mRNA expression by Scd6-MS2-F versus MS2-F seen in the ccr4Δ mutant (Fig 4B, Scd6-MS2-F vs. MS2-F, white bars) is >2-fold larger than that observed in WT cells (Fig 4B, Scd6-MS2-F vs. MS2-F, grey bars; Fig 4C, ΔGFP mRNA, ccr4Δ vs. WT; P = 0.004). These results suggest that Ccr4 is not only dispensable for the reduced abundance of GFP mRNA conferred by tethered Scd6-MS2-F, but actually appears to impede a more efficient repression pathway that can operate in its absence.

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Fig 4. Evidence that both translational repression and mRNA turnover evoked by tethered Scd6-MS2-F are intensified in ccr4Δ cells.

(A-D) Transformants of isogenic WT (BY4741) and ccr4Δ (387) strains (A-C), or WT (BY4741) and caf1Δ (7123) strains (D), harboring the GFP reporter and MS2 fusion expression plasmids described in Fig 1 were analyzed for reporter protein (A) and mRNA (B,D) expression as in Fig 1B–1D. Mean values (± S.E.M.s) were determined from at least three biological replicates. Calculations of S.E.M.s for changes in mean ratios of GFP protein and mRNA expression shown in (C & D), and determination of P-values from significance testing of differences in mean values in (A-D) using an unpaired Student’s t-test, were conducted as described in the supporting file S1 Text. P-values are summarized as: **, P <0.01; *, P <0.05. P-values for panel A are <0.0001 (WT) and 0.0012 (ccr4Δ); P-values for panel B are <0.0001 for both WT and ccr4Δ cells.

https://doi.org/10.1371/journal.pgen.1007806.g004

Interestingly, the absence of Ccr4 also dramatically increased the repression of GFP protein expression by Scd6-MS2–F (Fig 4A, Scd6-MS2-F vs. MS2-F, white vs. grey bars), reducing the Scd6-MS2-F:MS2-F repression ratio for GFP protein from 0.42 to ~0.07 (Fig 4C, ΔGFP protein, ccr4Δ vs. WT; P = 0.0001). Owing to greater repression of GFP protein versus GFP mRNA in ccr4Δ cells, tethering Scd6-MS2-F decreased the TE of GFP mRNA to ~40% of the corresponding TE of ~1.1 observed in WT cells (Fig 4C, ΔTE_GFP, ccr4Δ vs. WT; P<0.0001). These findings imply that the presence of Ccr4 also interferes with a more efficient mechanism for translational repression by tethered Scd6-MS2-F that can proceed in ccr4Δ cells. Similar findings were observed on tethering Dhh1-MS2 versus MS2-F to GFP mRNA, as repression of protein expression was greatly enhanced in ccr4Δ versus WT cells (Fig 4A and 4B, Dhh1-MS2-F vs. Scd6-MS2-F, grey vs. white bars), as previously observed with this same tethering system [6].

Recent findings suggest that the Caf1 subunit of the Ccr4-Not complex cooperates with Ccr4 in the deadenylation and degradation of a subset of yeast mRNAs with low codon optimality, functioning upstream of Dhh1-mediated decapping of such mRNAs [33]. Accordingly, we asked whether eliminating Caf1 from cells would diminish the repression of GFP mRNA abundance conferred by tethered Scd6-MS-F. At odds with this possibility, we observed a greater reduction in GFP mRNA abundance on tethering Scd6-MS2-F in caf1Δ compared to WT cells (Fig 4D; P = 0.01), similar to our findings with the ccr4Δ mutant (Fig 4C). Thus, the Ccr4-Not complex is dispensable for, and even seems to impede, the degradation of mRNAs promoted by tethered Scd6-MS2-F, as observed previously for tethered Dhh1 [6, 34].

Dhh1-dependent translational repression of lacZ reporter mRNA by tethered Scd6-MS2-F is enhanced in the absence of Ccr4

To examine further whether tethered Scd6-MS2-F can repress translation of reporter mRNA independently of Ccr4, we utilized an alternative reporter mRNA, in which the bacterial lacZ gene, encoding β-galactosidase, replaced the GFP coding sequences (Fig 5A), and assays of β-galactosidase activity in cell extracts replaced Western analysis for quantifying reporter protein expression. As observed with the GFP reporter, tethering Scd6-MS2-F conferred an ~2- to 2.5-fold repression of β-galactosidase activity compared to that measured with MS2-F alone in the two different WT strains described above (Fig 5B, Scd6-MS-F vs. MS2-F in WT(BY4741) (P<0.0001) and WT(W303) (P = 0.0005); Fig 5D, grey bars, WT strains). By contrast, β-galactosidase expression on tethering Npl3-MS2-F or Sbp1-MS2-F was indistinguishable from that observed for the corresponding MS2-only controls, or with empty vector in WT cells (S5C Fig). Thus, tethering Scd6-MS2-F, but not the corresponding Npl3 or Sbp1 fusions, confers similar repression of protein expressed from GFP and lacZ reporters. Repression of the lacZ reporter by Scd6-MS2-F was intact in an scd6Δ strain (S5A Fig; P<0.0001), ruling out a contribution of native Scd6 to the function of tethered Scd6-MS2-F. Expression of β-galactosidase activity from heterologous GCN4-lacZ or GAL1-lacZ reporters lacking MS2 binding sites was indistinguishable in cells expressing Scd6-MS-F or MS2–F (S5B Fig), indicating that repression by Scd6-MS-F requires its tethering to lacZ mRNA.

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Fig 5. Dhh1-dependent translational repression of lacZ reporter mRNA by tethered Scd6-MS2-F in vivo.

(A) Schema of the MS2CP tethering system, as in Fig 1A, for a lacZ versus GFP reporter mRNA. (B-C) Transformants of WT strains BY4741 or HFY114 (W303 background), as indicated, and ccr4Δ (387), dhh1Δ (3858), or dcp2Δ (CFY1016) strains, containing the MS2-F or Scd6-MS2-F expression plasmids from Fig 1 and lacZ reporter plasmid (pQZ131), were cultured as in Fig 1B. β-galactosidase activities (in units of nanomoles of o-nitrophenyl-b-D-galactopyranoside cleaved per min per mg) were measured in WCEs (B) and lacZ mRNA was quantified by RT-qPCR (C). The ccr4Δ and dhh1Δ strains are isogenic to BY4741; the dcp2Δ mutant is isogenic to HFY114. Mean values (± S.E.M.s) were determined from at least three biological replicates. Calculations of S.E.M.s for changes in mean ratios of β-galactosidase activity and lacZ mRNA expression shown in (D), and determination of P-values from significance testing of differences in mean values in (B-E) using an unpaired Student’s t-test, were conducted as described in the supporting file S1 Text. P-values are summarized as: **, P <0.01; *, P <0.05; n.s., not significant.

https://doi.org/10.1371/journal.pgen.1007806.g005

Interestingly, lacZ reporter mRNA abundance was not significantly altered, or reduced by only ~20%, by tethered Scd6-MS2-F in the two different WT strains (Fig 5C, Scd6-MS-F vs. MS2-F in WT(BY4741) and WT(W303) (P = 0.02); Fig 5D, ΔlacZ mRNA, WT strains). As a consequence of greater repression of protein versus mRNA expression in WT cells (Fig 5D, WT strains, grey vs. white bars), tethering Scd6-MS2-F reduced the TE of lacZ reporter mRNA by ~30–50% compared to MS2-F alone in the WT strains (Fig 5E, WT strains). Thus, unlike our findings for the GFP reporter (Fig 1F, ΔTE_GFP), the ability of tethered Scd6-MS2-F to repress translation of the lacZ reporter was observable in WT cells containing Dcp2.

Tethering Scd6-MS2-F in ccr4Δ cells conferred a dramatic ~9-fold reduction in β-galactosidase expression, and also a ~2.7-fold decrease in lacZ mRNA expression, in comparison to MS2-F alone (Fig 5B and 5C, Scd6-MS-F vs. MS2-F, ccr4Δ; P = 0.001 (panel B), P<0.0001 (panel C). The reductions in β-galactosidase expression and lacZ mRNA on tethering Scd6-MS-F were both greater in ccr4Δ versus WT(BY4741) cells (Fig 5D, grey and white bars, ccr4Δ vs. WT(BY4741); P = <0.0001 (grey), P = 0.0005 (white). However, the reduction in β-galactosidase was relatively larger, yielding an ~3-fold reduction in TE attributable to tethered Scd6-MS2-F, which exceeds the ~2-fold reduction in TE found in WT cells (Fig 5E, ccr4Δ; P = 0.002). Thus, eliminating Ccr4 enhances the repression of both mRNA abundance and translational efficiency of the lacZ reporter by tethered Scd6-MS2-F, as observed above for the GFP reporter (Fig 4C). The finding that tethered Scd6-MS2-F yields a larger reduction in GFP versus lacZ reporter mRNA abundance in WT cells might be explained by proposing that another step in mRNA turnover that is not accelerated by tethered Scd6-MS-F (eg. exonucleolytic degradation by Xrn1 or deadenylation) is more rate-limiting than decapping for the lacZ reporter.

Compared to the levels observed in the isogenic WT strain, the levels of the lacZ reporter mRNA and expression of β-galactosidase were substantially reduced in dhh1Δ cells, even in the presence of empty vector or MS2–F (S5E and S5F Fig, vector and MS2-F, grey vs. white bars). Expression of an unrelated GAL1-lacZ fusion was also diminished by dhh1Δ in cells lacking any MS2 proteins, albeit to a smaller degree (S5D Fig). While these effects of dhh1Δ complicated our analysis, we nevertheless obtained results consistent with the earlier conclusion that Dhh1 is required for translational repression by tethered Scd6-MS2-F. Despite the general reductions in lacZ mRNA levels in dhh1Δ cells, Scd6-MS2-F conferred ~2.5-fold lower mRNA levels versus MS2-F alone in this mutant (Fig 5C, Scd6-MS2-F vs. MS2-F, dhh1Δ; P = 0.0001). Thus, eliminating DHH1 uncovers a reduction in lacZ mRNA abundance on tethering Scd6-MS2-F not observed in the isogenic WT strain (Fig 5D, white bars, dhh1Δ vs. WT; P = 0.0002). Expression of β-galactosidase in dhh1Δ cells also showed an ~2-fold reduction on tethering Scd6-MS2-F versus MS2–F (Fig 5B, Scd6-MS2-F vs. MS2-F, dhh1Δ; P<0.0001), as in the WT strain (Fig 5D, white grey bars, dhh1Δ vs WT). Because tethered Scd6-MS2-F reduced both reporter mRNA and reporter protein by ~60% compared to MS2-F alone (Fig 5D, dhh1Δ), it did not confer any reduction in TE of lacZ mRNA, in contrast to the ~50% reduction in TE observed in WT cells (Fig 5E, dhh1Δ vs. WT; P = 0.001), which is consistent with Dhh1 being required for translational repression of the lacZ reporter.

Deletion of DCP2 led to an ~2.5-fold increase in lacZ mRNA levels in the presence of MS2-F alone (Fig 5C, MS2-F, dcp2Δ vs. WT(W303)), which also occurred in the presence of vector alone and, hence, is not a consequence of tethering (S5G Fig). Tethering Scd6-MS2-F evoked an ~27% additional increase in lacZ reporter mRNA versus tethering MS2-F in dcp2Δ cells (Fig 5C, Scd6-MS2-F vs. MS2-F, dcp2Δ; P = 0.03), eliminating the small reduction in lacZ mRNA abundance on tethering Scd6-MS2-F in the isogenic WT strain noted above (Fig 5C, Scd6-MS2-F vs. MS2-F, WT; Fig 5D, white bars, dcp2Δ vs. WT; P = 0.001). The fact that the ~1.3-fold increase in lacZ mRNA conferred by tethered Scd6-MS2-F versus MS2-F in dcp2Δ cells is smaller than the corresponding ~2-fold increase in GFP mRNA abundance conferred by tethered Scd6-MS2-F in dcp2Δ versus WT cells shown above (Fig 2C, black vs. dark grey bars) might reflect that tethering Scd6-MS2-F confers a much smaller reduction in lacZ mRNA (~1.1-fold) versus GFP mRNA abundance (~2.6-fold) in WT cells (Figs 5D vs. 2D, WT cells, white bars). Coupling the small increase in lacZ mRNA abundance with a slight reduction in β-galactosidase expressed on tethering Scd6-MS2-F versus MS2-F in dcp2Δ cells (Fig 5B and 5C, Scd6-MS2-F vs. MS2-F, dcp2Δ), results in an ~30% reduction in TE compared to tethering MS2-F alone, which is indistinguishable from that observed in the isogenic WT strain (Fig 5E, dcp2Δ vs. WT(W303)). These findings are consistent with our conclusion above that Dcp2 is dispensable for translational repression by tethered Scd6-MS2-F.

In summary, tethering Scd6-MS2-F to the lacZ reporter confers a decrease in translational efficiency that appears to be dependent on Dhh1, independent of Dcp2, and dampened by Ccr4; and Ccr4 also diminishes the repression of lacZ mRNA abundance by tethered Scd6-MS2-F. All of these observations are in agreement with our findings for the GFP reporter. Unlike our results for the GFP reporter, where deleting DCP2 reduced the TE on tethering Scd6-MS2–F (Fig 2E, cols. 1–2), dcp2Δ had little effect on TE of the lacZ reporter because it did not substantially reduce the apparent degradation of lacZ mRNA by tethered Scd6-MS2-F, conferring only a small increase in lacZ mRNA abundance (Fig 5D, WT vs. dcp2Δ, white bars.)

The LSm but not the RGG domain of Scd6 is required for repression of reporter expression by tethered Scd6-MS2-F

Scd6 interacts with eIF4G via the C-terminal region of Scd6 containing the RGG domain [22]. Interaction partners of the N-terminal LSm domain of Scd6 are unknown; however, the LSm domains in Scd6 homologs from different species mediate binding to Dcp2 in S. pombe [35], both Dcp1 and the translational repressor CUP in D. melanogaster [15], and decapping activators and translational repressors 4E-T and EDC4 in humans [36]. We examined the importance of the LSm and RGG domains of Scd6 for reporter mRNA repression by truncating the Scd6-MS2-F fusion at the N- or C-terminal ends to remove these domains individually (Fig 6A). Eliminating the LSm domain completely abrogated repression of both protein and mRNA expressed from the GFP reporter by tethered Scd6-MS2–F (Fig 6D and 6E, white vs. grey bars; P = 0.0001 (D), P<0.0001 (E) for ΔLSm-Scd6-MS2-F vs. Scd6-MS2-F), without detectably altering expression of the Scd6-MS2-F fusion protein (Fig 6B, upper blot, lanes 5–12). The fact that no reduction in GFP mRNA occurs on tethering the ΔLSm-Scd6-MS2-F variant implies that the LSm domain is required for accelerated mRNA turnover conferred by WT tethered Scd6-MS2-F. Because there is no reduction in GFP protein expression on tethering the ΔLSm-Scd6-MS2-F, despite high levels of the GFP reporter mRNA, we can also infer that translational repression of GFP mRNA is eliminated by removing the LSm domain. Repression of β-galactosidase expression from the lacZ reporter was also abolished by removing the LSm domain from Scd6-MS2–F (Fig 6G, white vs. grey bars; P<0.0001 for ΔLSm-Scd6-MS2-F vs. Scd6-MS2-F). Because there is little or no reduction in lacZ mRNA abundance on tethering WT Scd6-MS2-F, it seems likely that translational repression is abrogated by removing the LSm domain for this reporter mRNA as well. (However, we cannot discard the unlikely possibility that tethering ΔLSm-Scd6-MS2-F would substantially increase the abundance of lacZ mRNA and thereby mask efficient translational repression by the ΔLSm variant.)

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Fig 6. Evidence that the conserved N-terminal LSm domain is essential for translational repression and stimulation of mRNA decay by tethered Scd6-MS2-F in vivo.

(A) Diagrams representing the domain organization of full-length Scd6 (Scd6-MS2-F) or variants lacking amino acids 1–83 at its N-terminus (ΔLSm-Scd6-MS2-F) or amino acids 286–312 at its C-terminus (ΔRGG-Scd6-MS2-F), present in the corresponding fusions to MS2-F. MS2 and FLAG tags are not depicted. (B-F) Transformants of WT strain BY4741 containing the GFP reporter plasmid pJC429 and expression plasmids for MS2-F (pQZ130) and Scd6-MS2-F (pQZ127), and either ΔLSm-Scd6-MS2-F (pQZ139) (B, D-E) or ΔRGG-Scd6-MS2-F (pQZ142) (C & F) were analyzed for GFP protein (B-D & F) and mRNA (E) expression as in Fig 1B–1D. (G) Transformants of WT strain BY4741 harboring the lacZ reporter plasmid pQZ131 and the expression plasmids for MS2-F, Scd6-MS2-F, or ΔLSm-Scd6-MS2-F used in (B) were analyzed for β-galactosidase expression as in Fig 5B. Mean values (± S.E.M.s) were determined from at least four biological replicates. Determination of P-values from significance testing of differences in mean values in (D-G) using an unpaired Student’s t-test, were conducted as described in the supporting file S1 Text. P-values are summarized as: **, P <0.01; *, P <0.05.

https://doi.org/10.1371/journal.pgen.1007806.g006

In contrast to our findings on removing the LSm domain, eliminating the RGG domain from Scd6-MS2-F had no apparent effect on repression of the GFP reporter by Scd6-MS2–F (Fig 6C, GFP blot; Fig 6F, black vs. grey; P = 0.002), although an apparent increase in expression of the ΔRGG-Scd6-MS2-F versus WT Scd6-MS2-F fusion (Fig 6C, upper blot, lanes 5–12) might have obscured a reduced efficiency of reporter repression for the ΔRGG variant. These findings indicate that the LSm domain, and most likely interactions it mediates with effector proteins, is required for both enhanced degradation of GFP reporter mRNA and translational repression of both reporters by tethered Scd6-MS2-F. By contrast, interaction of Scd6-MS2-F with eIF4G via the Scd6 RGG domain might be dispensable for translational repression when Scd6 is tethered tightly to the mRNA; although we cannot eliminate the possibility that translational repression is impaired by the ΔRGG truncation and that the efficient repression of GFP reporter protein expression conferred by ΔRGG-Scd6-MS2-F occurs exclusively from accelerated mRNA turnover.

Scd6 and Dhh1 cooperate in regulating mRNA abundance and translational efficiencies of particular native mRNAs in vivo

To determine whether Scd6 and Dhh1 participate in regulating the abundance or translation of native yeast mRNAs, we conducted ribosome footprint profiling and RNA-Seq analyses on the WT, dcp2Δ, scd6Δ and dhh1Δ strains in the W303 genetic background, cultured in rich (YPD) medium. We also analyzed isogenic dcp2Δscd6Δ and dcp2Δdhh1Δ double mutants, anticipating that changes in translational efficiency might be more evident in the absence of mRNA decapping by Dcp2. Independent RNA-Seq analysis was also conducted in parallel on two isogenic scd6Δ strains, an additional isogenic dhh1Δ strain and isogenic mutants lacking the Dcp2-decapping activators Pat1 and Lsm1 [4] to determine whether Pat1 and Lsm1 contribute to Scd6-mediated repression of mRNA abundance. The results of biological replicates were highly correlated for both ribosome-protected fragments (RPFs) and mRNA sequences for all strains analyzed (S6A–S6L Fig).

RNA-Seq analysis of WT and scd6Δ strains identified 83 mRNAs whose abundance was significantly up-regulated in the mutant by ≥1.4-fold at an FDR of <0.01, with a median fold-change (FC) compared to WT of ~1.8, which is significantly higher than the median FC for all mRNAs (which is 1.0 (log₂(ΔmRNA) = 0)) owing to normalization for equal RNA read numbers for all genes in each strain) (Fig 7A, scd6Δ). Interestingly, this group of mRNAs also displayed significantly increased abundance in the isogenic dhh1Δ, pat1Δ, and lsm1Δ strains, with median FCs of ~3.3, ~1.9, and ~1.8, respectively (Fig 7A, cols. 2–4). Consistent with the latter, most of the 83 mRNAs up-regulated in scd6Δ cells are a subset of the larger group of 733 mRNAs elevated to the same degree in dhh1Δ cells (Fig 7B). Importantly however, derepression of these 83 mRNAs was not observed on deleting SCD6 or DHH1 in the strain lacking DCP2, ie. when comparing the dcp2Δscd6Δ and dcp2Δdhh1Δ double mutants to the dcp2Δ single mutant (Fig 7A, rows 5–6). These findings are consistent with the notions that: (i) Scd6 accelerates degradation of a subset of native mRNAs, (ii) that Dhh1, Pat1, and Lsm1 all participate in this down-regulation of mRNA abundance, and (iii) that the decapping enzyme subunit Dcp2 is required for both Scd6- and Dhh1-enhanced degradation of the set of Scd6-targeted mRNAs.

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Fig 7. Scd6 and Dhh1 cooperate in repressing mRNA abundance and translational efficiencies of a subset of native mRNAs.

(A) Notched box-plots of log₂ mRNA changes (ΔmRNA) in the indicated mutants for 83 mRNAs exhibiting ≥1.4-fold increased mRNA abundance in scd6Δ versus WT cells (at FDR<0.01). The 83 mRNAs were identified by RNA-Seq analysis of scd6Δ strains (SYY2352 and SYY2353) and isogenic WT strain (HFY114); and the RNA changes shown for the mutants were determined by RNA-Seq analysis of isogenic strains of the indicated genotypes: dhh1Δ (SYY2686), pat1Δ (SYY2674), lsm1Δ (SYY2680), all compared to WT strain (HFY114); and dcp2Δscd6Δ (FZY843) and dcp2Δdhh1Δ (QZY128) compared to the dcp2Δ strain (CFY1016). (In this and all subsequent plots, the double mutants carry an (*) to indicate that their values have been compared to the dcp2Δ single mutant rather than to WT.) Non-overlapping notches indicate that the true medians of the two groups are different with a 95% confidence level. (B) Overlap between 83 mRNAs exhibiting ≥1.4-fold increased mRNA abundance in scd6Δ (SYY2352 and SYY2353) vs. WT (HFY114) cells and 733 mRNAs similarly up-regulated in dhh1Δ cells (QZY126), both at FDR<0.01. (C) Notched box-plots of log₂ TE changes (ΔTE) in the indicated mutants for 78 mRNAs exhibiting ≥1.33-fold increased ribosome abundance across the CDS in scd6Δ versus WT cells (at P≤0.01). The 78 mRNAs were identified by ribosome profiling of scd6Δ strain (SYY2353); and TE changes were calculated from ribosome profiling/RNA-seq data conducted on this scd6Δ strain, dhh1Δ strain (QZY126, dhh1Δ(z)), and WT strain (HFY114); and on the dcp2Δscd6Δ, dcp2Δdhh1Δ and dcp2Δ strains mentioned in (A). TE changes for the dhh1Δ(r) and dhh1Δ(j) strains in the BY4743 background were calculated from published ribosome profiling/RNA-Seq data from Radhakrishnan et al. [10] and Jungfleisch et al. [37], respectively. (D) Overlap between 78 mRNAs exhibiting ≥1.33-fold increased ribosome occupancy (p<0.01) in scd6Δ (SYY2353) vs. WT (HFY114) cells and 968 mRNAs similarly up-regulated by ≥1.4-fold (FDR<0.01) in dhh1Δ cells (QZY126). (E) Notched box-plots of log₂(ΔTE) values in the indicated mutants for 53 mRNAs exhibiting ≥1.33-fold increased TE in scd6Δ versus WT cells (at P≤0.1), identified by ribosome profiling/RNA-Seq analysis of scd6Δ (SYY2353) and WT (HFY114) strains. The TE changes were calculated from the data sets described in (B). (F) Overlap between 53 mRNAs exhibiting ≥1.33-fold increased TE (p<0.10) in scd6Δ (SYY2353) vs. WT (HFY114) cells and 283 mRNAs similarly up-regulated by ≥1.33-fold (FDR<0.10) in dhh1Δ cells (QZY126). P-values in (B-F) were assigned based on the hypergeometric distribution. For the boxplots in panels A, C, & E, the changes in mRNA abundance or TE for the relevant group of mRNAs found in each of the indicated mutants were plotted irrespective of whether the changes exhibit statistical significance in that mutant, to allow a coherent comparison of the behavior of the complete cohort of mRNAs across the entire panel of mutants. Statistical significance is evaluated for differences in the median changes found in the different mutants, with non-overlapping notches indicating with 95% confidence that the median changes found for two mutants differ from one another.

https://doi.org/10.1371/journal.pgen.1007806.g007

We obtained complementary results for a group of 346 mRNAs whose abundance was significantly increased (at FDR<0.01) in an isogenic dhh1Δ mutant, exhibiting an ~3-fold increase in median mRNA abundance in dhh1Δ vs. WT cells (S7A Fig, dhh1Δ(z)), and also showing ~1.9-fold and ~1.8-fold increases in two published RNA-seq datasets for a dhh1Δ mutant in the BY4741 background [10, 37] (S7A Fig, dhh1Δ(r) and dhh1Δ(j)). This group of Dhh1 down-regulated mRNAs also displays a slight, but statistically significant, ~15% derepression in the scd6Δ mutant (S7A Fig, scd6Δ). Supporting these findings, hierarchical clustering analysis of expression changes for all mRNAs revealed that a large proportion of genes exhibit altered mRNA levels in the same direction in response to scd6Δ or dhh1Δ (S7B Fig), which is particularly evident for the mRNAs showing the largest fold-changes in dhh1Δ vs. WT cells (S7C Fig), with the magnitudes of these changes being generally greater in dhh1Δ vs. scd6Δ cells (S7B and S7C Fig). These results suggest that Scd6 contributes appreciably to Dhh1-enhanced degradation of a large fraction of the mRNAs whose abundance is repressed by Dhh1. As observed for the Scd6 down-regulated mRNAs, the derepression of mRNA levels for the group of 346 Dhh1-repressed mRNAs conferred by either scd6Δ or dhh1Δ was eliminated when these mutations were made in cells lacking DCP2 (S7A Fig, cf. cols. 1–2 and 5–6), indicating that Dcp2 is required for robust Dhh1-mediated mRNA turnover.

The two groups of 83 and 346 mRNAs whose abundance is derepressed in scd6Δ (Fig 7A) or dhh1Δ cells (S7A Fig), respectively, were interrogated next for changes in translation efficiency (TE) in different mutants by combining the results of ribosome profiling and RNA-Seq experiments conducted on the same cell cultures. The TE of each mRNA was calculated as the sum of RPFs divided by the sum of RNA reads across the CDS, and the change in TE (ΔTE) was calculated as the ratio of TE in mutant versus WT cells. (Because RPFs and mRNA reads for each gene are normalized to RPF or mRNA reads for all genes in each strain, the ΔTE for each gene is determined relative to the median ΔTE for all genes, which is ~1.0). Both groups of mRNAs showing derepression of mRNA abundance in scd6Δ or dhh1Δ cells also exhibited modest increases in median TE in our dhh1Δ mutant of ~12–15% (S7D and S7E Fig, col. 5 in each panel). Comparable or somewhat greater increases in TE were identified in the two published ribosome profiling/RNA-seq datasets for a dhh1Δ mutant in the BY4741 background [10, 37] (S7D and S7E Fig, cols. 3–4 in each panel); whereas the scd6Δ mutation increased the median TE for these groups of mRNAs by only ~5% (S7D and S7E Fig, col. 1). Despite the modest TE increases for these groups of mRNAs conferred by dhh1Δ, it is noteworthy that these changes were not observed on comparing the dcp2Δdhh1Δ double mutant to the dcp2Δ single mutant (S7D and S7E Fig, cols. 5–6 in each panel), providing genetic evidence that the TE changes are genuine, and indicating that translational repression by Dhh1 is dependent on Dcp2.

Evidence for Dcp2-dependent translational repression of native mRNAs by Scd6 was provided by examining a group of 78 mRNAs exhibiting significantly increased ribosome occupancies in the scd6Δ mutant, and for a second group of 53 mRNAs showing the largest TE increases conferred by scd6Δ. In response to scd6Δ, both groups of mRNAs exhibit increased median TEs of ~1.2- and ~1.5-fold, respectively, in otherwise WT cells, but not in the presence of dcp2Δ (Fig 7C & 7E, cf. cols. 1–2 in each panel). Comparable, or somewhat greater, increases in median TE were observed for both groups of mRNAs in all three dhh1Δ datasets (Fig 7C & 7E, cols. 3–5), indicating that Dhh1 contributes to translational repression of a substantial proportion of the mRNAs thus repressed by Scd6. This last inference is further supported by the significant overlaps between mRNAs exhibiting increased ribosome occupancies or TEs in response to scd6Δ and the larger groups of mRNAs showing comparable increases in ribosome occupancy or TE in response to dhh1Δ (Fig 7D & 7F). Moreover, clustering analysis for all mRNAs showed that the majority of mRNAs displaying increased TEs in response to scd6Δ also exhibit TE increases of generally greater degree in response to dhh1Δ; although it is also evident that many mRNAs translationally repressed by Dhh1 are not repressed by Scd6 (Fig 8A and 8B). Once again, comparing the dcp2Δdhh1Δ double mutant to the dcp2Δ single mutant revealed that dcp2Δ largely suppresses the TE increases conferred by dhh1Δ in DCP2 cells (Fig 7C & 7E, cf. cols. 5–6 in each panel), supporting a widespread requirement for Dcp2 in translational repression by Dhh1.

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Fig 8. Scd6 and Dhh1 cooperate in repressing translational efficiencies of a subset of native mRNAs in a manner associated with elevated Dhh1 occupancies.

(A-B) Hierachical clustering analysis conducted with the R heatmap.2 function from the R gplots library, using the default “hclust” hierarchical clustering algorithm, using ribosome profiling data from scd6Δ (SYY2353), dhh1Δ (QZY126, dhh1Δ(z)), and WT (HFY114) strains. Approximately 50 genes were removed for which no data were available in one of the strains, or where the log₂(ΔTE) value was >4 or <-4 in one of the mutant vs. WT comparisons, after which separate clustering analysis was performed on two sets of mRNAs in which all log₂(ΔTE) values fell between -2 or +2 (panel A, 5276 mRNAs), or in which the log₂(ΔTE) value in one of the mutants was < -2 or > +2 (panel B, 45 mRNAs). The color key for log₂(ΔTE) values is indicated above each analysis. (C) Exemplar gene exhibiting increased TE in both scd6Δ and dhh1Δ versus WT cells. Integrated Genomics Viewer (Broad Institute) display of ribosome-protected fragments (RPFs) and mRNA reads across the YOR173W gene from two biological replicates each for WT, scd6Δ and dhh1Δ strains, shown in units of rpkm (reads per 1000 million mapped reads). Position of the CDS (magenta) is at the bottom with the scale in bp; scales of rpkm for each track are on the left, and calculated ΔRPF, ΔmRNA and ΔTE values between each mutant and WT are on the right. (D-E) Boxplot analysis of changes in ribosome occupancy or TE versus Dhh1 occupancy. Dhh1 RIP-seq enrichment values from Miller et al (2018) were equally divided into five pentiles of 739 genes from lowest to highest values and plotted against the log₂(Δribo) values (D) or log₂(ΔTE) values (E) determined by ribosome profiling analysis of scd6Δ strain SYY2353 and WT strain HFY114. The Pearson correlation coefficients for the relationship between log₂(Δribo) values (panel D) or log₂(ΔTE) values (panel E) and Dhh1 enrichment for all mRNAs are 0.2 (P = 2 X 10⁻³⁴) and 0.16 (P = 2 X 10⁻²²), respectively.

https://doi.org/10.1371/journal.pgen.1007806.g008

The mRNAs encoded by YOR173W, YHR033W, YGR088W, YFR017C, and YNR034W-A exhibit TE increases conferred by scd6Δ, and they respond similarly to dhh1Δ, while exhibiting larger increases in mRNA and TE levels in dhh1Δ versus scd6Δ cells (Fig 8C and S8A–S8D Fig). Gene-ontology (GO) analysis revealed that mRNAs whose transcript abundance was derepressed in dhh1Δ or scd6Δ cells are enriched in related functional categories of genes involved in metabolism of energy reserves and other aspects of carbohydrate metabolism; and the Dhh1-repressed mRNAs are also enriched in stress-response genes (S9A and S9B Fig). These results suggest that Scd6 and Dhh1 cooperate in repressing both mRNA abundance and translational efficiency for a discrete subset of native mRNAs in nutrient-replete medium.

To provide evidence supporting a direct role for Dhh1 in translational repression of native mRNAs by Scd6, we interrogated published results from deep-sequencing of mRNAs specifically immunoprecipitated with epitope-tagged Dhh1 (RIP-Seq) from WT cells grown on rich medium [38]. The 3686 mRNAs for which both RIP-Seq and ribosome profiling data exist were sorted into 5 equal percentiles based on Dhh1 enrichment values in RIP-seq and compared for translation changes in response to dhh1Δ or scd6Δ. This analysis revealed a direct correlation between Dhh1 enrichment values and changes in both ribosome occupancies and TEs in response to dhh1Δ (S10A and S10B Fig), and a similar correlation exists between Dhh1 enrichment and changes in mRNA levels in dhh1Δ vs. WT cells (S10C Fig), as noted previously [38]. These correlations support previous conclusions that Dhh1 binding to mRNAs is associated with accelerated mRNA decay and translational repression. Interestingly, Dhh1 enrichment values are likewise correlated with changes in ribosome occupancies and TEs in response to scd6Δ (Fig 8D and 8E), supporting the notion that Scd6 translational repression of many native mRNAs involves recruitment of Dhh1. Finally, we considered the possible contribution of codon optimality in dictating susceptibility of mRNAs to Scd6. Previously, low codon optimality was associated with Dhh1-mediated mRNA decay in part by demonstrating an inverse correlation between the sTAI value, a measure of overall codon optimality of the mRNA [39], and the change in mRNA abundance in dhh1Δ versus WT cells [10]. This correlation is also evident in our dhh1Δ dataset and that of Jungfleisch et al. [37] (S11B and S11C Fig), although less pronounced than observed in the data from Radhakrishnan et al. [10] (S11A Fig). A similar, modest trend was also evident for mRNA changes observed here in scd6Δ cells (S12A Fig), suggesting that mRNAs exhibiting Scd6-dependent mRNA degradation have a tendency to exhibit poor codon optimality. Interestingly, however, the group of 82 mRNAs whose abundance is most strongly derepressed in scd6Δ cells (characterized in Fig 7A) exhibit sTAI values that are somewhat higher, not lower, than the genome average value (S12B Fig), indicating that poor codon optimality is not the key determinant of Scd6-dependent mRNA turnover for the transcripts that it most strongly represses.

Plasmid constructions

Plasmids employed in this study are listed in Table 1. Plasmids containing constructs encoding FLAG-tagged MS2-CP fusions to Npl3, Sbp1, and Scd6 (S13 Fig, left) were constructed by first PCR-amplifying NPL3, SBP1 or SCD6 respectively with their native endogenous promoter (~500 bp upstream flanking sequence) plus their coding sequence minus the stop codon, from genomic DNA of WT strain BY4741, with primers containing a gene-specific restriction site at the N-terminus, and an XhoI site and XbaI/SpeI site at the C-terminus. The following primers were used: (i) NPL3 (forward primer with SpeI site, 5’-ACGAGGACTAGTTATCAATATGCAAATGCTCGGC-3’; reverse primer with XhoI and SpeI sites, 5’-ACGAGCACTAGTCTCGAGCCTGGTTGGTGATCTTTCACG); (ii) SBP1 (forward primer with XbaI site, 5’-ACGAGCTCTAGATCATCGAGCGGAAAATATTG-3’; reverse primer with XhoI and XbaI sites, 5’-ACGAGCTCTAGACTCGAGTTCTTGCTTTTCTTCAGAACC-3’); (iii) SCD6 (forward primer with SpeI site, 5’-ACGAGGACTAGTTGCTCGTAACAATCTTGG-3’; reverse primer with XhoI and SpeI sites, 5’-ACGAGGACTAGTCTCGAGAAATTCAACGTTGGAAGGAGG-3’). The amplified fragments were inserted between the XbaI/SpeI sites of YCplac111 to generate YCplac111-NPL3, YCpLac111-SBP1, or YCpLac111-SCD6, respectively. The MS2-CP CDS was PCR-amplified from plasmid pJC236 with primers containing an XhoI site and encoding a flexible linker (Gly-Gly-Gly-Gly-Gly-Ser) at the N-terminus, 3xFLAG epitopes, a stop codon and an overlapping sequence (for fusion PCR) at the C-terminus, using forward primer 5’-ATTCATCTCGAGGGTGGTGGTGGTGGTTCTGCTTCTAACTTTACTCAGTTCGTT-3’ and reverse primer 5’-TTACTTGTCATCGTCATCCTTGTAGTCGATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCGTAGATGCCGGAGTTTGCTGCGAT-3’. Next, the 3’UTR from each gene was amplified from genomic DNA of BY4741, with primers containing an upstream overlapping sequence (for fusion PCR) and a downstream XmaI site, using primers: (i) NPL3 3’UTR, forward primer 5’-CATGACATCGACTACAAGGATGACGATGACAAGTAAGCCATTTATATAGTTGAGAAAAAA-3’; reverse primer 5’-ATTTATCCCGGGTACCTATTCTGGCGTGTAATCCTTATCA-3’); (ii) SBP1 3’UTR, forward primer 5’-CATGACATCGACTACAAGGATGACGATGACAAGTAATTACTTCTTACCCACATCCCTATT-3’; reverse primer 5’-ATTTATCCCGGGTACCTCTCCGAGGTAGTGAACCATTGAG-3’); and (iii) SCD6 3’UTR, forward primer 5’-CATGACATCGACTACAAGGATGACGATGACAAGTAAAATGATGTTTCTATGTAAATTAAGTA-3’; reverse primer 5’-ATTTATCCCGGGTACCCTTTTCTTGTAGTTTGTTGTTCTTAC-3’). Fragments containing linker-MS2CP-FLAG-3’UTR sequences for each gene were generated by fusion PCR using the amplified fragments above, and inserted between the XhoI/XmaI sites of YCplac111-NPL3, YCplac111-SBP1, or YCplac111-SCD6, to generate the constructs pQZ125 (NPL3-MS2-F), pQZ126 (SBP1-MS-F) and pQZ127 (SCD6-MS2-F). Plasmids encoding MS2-FLAG control proteins pQZ128 (P_NPL3-MS2-F), pQZ129 (P_SBP1-MS2-F) and pQZ130 (P_SCD6-MS2–F) (S13 Fig, right) were constructed by a strategy similar to that described above but with the NPL3, SBP1 and SCD6 CDSs absent from the final constructs and an ATG added at the beginning of the MS2 CP-encoding fragment. The specific primers for these constructions were: (i) P_NPL3-MS2-F: forward primer for NPL3 promoter with SphI site 5’-ACGAGGGCATGCTATCAATATGCAAATGCTCGGCTC-3’; reverse primer for NPL3 promoter with ATG 5’-CATTATCCTTATGGTTTTAGCGTAATT-3’; forward primer for MS2-NPL3 3’UTR with ATG 5’-AATTACGCTAAAACCATAAGGATAATGGGTGGTGGTGGTGGTTCTGCTTCT-3’; reverse primer for MS2-NPL3 3’UTR with KasI site 5’-ATTTATGGCGCCTATTCTGGCGTGTAATCCTTATCA-3’); (ii) P_SBP1-MS2-F: forward primer for SBP1 promoter with SphI site 5’-ACGAGGGCATGCTCATCGAGCGGAAAATATTGAAAA-3’; reverse primer for SBP1 promoter with ATG 5’-CATATTTTTCTTCGTTTGAGGGTTTTC-3’; forward primer for MS2-SBP1 3’UTR with ATG 5’-GAAAACCCTCAAACGAAGAAAAATATGGGTGGTGGTGGTGGTTCTGCTTCT-3’; reverse primer for MS2-SBP1 3’UTR with XmaI site 5’-ATTTATCCCGGGTACCTCTCCGAGGTAGTGAACCATTGAG-3’); (iii) P_SCD6-MS2-F: forward primer for SCD6 promoter with SphI site 5’-ACGAGGGCATGCTGCTCGTAACAATCTTGGCCTAGC-3’; reverse primer for SCD6 promoter with ATG 5’-CATTGCCTTGCTGCTGTTTTTCGATGA-3’; forward primer for MS2-SCD6 3’UTR with ATG 5’-TCATCGAAAAACAGCAGCAAGGCAATGGGTGGTGGTGGTGGTTCTGCTTCT-3’; reverse primer for MS2-SCD6 3’UTR with XmaI site 5’-ATTTATCCCGGGTACCCTTTTCTTGTAGTTTGTTGTTCTTAC-3’). The GFP reporter plasmid pJC429 was described previously [6]. The lacZ reporter plasmid pQZ131 was generated by PCR-amplifying the lacZ CDS sequence from GCN4-lacZ reporter plasmid p180, adding an SphI site and an ATG to the N-terminus (forward primer 5’-AAACTTGCATGCTTACGGAT-3’), and a PacI site to the C-terminus (reverse primer 5’-ACGAGCTTAATTAATTTTTGACACC-3’). The resulting fragment was inserted between the SphI/PacI sites of pJC429. Plasmid pQZ145 (DCP2 URA3) was generated by inserting into pRS316 a 4.3 kb XbaI-XmaI DCP2 fragment from pRS315-DCP2. Plasmids pQZ139 (ΔLSm-Scd6-MS2-F) and pQZ142 (ΔRGG-Scd6-MS2-F) were generated by deleting the CDS for amino acids Q3-D78 or S287-N318, respectively, of pQZ127 by site-directed mutagenesis (GenScript USA Inc). All plasmids were screened by restriction digestion and DNA sequencing was conducted to verify the presence of the intended inserts.

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Table 1. Plasmids used in this study.

https://doi.org/10.1371/journal.pgen.1007806.t001

Yeast strains and growth conditions

Yeast strains employed in this work are listed in Table 2. Strain QZY126 was constructed by transforming HFY114 with a DNA fragment containing dhh1Δ::kanMX4 and including ~400 bp of sequences from both upstream and downstream of DHH1 that was PCR-amplified from genomic DNA of strain 3858 and selecting on YPD medium containing G418. Strain QZY128 was constructed by transforming strain CFY1016 (dcp2Δ::HIS3) harboring pQZ145 (DCP2 URA3) with the dhh1Δ::kanMX4 cassette as above, and evicting pQZ145 by counter-selection growth on 5-FOA medium. FZY843 was constructed by transforming CFY1016 with a fragment containing scd6Δ::KanMX4 that was PCR-amplified from yeast strain 5544. Strains SYY2352 and SYY2353 were constructed by transforming HFY114 with a DNA fragment harboring the scd6::KanMX6 null allele, which contains 400 bp from both upstream and downstream of SCD6 with the coding region replaced by a 1447-bp KanMX6 cassette. Gene disruptions were confirmed by PCR analysis of chromosomal DNA using the appropriate primers. Unless otherwise noted, all strains were grown at 30°C in synthetic complete medium without leucine or uracil (SC-L-U) with 2% galactose/2% raffinose replacing dextrose. All cultures were grown for at least two cell divisions and harvested at mid-log phase (OD₆₀₀ = 0.6–0.7).

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Table 2. Yeast strains used in this study.

https://doi.org/10.1371/journal.pgen.1007806.t002

Biochemical analyses using yeast cell extracts

For Western blot analysis, whole-cell extracts (WCEs) from at least three biological replicates (independent transformants) were prepared by trichloroacetic acid (TCA) extraction as previously described [43]. Aliquots of WCEs were resolved by 4–20% SDS-PAGE, transferred to PVDF membrane and probed with antibodies against GFP (Covance), Prt1 [44], FLAG epitope (Sigma), or Gcd6 [45]. Immune complexes were detected using the Pierce enhanced chemiluminescence (ECL) system and autoradiography; and signal intensities were quantified by scanning densitometry using NIH ImageJ software. Assays of β-galactosidase activity in WCEs were performed as described previously [46]. At least four biological replicates (and two technical replicates per transformant) were employed for all β-galactosidase assays.

Polysome analysis and RNA extraction from sucrose gradients

For polysomes profiles, 300 mL of cells were treated with 50 μg/ml cycloheximide for 5 min prior to harvesting. WCEs were prepared in 1x breaking buffer (20 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 mM MgCl₂, 1 mM DTT, 50 μg/ml cycloheximide, 1 mM PMSF, Complete EDTA-free Protease Inhibitors, 1U/μl SUPERase-In RNase inhibitor) by vortexing with glass beads, followed by two cycles of centrifugation for 10 min at 15,000 rpm at 4°C. 15 OD₂₆₀ units of cleared lysate were loaded on 15%-45% (w/w) sucrose gradients prepared on a Biocomp Gradient Master in 1x breaking buffer and centrifuged at 41,000 rpm for 73 min at 4°C in a Beckman SW41Ti rotor. Gradients were fractionated with a Brandel Fractionation System and ribosomal peaks were visualized at 254 nm with an Isco UV detector. Fractions (0.7 mL) were precipitated overnight at -20°C using 1.5 volumes RNA precipitation mix (95% EtOH, 5% NaOAc), and centrifuged at 15,000 rpm for 30 min to pellet RNA/protein. Pellets were washed in 1 mL cold 80% EtOH, dried in a speed vacuum, and resuspended in 100 μl AE buffer (50 mM NaOAc, 10 mM EDTA, pH 5.2) with 1% SDS. Fractions were extracted by adding 350 μl QIAzol lysis reagent and incubating 5 min at room temperature, adding 70 μL chloroform and incubating 2–3 min at room temperature, and centrifuging at 15,000 rpm for 15 min at 4°C. The aqueous phase (~300 μl) was transferred to a new collection tube, thoroughly mixed with 1 volume of 100% EtOH, and applied to a purification column (RNA Clean and Concentrator™ kit, Zymo Research) to isolate RNA following the vendor’s instructions. RNA from each polysomal fraction was eluted with 25 μl RNase-free water, and 2 μl/reaction were used for first strand cDNA synthesis, as described below.

Total RNA isolation, RT-qPCR and Northern blot analysis

Yeast cultures (25 mL) were harvested by centrifugation, and the resulting pellets were resuspended in 400 μL AE buffer (50 mM NaOAc, 10 mM EDTA, pH 5.2) with 1% SDS. Cell suspensions were extracted twice with AE buffer-equilibrated phenol, twice with phenol/chloroform/isoamyl alcohol (25:24:1), once with chloroform/isoamyl alcohol (24:1), and then precipitated at -20°C with 2 volumes RNA precipitation mix (95% EtOH, 5% NaOAc). RNA pellets were recovered by centrifugation at 15,000 rpm for 30 min (4°C), washed once with 1 mL cold 80% EtOH, dried in a speed vacuum, and resuspended in 50 μL RNase-free water. Total RNA concentration was calculated at A₂₆₀ using a NanoDrop spectrophotometer. For RT-qPCR, reverse transcription was carried out using SuperScript III First-Strand cDNA Synthesis SuperMix (Invitrogen), with random primers and either 1 μg total cellular RNA from above or 2 μL polysomal RNA isolated from sucrose gradient fractions. qPCR was carried out using Brilliant III Ultra-Fast SYBR Green Master Mix (Agilent) in a Mx3000P System (Stratagene) and the following oligonucleotide pairs: GFP (5’-GGCTAGCAAAGGAGAAGAACTC-3’; 5’-CCGTATGTTGCATCACCTTC-3’), lacZ (5’-ACCAACGTAACCTATCCCATTAC-3’; 5’-TTCCTGTAGCCAGCTTTCATC-3’), ACT1 (5’-TGTGTAAAGCCGGTTTTGCC-3’; 5’-GATACCTCTCTTGGATTGAGCTTC-3’). Levels of reporter mRNA relative to actin were calculated using the ΔCt method. For Northern blot analysis, 10 μg of total RNA/lane were denatured with glyoxal/DMSO, separated on 1.4% agarose gels, transferred to nylon membranes, and probed with [³²P]-end-labeled DNA oligonucleotides complementary to GFP or yeast PYK1 transcripts. Blots were exposed to PhosphorImager screens and scanned using a Storm scanner.

Measurements of GFP mRNA half-lives. A previously described protocol [47] was employed with the following modifications. Yeast transformants were cultured in 330 mL of SC-U,-L with 2% Galacose and 2% Raffinose to an A₆₀₀ of ~0.8. An aliquot of 30 mL, representing the “0 min” time point, was poured onto ice and collected by centrifugation at 3,000 rpm in a Beckman JS-4.2 rotor for 5 min at 4°C. The cell pellet was resuspended and transferred to a 1.5 mL Eppendorf tube, and spun at 4°C in a microfuge at top speed (15,000 rpm) for 2 min. Residual medium was aspirated and cell pellets were immediately frozen on dry ice. The rest of the culture was collected by centrigugation at room temperature for 8 min at 7,000 rpm in an SLA3000 rotor. The cell pellet was resuspended in an equal volume of SC-U,-L 4% glucose medium pre-warmed to 30°C, and aliquots of 30 mL were harvested exactly as described for the “0 min” sample at 2-, 4-, 6-, 8-, 10-, 15-, -20, -30, -40, and 60 min after glucose containing medium was added. Frozen cells pellets were stored at -80°C until being thawed for isolation of total RNA and qRT-PCR analysis, which was conducted as described above.

Statistical analysis of tethering data

Changes in reporter protein expression (ΔGFP protein or Δβ-galactosidase) or reporter mRNA abundance (ΔGFP mRNA or ΔlacZ mRNA) were calculated as ratios of mean values of protein or mRNA expression measured in 3 or more biological replicates of transformants expressing Scd6-MS2-F vs. MS2-F alone. The propagated S.E.M.s for the resulting mean ratios were computed as (X/Y)*(√[(SE_x/x)²+(SE_y/y)²]), where X, SE_x, and x are the mean, standard error of the mean, and highest values for reporter protein input, respectively; and Y, SE_y, and y are the corresponding values for the mRNA input. Unpaired t-tests were performed to compare the mean changes in reporter protein or mRNA expression between wild type and mutant strains. Changes in TE of reporter mRNA on tethering Scd6-MS2 were determined by calculating the TE of the reporter, as the ratio of reporter protein to reporter mRNA expression, measured in 3 or more pairs of independent transformants expressing Scd6-MS2-F or MS2-F alone, from which the mean ΔTE and S.E.M. was calculated. Unpaired t-tests were performed to compare the resulting mean ΔTE values between wild type and mutant strains.

Ribosome footprint profiling and RNA-Seq

Ribosome profiling and RNA-Seq analysis were conducted in parallel essentially as described previously [48] on isogenic strains in the W303 background HFY114 (WT), CFY1016 (dcp2Δ), SYY2353 (scd6Δ), QZY126 (dhh1Δ), FZY843 (dcp2Δscd6Δ), and QZY128 (dcp2Δdhh1Δ), providing two biological replicates of each genotype. Strains growing exponentially in YPD medium at 30°C were harvested by vacuum filtration at room temperature, and quick-frozen in liquid nitrogen. Cells were lysed in a freezer mill with lysis buffer (20 mM Tris [pH 8.0], 140 mM KCl, 1.5 mM MgCl₂, 1% Triton, 500 μg/mL cycloheximide). For ribosome footprint library preparation, approximately 60 A₂₆₀ units of extract were treated with RNAse I at 15 U per OD₂₆₀ unit (Ambion, #AM2295) for 1h at 25°C on a Thermomixer at 700 rpm, and 80S ribosomes were purified by sedimentation through a sucrose density gradient as described [49]. Ribosome-protected mRNA fragments (footprints) were purified by phenol and chloroform extractions [49]. Following size selection and dephosphorylation, a Universal miRNA cloning linker (Synthesized by Integrated DNA Technologies) was ligated to the 3’ ends of footprints, followed by reverse transcription, circular ligation, rRNA subtraction, PCR amplification of the cDNA library, and DNA sequencing with an Illumina HiSeq system. For RNA-Seq library preparation, total RNA was purified using miRNeasy Mini kit (Qiagen) from aliquots of the same extracts (30 OD₂₆₀ units) used for footprint library preparation, 5 μg total RNA was randomly fragmented at 70°C for 12 min in fragmentation reagent (Ambion #AM8740). Fragment size selection, library generation and sequencing were carried out as above, except Ribo-Zero Gold rRNA Removal Kit (Yeast) was employed to remove rRNAs after linker-ligation in lieu of poly(A) selection. As described [48], linker sequences were trimmed from Illumina reads and the trimmed fasta sequences were aligned to the S. cerevisiae ribosomal database using Bowtie [50]. The non-rRNA reads (unaligned reads) were then mapped to the S. cerevisiae genome using TopHat [51]. Wiggle track normalization for viewing RPF or RNA reads in the IGV browser was conducted as follows. Wiggle files were produced from the alignment file, one each for genes on the Watson or Crick strand. The total reads on both strands were summed and a normalization factor q was calculated as 1,000,000,000/(total reads on W+C strands). Wiggle files were then regenerated by multiplying all reads by the factor q, yielding the number of reads per 1,000 million total reads (rpkm). Statistical analysis of changes in mRNA, RPFs, or TE values between two replicates each of any two strains being compared was conducted using DESeq2 [52], excluding any genes with less than 10 total mRNA reads in the 4 samples combined.

RNA-Seq analysis of strains SYY2352 (scd6Δ), SYY2353 (scd6Δ), SYY2686 (dhh1Δ), SYY2674 (pat1Δ), and SYY2680 (lsm1Δ) was conducted as described previously [53] after culturing cells in YPD at 30°C; and the results will be described in full in a future publication.

For all notched box-plots, constructed using a web-based tool at http://shiny.chemgrid.org/boxplotr/, the upper and lower boxes contain the 2^nd and 3^rd quartiles and the band gives the median. If the notches in two plots do not overlap, there is roughly 95% confidence that their medians are different.

Accession numbers of deposited data

RNA-Seq data employed in the analysis of mRNA changes in scd6Δ, dhh1Δ, pat1Δ, and lsm1Δ strains for the group of 83 mRNAs derepressed in scd6Δ cells have been deposited in the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) under the accessions numbers GEO:GSE107841 and GEO:GSE114428. All other RNA-Seq or Ribo-Seq data generated in this study are deposited separately in GEO:GSE114892.