Research ArticleCOMPUTATIONAL BIOLOGY

Multiplexed gene control reveals rapid mRNA turnover

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Science Advances  12 Jul 2017:
Vol. 3, no. 7, e1700006
DOI: 10.1126/sciadv.1700006
  • Fig. 1 Design of MGC to measure mRNA half-lives.

    (A) The endogenous promoter is substituted with P[tetO]4inGAL1, and silent mutations mark the RNA expressed under the control of tTA. (B) The introduction of silent mutations permits multiplexed measurements so that the expression of the endogenous gene allele is maintained to prevent the pleiotropic effects of shutting down expression. (C and D) Time course of mRNA decay in experiments using single (C) or multiplexed (D) shutdown. The two half-lives are in good agreement (1.67 and 1.66 min). Five decay curves are shown from a single multiplexed experiment pooling 10 strains. (E) The silent mutations (SM) do not alter half-lives, as evidenced by comparing the decay of mRNAs with and without silent mutations in haploid cells. The dashed gray line in the left panel indicates the expression of endogenous COG8. The gray dashed line in the right panel indicates the expected equality of the two decay rates, and the dotted lines indicate a twofold departure from this equality. Error bars represent SD (n = 3 or 4).

  • Fig. 2 MGC yields short mRNA half-lives and correlates with only one of the global methods.

    (A) Distribution of half-life using MGC. (B) Correlation matrix of published data sets and half-lives measured by MGC with the Spearman’s rank correlation coefficient. Data from the following studies were compared (the specific method names, when available, are indicated as given in the respective study): rbp1-1, Wang et al. (14), Munchel et al. (16), phenanthroline (15), DTA (17), cDTA (11), the anchor-away (7), Genomic Run-On (GRO) (19), RATE-seq (18), and the study that measured the half-lives of total and poly(A)+ RNA fractions with rbp1-1 (13). The methods using metabolic labeling and transcriptional inhibition are shown in magenta and green, respectively. (C) Correlation of half-lives measured by MGC and the indicated methods (Md, median; rs, Spearman’s rho; P, P value).

  • Fig. 3 Lack of promoter dependence of mRNA decay assessed by endogenous and insertional promoters.

    (A) Scheme of the endogenous, substitutional, and insertional promoters. (B) Time course of the decay of the YLR081W (GAL2) mRNA driven by its endogenous promoter (shut down by galactose removal; t1/2 = 2.6 min) or driven by the substitutional promoter (t1/2 = 2.6 min). (C) Two tet operators were introduced upstream of the TATA box in the endogenous promoter to create the insertional promoters. (D) USA1 mRNA expression driven by the respective promoters. Dox, doxycycline. (E) Decay profiles of the precursor and mature mRNAs of the GLT1 and USA1 genes upon dissociation of TetR-Sum1 (blue). The addition of doxycycline results in a slow deinduction kinetics with TetR-Ssn6, evidenced by the long half-time (5.2 min; green) of the GLT1 precursor mRNA. (F) Comparison of half-lives obtained with substitutional promoters to that with the endogenous (GAL2) or insertional promoters (USA1 and GLT1 mRNAs). The decay of the mature mRNA was not affected by splicing (USA1 with intron, t1/2 = 2.6 min; USA1 without intron, t1/2 = 2.0 min).

  • Fig. 4 The cytoplasmic decay rate of mRNA is not affected by promoter replacement, as determined by smFISH.

    (Left) Representative pictures of smFISH for mRNAs driven by the substitutional promoter (blue) or by their endogenous promoters (magenta). Scale bar, 5 μm. (Right) Ratio of nascent to mature mRNAs expressed under the control of endogenous and substitutional promoters. Error bars are SD of duplicate measurements.

  • Fig. 5 Correct mRNA half-life estimation is of critical importance for stochastic models.

    (A and B) Histogram of USA1 mRNA copy number distribution (smFISH) under the control of the endogenous (A) or substitutional (B) promoter and fitted distribution using either MGC (red) or cDTA (blue) half-life estimates. (C and D) Parameters estimated for the two-state promoter model (kON, kOFF, and kTr rates) are shown for models assuming the two different decay rate constants for the endogenous (C) or substitutional (D) promoter.

  • Fig. 6 Determinants of variability in the mRNA decay rate and promoter-independent variation in RNA synthesis rate.

    (A and B) Time courses for and half-lives in xrn1Δ and wt cells, determined using MGC. The dashed line represents the expected equality, and the dotted lines are twofold deviations from this equality. Error bars represent the SD of duplicate measurements, except for DAK1, USA1, and COG8, which were triplicated. (C) Comparison of half-lives of the endogenous GAL mRNAs in xrn1Δ and wt cells after galactose washout (right). (D) Scheme of processes contributing to variations of promoter-dependent (green) and promoter-independent (blue-magenta) mRNA synthesis rate. RNA pol II, RNA polymerase II. (E) mRNA synthesis rates under the control of the substitutional and endogenous promoters. The dashed orange lines represent the 0.1 and 0.9 quantiles of the synthesis rates (rs = 0.16; P = 0.24). a.u., arbitrary units. (F) The bases of the magenta triangles are scaled to represent the dynamic range of promoter-dependent and promoter-independent mRNA synthesis rates and the mRNA decay rates, expressed as the 90:10 quantile ratio. The width of the black arrows indicates the turnover rates.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/7/e1700006/DC1

    fig. S1. The cell ploidy and the choice of reference mRNAs have no impact on measured half-lives.

    fig. S2. Pop-out of the chromosomally integrated plasmid is dispensable for half-life estimation of mature mRNA.

    fig. S3. The doubling time of the MGC strains (red dots) is similar to that of the parent diploid strain (black line).

    fig. S4. Insertional promoter strategy.

    fig. S5. Phenanthroline increases the number of P bodies.

    fig. S6. mRNA half-life has a major impact on response time of a protein in a negative feedback loop.

    fig. S7. Degradation rates and not synthesis rates affect the response time.

    fig. S8. Parameter estimation of the two state model to fit the USA1 RNA copy number distributions.

    fig. S9. Impact of 5’UTR length on the decay profiles of mRNAs with varying 5’UTR lengths.

    fig. S10. Design of plasmids with substitutional promoters and the strategy for chromosomal integration.

    fig. S11. Reverse transcription strategy.

    table S1. Plasmid list.

    table S2. Strain list.

    table S3. Cloned 5′UTR lengths of genes.

    table S4. qPCR primers detecting marked mRNAs and their amplification efficiencies.

    table S5. qPCR primers detecting endogenous mRNAs with their efficiencies.

    table S6. Cross-reaction of primers to detect the marked mRNA with the endogenous mRNAs.

    table S7. Mean, Fano factor, and CV of the RNA molecule copy number distributions.

    data file S1. Ct values for one of the replicate experiments of pooled strains in the MGC experiment.

    data file S2. Time courses in the replicate measurements.

    data file S3. mRNA (relative) levels and decay rates.

    data file S4. Time courses for YJR139C and YDR032C mRNAs.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. The cell ploidy and the choice of reference mRNAs have no impact on measured half-lives.
    • fig. S2. Pop-out of the chromosomally integrated plasmid is dispensable for half-life estimation of mature mRNA.
    • fig. S3. The doubling time of the MGC strains (red dots) is similar to that of the parent diploid strain (black line).
    • fig. S4. Insertional promoter strategy.
    • fig. S5. Phenanthroline increases the number of P bodies.
    • fig. S6. mRNA half-life has a major impact on response time of a protein in a negative feedback loop.
    • fig. S7. Degradation rates and not synthesis rates affect the response time.
    • fig. S8. Parameter estimation of the two-state model to fit the USA1 RNA copy number distributions.
    • fig. S9. Impact of 5′UTR length on the decay profiles of mRNAs with varying 5′UTR lengths.
    • fig. S10. Design of the plasmids with the substitutional promoters and the strategy for chromosomal integration.
    • fig. S11. Reverse transcription strategy.
    • table S1. Plasmid list.
    • table S2. Strain list.
    • table S3. Cloned 5′UTR lengths of genes.
    • table S4. qPCR primers detecting marked mRNAs and their amplification efficiencies.
    • table S5. qPCR primers detecting endogenous mRNAs with their efficiencies.
    • table S6. Cross-reaction of primers to detect the marked mRNA with the endogenous mRNAs.
    • table S7. Mean, Fano factor, and CV of the RNA molecule copy number distributions.

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    Other Supplementary Material for this manuscript includes the following:

    • data file S1 (Microsoft Excel format). Ct values for one of the replicate experiments of pooled strains in the MGC experiment.
    • data file S2 (Microsoft Excel format). Time courses in the replicate measurements.
    • data file S3 (Microsoft Excel format). mRNA (relative) levels and decay rates.
    • data file S4 (Microsoft Excel format). Time courses for YJR139C and YDR032C mRNAs.

    Download data files S1 to S4

    Files in this Data Supplement:

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