Single-Molecule Analysis of Gene Expression Using Two-Color RNA- Labeling in Live Yeast

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1 Supplemental Figures, Tables and Results Single-Molecule Analysis of Gene Expression Using Two-Color RNA- Labeling in Live Yeast Sami Hocine 1, Pascal Raymond 2, Daniel Zenklusen 2, Jeffrey A. Chao 1 & Robert H. Singer 1 1 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 2 Department of Biochemistry, Université de Montréal, Montreal, Canada Correspondance should be addressed to R.H.S. (robert.singer@einstein.yu.edu).

2 a 24 PP7 KAN R loxp loxp MDN1 UTR MDN1 24 PP7 KAN R loxp loxp UTR MDN1 24 PP7 loxp UTR (tagged mrna) b MATa MDN1-24PP7 MET25 promoter PCP 2yEGFP HIS3 c MAT a (labeled mrna) MAT α Supplementary Figure 1. Schematic for tagging of yeast genes (a) Targeted integration of the 24PP7 cassette including the kanamycin selectable marker is performed by transformation and homologous recombination. The marker is removed by inducing expression of the CRE recombinase yielding mrnas that are precisely tagged in the 3 UTR. (b) PP7 tagged strains are then transformed with a plasmid encoding PCP-2yEGFP and mrnas become fluorescently labeled, allowing visualization of single molecules. (c) Two-color strains were generated by mating one-color haploids, generating yeast that express green or red MDN1 mrnas depending upon which allele they were transcribed from.

bps a 24MS2-GAL7 24PP7-GAL7 3054 1636 1018 b Frequency of Loss of Repeats (%) 24MS2-GAL 24PP7-GAL 24MS2 bps 3054 1636 1018 c 24PP7 bps 3054 1636 1018 d

Hairpin copy number is determined by PCR product size (24x = 2039bps) for colonies resulting from independent integration events (24x = blue arrowhead; 1-23x =

3 bps a 24MS2-GAL7 24PP7-GAL b Frequency of Loss of Repeats (%) 24MS2-GAL 24PP7-GAL 24MS2 bps c 24PP7 bps d Supplementary Figure 2. Assessing PP7 and MS2 hairpin copy number by PCR (a) GAL gene tagged in 5 UTR by 24MS2 and 24PP7. Hairpin copy number is determined by PCR product size (24x = 2039bps) for colonies resulting from independent integration events (24x = blue arrowhead; 1-23x = yellow arrowhead, 0x = red arrowhead). (b) 5 Taggings of GAL1, GAL7 and GAL10 indicates the occurrence of hairpin loss as 53 ± 7% for MS2 vs. 27 ± 7% PP7 (n = 15 colonies). (c,d) Single colonies confirmed to have the full complement of MS2 or PP7 hairpins were repeatedly diluted and grown in log-phase for approximately 80 generations. Cultured cells were plated and colonies were again screened for hairpin copy number. Both MS2 and PP7 hairpins remain stable (retain 24 stem loops) in culture following integration. Error bars indicate s.e.m.

4 a + TAG TAG MDN1-24PP7 b WT MDN1 + PCP-FP c MDN1-24MS2 d WT MDN1 + MCP-FP Supplementary Figure 3. Fluorescent spots require RNA tags (a-d) Maximum intensity projections highlight single MDN1 mrnas within the cell volume. MDN1 mrnas are labeled by co-expression of CP-FP driven off the MET25 promoter. Fluorescent spots require the mrna tag and do not result from CP-FP aggregation alone. Scale bars, 3 µm.

a z-sweep Acquisition Exposure (ms) b z-sweep c z-sections MDN1-24PP7 Stage Position (um) EMCCD Read-out Intervals Acquisition time: < 700ms 4000+ms Supplementary Figure 4.

5 a z-sweep Acquisition Exposure (ms) b z-sweep c z-sections MDN1-24PP7 Stage Position (um) EMCCD Read-out Intervals Acquisition time: < 700ms 4000+ms Supplementary Figure 4. Visualization of single MDN1 mrnas (a) Detection of diffusing mrnas depends on rapid acquisition, such that the fluorescent signal is not spatially distributed during acquisition. This is achieved by a continuous z-sweep in which the stage continually moves along the z-axis (green lines). The sample receives constant 488 nm laser illumination (1.9 mw measured from back focal plane) and the EMCCD camera maintains a read-out interval of 0.75 µm (red arrows) over a total range of 4.5 µm. (b) Maximum intensity projection of MDN1-24PP7 mrnas acquired as z-sweep. Optimized conditions: 40% 488nm laser power; 90 ms exposure every 0.75 µm. (c) Maximum intensity projection of MDN1-24PP7 mrnas acquired as z-stack. Optimized conditions: 60% 488nm laser power; 50 ms exposure every 0.25 µm. Acquisition by z-sweep is ~6 times faster than standard z-sections, providing a snapshot of single mrnas within the cell volume. Scale bars, 3 µm.

] a WT MDN1 MDN1-24MS2 MDN1-24PP7 b MDN1-24MS2 c g h ]FISHLive-cell MDN1-24PP7 Average MDN1 mrnas per

Average MDN1 nascent chains per cell WT MDN1 MDN1-24MS2 MDN1-24PP7 WT MDN1 MDN1-24MS2 MDN1-24PP7

MS2 and PP7 3 UTR tagging does not disrupt MDN1 expression Single-molecule FISH performed on haploid

Cells lacking CP-GFP expression are grown in YPD rich media while those expressing CP-GFP are grown

16 per cell (n = 269 cells) whereas MDN1-24MS2 mrnas average 5.59 ± 0.

(d-f) When expressing CP-GFP, WT MDN1 mrnas average 5.71 ± 0.

16 per cell (n = 259 cells) and MDN1-24PP7 mrnas average 5.56 ± 0.16 per cell (n = 292 cells).

46 per cell (n = 85 cells) for MDN1-24MS2 and 5.54 ± 0.42 per cell (n = 92 cells) for MDN1-24PP7.

6 ] a WT MDN1 MDN1-24MS2 MDN1-24PP7 b MDN1-24MS2 c g h ]FISHLive-cell MDN1-24PP7 Average MDN1 mrnas per cell + CP-FP CP-FP d e f i STEADY-STATE mrna FISH ( CP-FP) FISH (+ CP-FP) j NASCENT mrna Live-cell Average MDN1 nascent chains per cell WT MDN1 MDN1-24MS2 MDN1-24PP7 WT MDN1 MDN1-24MS2 MDN1-24PP7 MDN1-24MS2 MDN1-24PP7 WT MDN1 MDN1-24MS2 MDN1-24PP7 Supplementary Figure 5. MS2 and PP7 3 UTR tagging does not disrupt MDN1 expression Single-molecule FISH performed on haploid yeast using Cy3 labeled MDN1 probes (red) with nuclei labeled by DAPI (blue). Cells lacking CP-GFP expression are grown in YPD rich media while those expressing CP-GFP are grown in the appropriate synthetic minimal media. (a-c) WT MDN1 mrnas average 5.94 ± 0.16 per cell (n = 269 cells) whereas MDN1-24MS2 mrnas average 5.59 ± 0.17 per cell (n = 305 cells) and MDN1-24PP7 mrnas average 5.41 ± 0.15 per cell (n = 319 cells). (d-f) When expressing CP-GFP, WT MDN1 mrnas average 5.71 ± 0.19 per cell (n = 248 cells) whereas MDN1-24MS2 mrnas average 5.25 ± 0.16 per cell (n = 259 cells) and MDN1-24PP7 mrnas average 5.56 ± 0.16 per cell (n = 292 cells). (g,h) Live cell mrna counting yields an average of 5.59 ± 0.46 per cell (n = 85 cells) for MDN1-24MS2 and 5.54 ± 0.42 per cell (n = 92 cells) for MDN1-24PP7. (i) Quantification of mean MDN1 steady-state expression levels for all conditions. (j) WT MDN1 shows an average of 1.82 ± 0.16 nascent mrnas per cell (n = 61 cells), as compared to 1.85 ± 0.15 nascent mrnas for MDN1-24MS2 (n = 59 cells) and 1.78 ± 0.15 nascent mrnas for MDN1-24PP7 (n = 65 cells). Scale bars, 3 µm. Error bars indicate s.e.m.

7 MDN1-24PP7 + PCP-FP + MCP-FP a b MDN1-24MS2 c d Supplementary Figure 6. PP7 and MS2 are orthogonal (a-d) PCP does not bind to MS2 hairpins and MCP does not bind to PP7 hairpins. MDN1-24PP7 mrnas are only visualized by co-expressing the appropriate fluorescent coat protein, PCP-2yEGFP, and MDN1-24MS2 mrnas are only visualized by co-expressing the appropriate fluorescent coat protein, MCP-2yEGFP. Scale bars, 3 µm.

S G2 Pre-Mitotis Late-Mitotis G1 a b c d e 0 min 20 min 40 min 60 min 80 min Mitotic Progression f 0 min g

Assessment of cell cycle stage MDN1-24PP7 mrnas are labeled with PCP-2yEGFP and nuclei are visualized by

(a-e) Cells can be imaged every 20 minutes in order to assess MDN1-24PP7 expression as the cell progresses

8 S G2 Pre-Mitotis Late-Mitotis G1 a b c d e 0 min 20 min 40 min 60 min 80 min Mitotic Progression f 0 min g 5 min h 10 min i 15 min j 20 min Supplementary Figure 7. Assessment of cell cycle stage MDN1-24PP7 mrnas are labeled with PCP-2yEGFP and nuclei are visualized by H2A2-yECFP. Cell cycle stage is determined by nuclear signal and cell morphology. (a-e) Cells can be imaged every 20 minutes in order to assess MDN1-24PP7 expression as the cell progresses through the cell cycle. (f-j) Cells can also be imaged at shorter timescales (5 min) in order to assess MDN1-24PP7 expression at different stages in the mitotic cycle. Scale bars, 3 µm.

H2A2 such that cell nuclei can be easily

9 MDN1-24PP7 + PCP-2yEGFP MDN1-24MS2 + MCP-mCherry H2A2-yECFP Merge a b c d e f g 0 min 20 min 40 min h i j 60 min 80 min 100 min Supplementary Figure 8. Two-color system paired with a nuclear marker (a) MDN1-24PP7 mrnas labeled with PCP-2yEGFP. (b) MDN1-24MS2 mrnas labeled with MCP-mCherry. (c) yecfp is fused to endogenous histone H2A2 such that cell nuclei can be easily visualized by low-power excitation with 436 nm light. (d) Merge. (e-j) Cells are imaged every 20 minutes and MDN1 expression can be quantified for each allele within the context of cell cycle stage. Scale bars, 3 µm.

10 a b transcript abundance transcript abundance time (min) time (min) Supplementary Figure 9. Cells exhibit different expression profiles over time Transcript counts for two cells (MDN1-24PP7 in green and MDN1-24MS2 in red) over 80 minutes. (a) Cell in which both alleles fluctuate around the mean. (b) Cell in which one allele (MDN1-24PP7 in green) exhibits persistently high expression while the other allele (MDN1-24MS2 in red) exhibits persistently low expression.

a MDN1 24 PP7 b c PP7-Cy3 24 MS2 d MS2-Cy5 e

Intramolecular tagging of MDN1 (a) Schematic

probes in green, demonstrating that both tags are

11 a MDN1 24 PP7 b c PP7-Cy3 24 MS2 d MS2-Cy5 e Merge DIC Supplementary Figure 10. Intramolecular tagging of MDN1 (a) Schematic showing MDN1 being 5 -tagged with 24PP7 and 3 -tagged with 24MS2. (b-e) PP7 probes in red colocalize with MS2 probes in green, demonstrating that both tags are present within the same molecules. DAPI signal (blue) highlights the nucleus and DIC images show cell morphology.

12 Supplementary Table 1. Sequences for mrna tagging PP7V3 TAAGGTACCTAATTGCCTAGAAAGGAGCAGACGATATGGCGTCGCTCCCTGCAGGTCGACTCTAGAAACCAG CAGAGCATATGGGCTCGCTGGCTGCAGTATTCCCGGGTTCATT MS2ORF TACGGTACTTATTGCCAAGAAAGCACGAGCATCAGCCGTGCCTCCAGGTCGAATCTTCA AACGACGACGATCACGCGTCGCTCCAGTA TTCCAGGGTTCATC *bold regions indicate hairpin sequences

13 Supplementary Table 2. PCR primer pairs Forward Primer Reverse Primer a TTCACTGATTTTGCGTCAATACTTTACAGACCTGGCA TCCAGCTAAGCCGCTCTAGAACTAGTGGATCC CCTTTGATTCGTGTAGTAAACCTCCTCTTCTTGGTTTTCA CGATATAGCATAGGCCACTAGTGGATCTG b CCTCGGTGAGTTTTCTCCTTCA ATATATTTAATTAACCTATAGAACTGAATGGGAAACT c GCGTCAATACTTTACAGACCTGGC ATATAT TTAATTAACCTATAGAACTGAATGGGAAACT d ACTCCACTTCAAGTAAGAGTTTG AGTCACATCAAGATCGTTTATGG e GCACGGAATATGGGACTACTTCG AGTCACATCAAGATCGTTTATGG

14 Supplementary Table 3. FISH probes MDN1 794 TTT GTC GTG GAT AGT GTG GAC CTT AGG GAC GAT AAC GCC ACA GAT TGA CG MDN1 860 CTC CCG AGT TGA CGA AGA GAG GAA ACC GTT TTA TGA GTA GGG ACA AAG GTT MDN CTA TAA GTA CCC ATC TCC CTT CTT TGA CCG CGG TAG CGA GAA CAC CAG CTC MDN TTT GCA GCC TTT ACA GTC TCT CCT CTG GAT GGA ATG GTT AGT TCG CGC TT PP7 GTA CCT TAG GAT CTA ATG AAC CCG GGA ATA CTG CAG CCA GCG AGC CCA TA MS2 TCT TGG CAA TAA GTA CCG TAG GAT CTG ATG AAC CCT GGA ATA CTG GAG CG *bold nucleotides indicate dye conjugation sites (Cy3, Cy3.5 or Cy5)

15 Supplemental Results Strain preparation for visualization of MDN1 mrnas Strains were prepared by site-specific genomic integration of 24PP7 or 24MS2 into the 3 UTR of one MDN1 allele (Supplementary Fig. 1a). Each stem loop binds two viral coat proteins, so RNA molecules become labeled following transformation with a plasmid that encodes fluorescent coat protein (Supplementary Fig. 1b). Two-color strains were obtained by mating one-color haploids, each expressing a different CP-FP (Supplementary Fig. 1c). MS2 and PP7 hairpin sequences were also optimized to minimize loss of hairpins during propagation and cloning in bacteria, a phenomenon observed with the use of prior MS2 versions. In addition to the hairpin array, the integration cassettes include a kanamycin selectable marker flanked by LoxP sites. The cassette was integrated into the 3 UTR of MDN1 immediately downstream of the stop codon, leaving the 3 UTR fully intact. Tagged strains were selected for by kanamycin resistance and integration sites were confirmed by PCR. Interestingly, fluorescent mrnas were not visible in all kanamycin-resistant colonies. Further PCR analysis revealed that during genomic integration by homologous recombination, varying numbers of hairpins could be lost (Supplementary Fig. 2a). Because hairpin copy number is directly proportional to the fluorescent signal of single mrnas, it was necessary to use only those strains that retained the full complement of hairpins for imaging experiments. MS2 hairpins were found to be twice as likely to undergo loss of hairpins compared to PP7 hairpins during homologous recombination in yeast (Supplementary Fig. 2b), however both were found to be stable once integrated (Supplementary Fig. 2c,d). In vivo imaging of single MDN1 mrnas We visualized fluorescent MDN1 mrnas for both the MDN1-24PP7 and MDN1-MS2 haploid strains by epifluorescence. Fluorescent spots that could be observed in yeast cells were shown to require the RNA tags, and did not result from CP-FP aggregation (Supplementary Fig. 3a-d).

16 Achieving the appropriate signal-to-noise levels in vivo depends on several factors. Yeast cells exhibit auto-fluorescence in the green and red channels and we reduced auto-fluorescence by culturing cells at very low log-phase growth for hours. Rapid diffusion of single mrnas in the x, y and z directions is also a concern, as it results in blurred fluorescence signal due to photons being spatially distributed on the EMCCD chip during exposure. Diffusion of individual mrnas in and out of z focal planes during image acquisition can also lead to oversampling of the total number of cellular mrnas. Therefore, a balance of high-intensity laser excitation and rapid acquisition through the cell volume is critical for accurately counting MDN1 steady-state levels. Cells were illuminated as the stage moved continuously in the z-axis, with the EMCCD camera reading out at 90ms intervals (Supplementary Fig. 4a). We have achieved ~6-fold faster acquisition using this z-sweeps acquisition as compared to traditional z-sections, minimizing blurring by providing a snap-shot of diffusing mrnas (Supplementary Fig. 4b,c). Tagging does not disrupt MDN1 expression Theoretically, PP7 and MS2 systems can be used to tag yeast genes at any position, however the addition of each cassette (approximately 1.3 kb) and the binding of fluorescent coat proteins to a transcript has the potential to alter a gene s expression level. Since MDN1 is an essential gene, we created MDN1-24PP7 and MDN1-24MS2 haploid strains by first tagging diploid yeast and then dissecting and screening tetrads. We observed that sporulation always resulted in four tetrads indicating that tagging did not render cells inviable. Haploid strains containing either tag also exhibited wild type doubling times in liquid media, indicating that tagging did not confer any growth defect. There have been mixed reports regarding how exactly the use of these tags may affect gene expression 1,2. In order to more closely examine the effects of tagging on MDN1 expression levels and whether such effects might differ based on which tag was used, we performed single-molecule FISH to

17 quantify MDN1 expression for WT and tagged strains (Supplementary Fig. 5a-c). WT MDN1 was expressed on average at 5.94 ± 0.16 mrnas per cell compared to 5.59 ± 0.17 mrnas per cell for MDN1-24MS2 and 5.41 ± 0.15 mrnas per cell for MDN1-24PP7. We performed the same experiments on WT and tagged strains expressing either PCP-2yEGFP or MCP-2yEGFP in order to determine if labeling of the mrna by the appropriate CP-FP affected transcript numbers. (Supplementary Fig. 5d-f). Under these conditions, FISH experiments revealed that WT MDN1 was expressed on average at 5.71 ± 0.19 mrnas per cell compared to 5.25 ± 0.16 mrnas per cell for MDN1-24MS2 + MCP-2yEGFP and 5.56 ± 0.16 mrnas per cell for MDN1-24PP7 + PCP-2yEGFP. We used the same analysis to quantify MDN1 expression in living cells (Supplementary Fig. 5g,h) and counted an average of 5.59 ± 0.46 mrnas per cell for MDN1-24MS2 + MCP-2yEGFP and 5.54 ± 0.42 mrnas per cell for MDN1-24PP7 + PCP-2yEGFP.. Taken together, it appears that tagging of the 3 UTR of MDN1 did not perturb steady-state expression levels and that live cell experiments had detection sensitivities similar to those of FISH (Supplementary Fig. 5i). Furthermore, we see by FISH that MDN1 remains transcriptionally active in nearly all cells regardless of whether it is tagged or not. More specifically, WT MDN1 shows an average of 1.82 ± 0.16 nascent mrnas per cell, as compared to 1.85 ± 0.15 nascent mrnas for MDN1-24MS2 and 1.78 ± 0.15 nascent mrnas for MDN1-24PP7 (Supplementary Fig. 5j), suggesting that transcriptional activity is also unaffected. These experiments provided a foundation for two-color quantitative analysis of gene expression with physiological relevance. MS2 and PP7 labeling systems are orthogonal The prospect of a two-color imaging utilizing both PP7 and MS2 tags requires that the individual imaging systems be orthogonal. The RNA-binding surfaces of PCP and MCP have evolved to recognize distinct RNA hairpins and have been shown in vitro to discriminately bind their own hairpin with ~1000- fold difference in affinity 3-5. In order to confirm this specificity in living yeast cells, we expressed MCP-

18 2yEGFP in MDN1-24PP7 haploids and PCP-2yECFP in MDN1-24MS2 haploids (Supplementary Fig 6a-d). We did not observe fluorescently-labeled mrnas in these strains, indicating that each CP maintains high specificity for its cognate hairpin sequence at concentrations required for live cell imaging. Determining transcript counts within the context of cell cycle As mentioned, we were interested in applying this two-color system to correlate the mrna expression for two identical MDN1 alleles. However, partitioning of mrnas during cell division can complicate the evaluation of MDN1 allelic correlation, as the division and partitioning of transcripts will automatically translate to a decrease in steady-state numbers for both alleles within the parental cell. We therefore fused yecfp to endogenous histone H2A2 in order to visualize nuclei and used cell and nuclear morphology to determine cell cycle stage (Supplementary Fig. 7a-e). Imaging at a faster acquisition rate, we can observe different time points during progression through mitosis (Supplementary Fig. 7f-j). We applied this to generate three-color strains (Supplementary Fig. 8a-d) in which we quantified expression of each allele at 20 minute time points up until mitosis (Supplementary Fig. 8e-j, Supplementary Movie 1-4). Single-cell resolution provides a window into different expression behaviors This system also provides a window into the different behaviors that cells exhibit in terms of allelic coordination over time. While single-molecule FISH has demonstrated cell-to-cell variability in expression levels, it was not previously possible to determine whether this variation resulted from subpopulations of cells that vary in expression levels that persist or whether expression levels fluctuate rapidly and equally in all cells over time. While most cells within a population exhibit MDN1 expression that fluctuates about the mean through time (Supplementary Fig. 9a), we also observe cells that exhibit persistently high or low expression as compared to the mean for the population. In fact, certain cells show one allele exhibiting persistently high expression and the other allele exhibiting persistently low

19 expression (Supplementary Fig. 9b). These expression regimes are not likely to result from changes in transcription factor binding, and may instead point to inherent differences in chromatin states at these two loci. Dual-tagged MDN1 is transcribed into mrnas that contain both tags within the same molecule Before determining elongation rates using dual-tagged MDN1, it was necessary to first confirm that both tags were indeed present within the same molecule. We prepared this strain by integrating 24PP7 into the 5 UTR of a strain in which MDN1 was already tagged in the 3 UTR with 24MS2 (Supplementary Fig. 10a). Because it was a diploid, it was necessary to verify that this 5 tag was integrated into the appropriate allele. We used single-molecule FISH to confirm dual-tagging of MDN1, showing that PP7 and MS2 probes colocalize to single transcripts in this intramolecular strain (Supplementary Fig. 10b-e). We induced transcription in confirmed strains by adding galactose, and acquired at 2 minute intervals to visualize 3 and 5 signals (Supplementary Movie 5-8).

20 1. A. B. Muthukrishnan, M. Kandhavelu, J. Lloyd-Price et al., Nucleic Acids Res 40 (17), 8472 (2012). 2. J. R. Chubb, T. Trcek, S. M. Shenoy et al., Curr Biol 16 (10), 1018 (2006). 3. J. Carey, V. Cameron, P. L. de Haseth et al., Biochemistry 22 (11), 2601 (1983). 4. F. Lim, T. P. Downey, and D. S. Peabody, J Biol Chem 276 (25), (2001). 5. J. A. Chao, Y. Patskovsky, S. C. Almo et al., Nat Struct Mol Biol 15 (1), 103 (2008).

c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP

c Tuj1(-) apoptotic live 1 DIV 2 DIV 1 DIV 2 DIV Tuj1(+) Tuj1/GFP/DAPI Tuj1 DAPI GFP Supplementary Figure 1 Establishment of the gain- and loss-of-function experiments and cell survival assays. a Relative expression of mature mir-484 30 20 10 0 **** **** NCP mir- 484P NCP mir- 484P b Relative