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An improved MS2 system for accurate reporting of the mRNA life cycle

Nature Methods volume 15, pages 81–89 (2018)Cite this article

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Abstract

The MS2–MCP system enables researchers to image multiple steps of the mRNA life cycle with high temporal and spatial resolution. However, for short-lived mRNAs, the tight binding of the MS2 coat protein (MCP) to the MS2 binding sites (MBS) protects the RNA from being efficiently degraded, and this confounds the study of mRNA regulation. Here, we describe a reporter system (MBSV6) with reduced affinity for the MCP, which allows mRNA degradation while preserving single-molecule detection determined by single-molecule FISH (smFISH) or live imaging. Constitutive mRNAs (MDN1 and DOA1) and highly-regulated mRNAs (GAL1 and ASH1) endogenously tagged with MBSV6 in Saccharomyces cerevisiae degrade normally. As a result, short-lived mRNAs were imaged throughout their complete life cycle. The MBSV6 reporter revealed that, in contrast to previous findings, coordinated recruitment of mRNAs at specialized structures such as P-bodies during stress did not occur, and mRNA degradation was heterogeneously distributed in the cytoplasm.

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Figure 1: Previous MBS systems resist degradation in yeast.
Figure 2: Design and characterization of a new MS2–MCP system.
Figure 3: 12xMBSV6 faithfully reports the rapid degradation of GAL1 mRNA.
Figure 4: MBSV6–MCP maintains single-mRNA resolution in living cells.
Figure 5: MBSV6–MCP enables single-mRNA imaging under stress conditions.
Figure 6: MBSV6–MCP quantitatively reports ASH1 mRNA levels throughout the cell cycle.

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Acknowledgements

We thank X. Meng for help with cloning; D. Muhlrad for help with the northern blots; B. Wu for writing the script to generate the new MBS system; and C. Eliscovich, Y. Yoon and S. Das for critical reading. This work was supported by NIH grant GM57071 to R.H.S. R.P. was supported by HHMI. E.T. was supported by Swiss National Science Foundation Fellowships P2GEP3_155692 and P300PA_164717. J.B. was supported by training support for AECOM (T32GM007288) and a predoctoral NIH fellowship (F30CA214009). J.G. was supported by NIH grant F32 GM10807.

Author information

Author notes

  1. Evelina Tutucci and Maria Vera: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, USA

    Evelina Tutucci, Maria Vera, Jeetayu Biswas & Robert H Singer

  2. Department of Molecular Biology, Colorado College, Colorado Springs, Colorado, USA

    Jennifer Garcia

  3. Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, Colorado, USA

    Roy Parker

  4. Howard Hughes Medical Institute, University of Colorado Boulder, Boulder, Colorado, USA

    Roy Parker

  5. Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA

    Robert H Singer

  6. Janelia Research Campus of the HHMI, Ashburn, Virginia, USA

    Robert H Singer

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  1. Evelina Tutucci
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Contributions

E.T. and M.V. designed and performed the experiments and analyzed the data. J.B. performed EMSAs. J.G. performed the northern blot. E.T., M.V. and R.H.S. conceived ideas and wrote the manuscript with input from R.P. R.H.S. supervised the research.

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Correspondence to Robert H Singer.

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Competing interests

The material in this manuscript is the subject of a provisional application to the US Patent and Trademark Office (no. 62/487,058). It has not been licensed to any corporation, and the authors (R.H.S., E.T. and M.V.) are the sole inventors.

Integrated supplementary information

Supplementary Figure 1 The use of previous MBS systems to tag endogenous mRNAs affects their degradation but not their localization.

(a) Frequency distribution of mature ASH1 mRNAs per cell in an asynchronous population of wt cells. Single mRNA molecules quantified with probes recognizing the CDS. Mean and s.d. of two independent cultures, n ≈500 cells per experiment.

(b) Frequency distribution of mature wt MDN1 mRNAs per cell in a wt strain. Single mRNA molecules quantified with probes recognizing the CDS. Mean and s.d. of two biological replicates, n ≈ 500 cells per experiment.

(c) Correlation between the number of single CDS (ASH1 or MDN1) and MBS (MBSV5 or MBSORF) molecules per cell in presence or absence of MCP. Pearson r values calculated by combining two independent experiments (n≍1000).

(d) Two color smFISH for ASH1 mRNA tagged with 12xMBSV5 in cells expressing MCP. MERGE shows the overlap of the DAPI signal (blue), smFISH for the ASH1 CDS (green) and MBS (red). (DIC) differential interference contrast image. Yellow arrowhead, MBS aggregates. Scale bar = 5 μm.

(e) MCP expression in the wt strains does not lead to the formation of aggregates. (Left) BY4671 background transformed with the yeast expression plasmid YcpLac111 or with YcpLac111 CYC1p-MCP-NLS-2xyeGFP. (Right) W303-BMA64-1A background transformed with the yeast expression plasmid YcpLac111 or with YcpLac111 CYC1p-MCP-NLS-2xyeGFP. Scale bar = 5 μm.

(f) Quantification of single ASH1 24xMBSV5 mRNAs in the bud tip of cells expressing MCP or the vector alone. Each dot represents the quantification obtained from a single cell by two color smFISH with probes binding the CDS (green) or the MBSV5 (red). Bars indicate the mean and the s.d. from two independent cultures. P = ns indicates a non-significant difference (non-parametric test p>0.05) between the quantification ns of ASH1 and MBSV5 probes.

Supplementary Figure 2 MCP has reduced affinity for the MS2 U-variant compared to the MS2 C-variant.

(a-c) Schematic representation of different MS2 Stem Loops. (a) MS2 wt sequence from the bacteriophage. Nucleotide positions are relative to the translation start codon AUG (+1). (b) MBSORF loops are dimers of 2 different loop sequences distanced by 20 nts, repeated every 50 nts. MBSORF is C-variant (cytosine at position -5). The stem loop is 7 nts long. (c) MBSV5 loops have non-repeated sequences and 30 nt linkers with stem loops of 9 nts. All loops are C-variant.

(d) MS2 is the original bacteriophage sequence. MS2 was synthetized either as a U-variant or C-variant for the EMSA.

(e) Binding affinity of MCP for the MS2 C-variants and U-variants. Plot of the fraction of RNA bound as a function of MCP concentration and its fit to the Hill equation. The Kd from three independent measurements is indicated on the plots for the MS2, either U or C-variant.

(f-h) Representative EMSA gels used to plot the binding curves and calculate the Kd in Figures 2b, 2c and Supplementary 2e (Full gel pictures are provided separately). EMSAs performed using labelled free 19nt RNA hairpin and RNA-protein complexes. Filled triangle on top of each panel represents MCP concentration increasing by a factor of two per lane. MBS-MCP-MBS (U-variant) or MBS-MCP(C-variant) are shown for: (f) Stem1 (g) Stem2 and (h) original MS2 sequence.

Supplementary Figure 3 Tagging endogenous mRNAs with the new MBS systems reduces MBS fragments accumulation.

(a, b) Scheme of new MBS systems. MBSV6 and MBSV7 have a randomized sequence of twelve stem loops and linkers. The length of the stem is 7 nts and the linkers are 50 nt (MBSV6) or 40 nts (MBSV7). MBSV6 and MBSV7 were synthetized in two versions: C-variant (12 loops with C) or a U-variant (12 loops with U).

(c) Scheme of the newly generated constructs integrated in the 3′ UTR of ASH1 or MDN1: (1) 12xMBSV7 U-variant with 12 loops with random stem sequence but all with a U at position -5, interspaced by 40 nucleotides linkers; (2) 12xMBSV7 C-variant, with the sequence of (1) but with C instead of U at position -5 in the 12 loops; (3) 12xMBSV6 U-variant, the same loop sequence of MBSV7 but with 50 nucleotides linkers; (4) 12xMBSV6 C-variant, with the same sequence of (3) but with C instead of U at position -5 in the 12 loops. For all constructs a 24xMBS variant was generated by duplication of the sequence (1b, 2b, 3b, 4b). For all constructs STOP codons were avoided in all frames.

(d, e) Quantification of two color smFISH for (d) ASH1 mRNAs and (e) MDN1 mRNAs tagged with 24xMBSV7, in cells expressing MCP or the vector alone. Quantification with CDS probes (green plots) or MBS probes (red plots) reported as frequency distribution of mature ASH1 (d) and MDN1 (e) mRNAs per cell. Mean and s.d. of two independent cell cultures, n ≈500 cells per experiment.

(f, g) Correlation between the number of single CDS (ASH1 (f) or MDN1 (g)) and (MBSV7 (top) or MBSV6 (bottom) molecules per cell in presence or absence of MCP. Pearson r values calculated by combining two independent experiments (total n ≈ 1000).

Supplementary Figure 4 Endogenous mRNAs tagged with the MBSV6 system are rapidly degraded.

(a, b) Two color smFISH for (a) ASH1 mRNAs and (b) MDN1 mRNAs tagged with 12xMBSV6, in cells expressing MCP or the vector alone. (a) DIC/MERGE shows the overlap of the DAPI (blue), smFISH for the ASH1 CDS (green) and the MBSV6 (red) with the DIC image. (b) MERGE shows the overlap of the DAPI signal (blue), smFISH for the MDN1 CDS (green) and the MBSV6 (red). Yellow lines define the shape of a single cell. For each cell is indicated the stage of the cell cycle. Scale bar = 5 μm.

(c, d) Quantification of smFISH represented in Supplementary Figure 4e-b with CDS probes (green plots) or MBSV6 probes (red plots) reported as frequency distribution of mature ASH1 (c) and MDN1 (d) mRNAs per cell. Mean and s.d. of two independent cultures, n≍ 500 cells per experiment.

(e) Correlation between the number of single CDS (ASH1 or MDN1) and 12xMBSV6 molecules per cell in presence or absence of MCP. ASH1 (top) or MDN1 (bottom) mRNAs tagged with 12xMBSV6, in cells expressing MCP or the vector alone. Pearson r values calculated by combining two independent experiments (total n ≈ 1000).

Supplementary Figure 5 The MBSV6 system solves the formation of aggregates

(a) Northern-blot analysis of ASH1 mRNA from cells expressing MCP or a control plasmid. Scheme represents the ASH1 mRNA tagged with different MBS systems and the position of the probe used to detect it. Lane 1 cells with the ASH1 gene deleted. Lane 2-3 wt cells expressing endogenous ASH1; Lane 4-5 cells expressing ASH1 endogenously tagged with 24xMBSV5; Lane 6-7 cells expressing ASH1 endogenously tagged with 24xMS2V7; Lanes 8-9 cells expressing ASH1 endogenously tagged with 24xMS2V6; Lane 10-11 cells expressing ASH1 endogenously tagged with 12xMS2V5; Lane 12-13 cells expressing ASH1 endogenously tagged with 12xMS2V7; Lanes 14-15 cells expressing ASH1 endogenously tagged with 12xMS2V6. Loading was controlled by hybridization with a probe recognizing the 7S RNA. FL: Full length mRNA. The asterisks mark non-specific bands also present in the ash1∆ control strain (lane 1), with the predominant one larger than the mRNA decay fragments stabilized by the MBSV5 constructs.

(b) Quantification of the Northern-blot. Intensity of MBS fragments in MCP expressing cells is relative to the values obtained in cells not expressing MCP and normalized to 7S levels.

(c) Northern-blot analysis of GAL1 mRNA from cells expressing MCP or a control plasmid. Lane 1-2 wt cells expressing endogenous GAL1. Lane 3-4 cells expressing GAL1 endogenously tagged with 12xMS2V5; Lane 5-6 cells expressing GAL1 endogenously tagged with 12xMS2V6. Loading was controlled by hybridization with a probe recognizing the 7s RNA. FL: Full length mRNA.

Supplementary Figure 6 Formation of MBS aggregates correlates with the rapid degradation of GAL1 mRNA.

(a) Representative two color smFISH for GAL1 mRNAs tagged with 24xMBSV6 in cells expressing MCP. MERGE shows the overlap of the DAPI signal (blue), smFISH for the GAL1 CDS (green) and the MBSV6 (red). Yellow arrowhead, MBS aggregate. White arrowhead, single mRNA. Scale bar = 5 μm.

(b, c) Quantification of smFISH for (b) GAL1 12xMBSV6 and (c) GAL1 12xMBSV5 mRNAs in cells expressing left: vector alone or right: MCP. Each dot represents the quantification obtained from a single cell with probes binding the CDS (green) or the MBSV6 (red). Error bars indicate the Mean and s.d. from two independent cell cultures.

(d) Live imaging of cells expressing GAL1 12xMBSV5-MCP upon thirty minutes of glucose addition. Yellow arrowhead, MBS aggregate. White arrowhead, single GAL1 mRNA. Scale bar = 5 μm.

(e) Quantification of single mRNAs in MBS aggregates probed with CDS probes (green) or MBS probes (red) in cells expressing GAL1 24xMBSV6-MCP. Mean and s.d. from three independent cell cultures.

Supplementary Figure 7 24xMBSV6 allows visualizing single mRNAs in eukaryotic cells without loss of brightness

(a) Live imaging of U2OS cells co-expressing a reporter (BFP) and tdMCP. Schematic representation of the plasmids, with 24xMBSV5 or 24xMBSV6 inserted in the 3’UTR of the BFP CDS, transiently transfected in U2OS cells. Green arrowhead, transcription sites in the nucleus. White arrowhead, single mRNAs in the cytoplasm. The nucleus accumulates GFP fluorescence because the tdMCP is fused to a nuclear localization signal. Scale bar = 5 μm.

(b) Frequency distribution of mRNAs intensities tagged with either 24xMBSV5 or 24xMBSV6. Single mRNA molecules counted from Supplementary Figure 7a.

(c) Plot of the smFISH intensities of single MDN1 mRNAs tagged with either 24xMBSV6 or 12xMBSORF. Single molecules were detected and analyzed using FISH-quant. Mean and s.d. of two independent cultures, n ≈ 150 cells per experiment. Non-parametric Mann-Whitney test indicates significant difference in brightness between 24xMBSV6 and 12xMBSV6 populations, P<0.001.

(d) Quantification of smFISH with MDN1 probes (green plots) or MBS probes (red) reported as frequency distribution of mature MDN1 mRNAs per cell tagged with NUP49-tdTomato 24xMBSV6-MCP or 12xMBSV6. Mean and s.d. of two independent cell cultures, the same used to measure the brightness of single mRNAs by live imaging (Fig. 4e-h, n ≈ 150 cells per experiment).

Supplementary Figure 8 The MBSV6-MCP system allows mRNA degradation during stress conditions.

(a) Two color smFISH confirmed that ASH1 and MDN1 mRNAs tagged with 24xMBSV6-MCP were full length throughout glucose starvation. Two color smFISH to analyze endogenous mRNAs tagged with 24xMBSV6 system upon glucose starvation. (Upper panels) Two color smFISH for ASH1 or MDN1 mRNAs tagged with 24xMBSV6 in cells expressing MCP upon 30 minutes of glucose starvation. (Lower panels) To confirm the induction of the stress response smFISH for HSP104 mRNA (gray) and DAPI (blue) was performed for cells expressing ASH1 (left) or MDN1 (right) mRNAs tagged with 24xMBSV6. Scale bar = 5 μm.

(b) Quantification of smFISH represented in Supplementary Figure 8a with CDS probes (green) or MBS probes (red). Each dot represents the quantifications obtained for a single cell. Bars indicate Mean and s.d. for two independent cell cultures.

(c) Cumulative number of PB detected in 150 cells with the marker Edc3-mCherry (red) and PB co-localizing with MBS aggregates (gray) in the strain ASH1 24xMBSV5-MCP. Particles were identified and counted with ImageJ. Quantifications from three independent cultures (total n = 150 cells) were combined in a single plot.

(d) Cumulative number of PB detected in 150 cells with the marker Edc3-mCherry (red) and PB co-localizing with MBS aggregates (gray) in the strain MDN1 24xMBSORF-MCP. Particles were identified and counted with ImageJ. Quantifications from three independent cultures (n = 150 cells) were combined in a single plot.

Supplementary Figure 9 The MBSV6-MCP system reporters the expression of ASH1 and DOA1 mRNAs tagged with 24xMBSV6.

(a) Quantification of single ASH1 mRNAs of cell shown in Figure 6b during the cell cycle (black dots connected by green line). Black curve indicates cytoplasmic ASH1 mRNA profiles fitted to a single exponential decay model.t1/2 = 5.6 min.

(b) Top, schematic representation of ASH1 locus with 24xMBSV6 followed by the kanamycin coding sequence inserted in the 3’UTR. As RNAPII transcribes, the MBS are bound by MCP, marking nascent mRNAs at the transcription site. Bottom, representative images of Supplementary Video 5. Simultaneous two-color imaging of cells co-expressing ASH1 24xMBSV6-KAN-MCP (gray) and NLS-2xmCherry (red). Time indicates the minutes after imaging starts. Images were acquired every 2 minutes.

(c) Schematic representation of DOA1 locus tagged with 24xMBSV6 inserted in the 3’UTR. The dotted lines designate the position of the probes that recognize the CDS (green) or the MBS sequence (red).

(d) Two color smFISH for DOA1 mRNA tagged with 24xMBSV6 in cells expressing MCP or a control plasmid. DIC/MERGE shows the overlap of the DAPI (blue), smFISH for the DOA1 CDS (green) and the MBS (red) with the DIC image. Yellow lines define the shape of a single cell. For each cell is indicated the stage of the cell cycle. Scale bar = 5 μm.

(e) Quantification of smFISH represented in Figure Supplementary 9d with CDS probes (green plots) or MBS probes (red plots) reported as frequency distribution of mature DOA1 mRNAs per cell. Mean and s.d. of two independent cell cultures, n≍500 cells per experiment, distribution of the mRNAs was generated using the same binning.

Supplementary Figure 10 Summary of the improved characteristics of the new MBSV6 system.

Previous MS2-MCP systems have been resistant to degradation in S. cerevisiae. We have modified the MS2 system and it accurately reports the life cycle of mRNA. This new MS2 (V6) empowers imaging the decay kinetics of single mRNA molecules. During stress, single mRNA molecules were not sequestered in any cellular compartment.

Supplementary Figure 11 EMSA Uncropped Gels

(a)Original gel for Supplementary Figure 2f (left panel)

(b)Original gel for Supplementary Figure 2f (right panel)

(c)Original gel for Supplementary Figure 2g-h

Supplementary Figure 12 Northern Uncropped Blots

(a)Original blot for Supplementary Figure 5a (top)

(b) Original blot for Supplementary Figure 5a (bottom)

(c) Original blot for Supplementary Figure 5c (top)

(d) Original blot for Supplementary Figure 5c (bottom)

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12, Supplementary Notes 1–8 and Supplementary Tables 1–4 (PDF 4762 kb)

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GAL1 mRNAs tagged with 12xMBSV6 is degraded upon glucose supplementation.

Comparison of live imaging of cells expressing GAL1mRNAs tagged with 24xMBSV6-MCP or 12xMBSV6-MCP. (Left) Cell tagged with GAL1 24xMBSV6-MCP grown in raffinose were induced with 0.2% of galactose for 20 minutes. Cells were washed and supplemented with 4% glucose containing media. 11 Z-stacks (0.5 μm) were acquired every two minutes. GAL1 single mRNAs were observed after 4 minutes of induction (time 0) and degraded or forming small MBS aggregates after 10 minutes of glucose supplementation (time 30). (Middle) Cells tagged with GAL1 12xMBSV6-MCP grown in raffinose, were induced with 0.2% of galactose for 20 minutes. Cells were washed and supplemented with 4% glucose containing media. 11 Z-stacks (0.5 μm) were acquired every two minutes. (Right) Cells tagged with GAL1 12xMBSV6-MCP grown in raffinose, were shifted to 0.2% galactose containing media for 100 minutes. The GAL1 mRNA signal disappearance was due to degradation instead of photo-bleaching because the mRNAs in the galactose controls did not disappear. Acquisition was started 4 minutes after induction. 11 Z-stacks (0.5 μm) were acquired every two minutes. (AVI 648 kb)

MBSV6-MCP enables ASH1 single mRNA imaging under stress conditions.

Comparison of live imaging of cells co-expressing the PB marker Edc3-mCherry and ASH1 mRNAs tagged either with 24xMBSV5-MCP (left) or 24xMBSV6-MCP (right). Cells grown in media supplemented with 2% glucose were shifted to glucose lacking medium to induce starvation (time=0). We acquired 13 Z- stacks (0.5 μm) every minute using simultaneous two-color imaging. Bright MBS aggregates were observed before glucose starvation in cells expressing 24xMBSV5-MCP (left). No MBS aggregates were observed in cells expressing 24xMBSV6-MCP (right). Edc3-mCherry clustered in PBs after ~10 minutes of starvation and accumulated as a bright red signal during glucose starvation. (AVI 875 kb)

MBSV6-MCP enables MDN1 single mRNA imaging under stress conditions.

We acquired 13 Z- stacks (0.5 μm) every minute using simultaneous two-color imaging. Bright MBS aggregates were observed before glucose starvation in cells expressing 24xMBSORF-MCP (left). No MBS aggregates were observed in cells expressing 24xMBSV6-MCP (right). Edc3-mCherry clustered in PBs after ~10 minutes of starvation and accumulated as a bright red signal during glucose starvation. (AVI 996 kb)

The MBSV6-MCP system reports the expression ASH1 and DOA1 mRNAs during the complete cell cycle.

Comparison of live imaging of cells co-expressing the cellcycle marker mRuby-Tub1 either with ASH1-24xMBSV6-MCP (left) or DOA1-24xMBSV6-MCP (right). Cells were grown in selective media at a stable temperature of 30°C. 13 Z- stacks (0.5 μm) were acquired every 2 minutes using simultaneous two-color imaging. The ASH1 mRNA is expressed during anaphase and localizes to the bud tip, where it is degraded (left). Cells expressing the stable DOA1 mRNA, with same imaging conditions used for ASH1, show steady number of single mRNAs during the cell cycle (right), demonstrating that the disappearance of ASH1 (left) is not due to photobleaching. (AVI 364 kb)

The MBSV6-MCP system reports the transcription of ASH1 mRNAs.

Live imaging of cells co-expressing the nuclear marker NLS-2xmCherry with ASH1-24xMBSV6-MCP. Cells were grown in selective media at a stable temperature of 30oC. 13 Zstacks (0.5 μm) were acquired every 2 minutes using simultaneous two-color imaging. The ASH1 mRNA is expressed during anaphase and localizes to the bud tip, transcription sites are observed both in the mother and the daughter cell (white arrows). (AVI 247 kb)

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Tutucci, E., Vera, M., Biswas, J. et al. An improved MS2 system for accurate reporting of the mRNA life cycle. Nat Methods 15, 81–89 (2018). https://doi.org/10.1038/nmeth.4502

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