Supplementary Information. A superfolding Spinach2 reveals the dynamic nature of. trinucleotide repeat RNA

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1 Supplementary Information A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat RNA Rita L. Strack 1, Matthew D. Disney 2 & Samie R. Jaffrey 1 1 Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10065, USA. 2 Department of Chemistry, The Scripps Research Institute, Scripps Florida, Jupiter, Florida 33458, USA. Correspondence to: Samie R. Jaffrey (srj2003@med.cornell.edu). List of supplementary information Supplementary Figure 1 Spinach-tagged CGG-repeat containing RNAs form nuclear foci Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Figure 6 DFHBI is permeable in mammalian cells Alignment of Spinach and derivatives COS-7 cells form heterogeneous CGG aggregates Extended 1a treatment does not disrupt existing foci CGG-Spinach2 remains nucleoplasmic after tautomycin removal 1

2 Supplementary Figure 7 The PP1 and PP2A inhibitor okadaic acid does not disrupt existing foci Supplementary Table 1 Supplementary Table 2 Supplementary Video 1 Supplementary Video 2 Supplementary Note 1 Properties of Spinach vs. Spinach2 Brightness of Spinach1.2 mutants at 25 and 42 o C Foci formation after transient transfection Foci disaggregation after treatment with tautomycin MS2-GFP based imaging approaches for studying toxic RNAs 2

3 SUPPLEMENTARY FIGURES Supplementary Figure 1. Spinach-tagged CGG-repeat containing RNAs form nuclear foci. (a) To test whether (CGG) 60 -Spinach formed RNA aggregates, COS-7 cells were transfected with plasmids encoding either a 60 CGG-repeat containing transcript or (CGG) 60 -Spinach. At 24 3

4 h post-transfection, cells were analyzed for nuclear foci using FISH with a Texas Red-labeled probe against CGG repeats to demonstrate that Spinach insertion did not perturb foci formation (top two rows). FISH with a Texas Red-labeled probe against Spinach also labeled nuclear aggregates (bottom row), indicating that Spinach is present in these foci. DNA is labeled with DAPI. Scale bar 10 µm. (b) To test the stability of CGG-repeat containing RNA, COS-7 cells were cotransfected to express ptet-off and either (CGG) 30, (CGG) 30 -Spinach2, (CGG) 60, (CGG) 60 -Spinach, or (CGG) 60 -Spinach2. 24 h post-transfection, transcription was inhibited by addition of doxycycline. Transcript abundance was then measured using qrt-pcr at 0, 6, 12, and 24 h post-doxycycline addition. Results shown represent mean and s.e.m. values for three independent replicates. We observed that greater than 80% of the initial signal is still present 24 h after transcription was silenced by the addition of doxycycline for all (CGG) 60 -containing transcripts. In this case, the presence of a Spinach-tag did not affect RNA stability. In addition, we tested whether RNA aggregation is responsible for the stability of these constructs. For this experiment, we compared the stability of (CGG) 60 RNA to that of (CGG) 30 RNA, which does not form nuclear aggregates and is exported from the nucleus. Here we observed that (CGG) 30 RNA has a half-time of ~6 h, which is substantially lower than that of (CGG) 60 RNA. The stability of (CGG) 30 RNA was not substantially affected by tagging with Spinach2. Taken together, these results indicate that 60 CGG-repeat RNA is highly stable, and that the stability is due to formation of nuclear aggregates. By comparing qrt-pcr signal from samples to a standard curve of known concentration, we also determined that the CGG-repeat RNA is highly concentrated in these foci, with up to ~ copies of RNA per aggregate. 4

5 Supplementary Figure 2. DFHBI is permeable in mammalian cells. Hoeschst (1 µg/ml) and DFHBI (20 µm) were added to COS-7 cells expressing (CGG) 60 - Spinach2. Images were taken at regular intervals after dye addition. Signal in the blue and green channels were recorded for 20 nuclei, and maximum brightness was normalized to 100% for each cell. Hoechst and Spinach2 signal increased linearly over time and reached a plateau approximately 30 min after dye addition. These data indicate that DFHBI shows similar permeability kinetics to Hoechst. 5

6 Supplementary Figure 3. Alignment of Spinach and derivatives. The aligned DNA sequences of Spinach, Spinach1.1, Spinach1.2, and Spinach2 are shown. Green shaded positions represent sites that were mutated to generate Spinach2 from Spinach1.2. Underlined regions correspond to the designated stem or stem loop. 6

7 Supplementary Figure 4. COS-7 cells form heterogeneous CGG aggregates. (a) Schematic diagram of (CGG) 60 -Spinach2. (b) Shown are live-cell images of representative foci-containing nuclei from cells expressing (CGG) 60 -Spinach2. Aggregates vary in size, number, and distribution. These differences may be due to the heterogeneous nature of transient transfection, the specific cell-cycle position of a given cell, or other factors. Typical nuclei (left panel, upper left) contain 5-10 large foci with additional smaller foci and moderatelow nucleoplasmic signal. The foci change during cell division (left panel, lower right, dividing cell) to become a large mass that is partitioned between daughter cells. Occasionally, cells contain both large aggregates and numerous small punctate aggregates (upper right panel). Nuclear size is also highly variable after transfection (middle panel, lower right panel), ranging from 5-25 µm. Scale bar, 10 µm. 7

8 Supplementary Figure 5. Extended 1a treatment does not disrupt existing foci. To determine whether longer treatments were required for 1a to disrupt foci, COS-7 cells were cotransfected with the (CGG) 60 -Spinach2 vector and pdsred-max 2 as a transfection control. Approximately 24 h post-transfection, cells were treated with either vehicle or 20 µm 1a. After 48 h, nuclei from 100 DsRed-positive cells were examined for foci. (a) Percentage of cells retaining foci after treatment with 1a. At 0 and 48 h post-drug treatment, 94 ± 2.8 and 86 ± 3.5% of nuclei contained foci, respectively, indicating that 1a does not disrupt foci under these conditions. Data shown represent the mean and s.e.m. values for three independent replicates. (b) Representative nuclei for cells treated with either vehicle or 20 µm 1a at 0 and 48 h postdrug treatment. Nuclei from typical COS-7 cells expressing both (CGG) 60 -Spinach2 and DsRed- Max after drug treatment are shown (top panels). The experiment was repeated to determine whether 1a could disrupt protein binding to foci. Here, Spinach2 and mcherry-hsam68 localization was examined at 0 and 48 h post-drug treatment (bottom panels). No changes were observed for Sam68 localization, indicating that 1a does not disrupt protein binding from foci. Scale bar, 10 µm. 8

9 Supplementary Figure 6. CGG-Spinach2 remains nucleoplasmic after tautomycin removal. To determine whether foci formation after tautomycin is rapidly reversible, cells treated with 5 µm tautomycin for 4 h were then changed into medium lacking tautomycin. Cells were monitored for 10 h. No foci reformation was observed during this time period. This suggests that tautomycin induces changes in cells that inhibit CGG repeat aggregation, at least within the time frame examined. Scale bar, 10 µm. 9

10 Supplementary Figure 7. The PP1 and PP2A inhibitor okadaic acid does not disrupt existing foci. To test whether tautomycin disrupts foci by inhibiting PP1 or PP2A, cells with CGG-Spinach2 foci were treated with 1 µm okadaic acid and analyzed for changes in foci. Okadaic acid at this concentration inhibits both protein phosphatases. However, no change in foci number was observed over the 4 h time course of the experiment. These data suggest that a different tautomycin target mediates its effects on CGG repeat foci. Okadaic acid treatment was limited to 4 h because cell death was observed between 4-6 h post-treatment. Scale bar, 10 µm. 10

11 Supplementary Table 1. Properties of Spinach vs. Spinach2 Excitation / Emission max (nm) Quantum yield Extinction coefficient (M -1 cm -1 ) Relative K D (nm) c T m ( o C) c brightness a Spinach 452 / 496 b 0.70 b 25,400 b ± ± 0.6 Spinach1.1 ND d ND ND ND ND 35 ± 0.5 Spinach1.2 ND ND ND ND ND 38 ± 0.3 Spinach2 454 / , ± ± 0.4 a Brightness is reported relative to Spinach. b Values obtained from ref. 1. c Data represent the mean and s.e.m. for three independent measurements. d ND indicates not determined for this study. 11

12 Supplementary Table 2. Brightness of Spinach1.2 mutants at 25 and 42 o C Mutations a Relative brightness at 25 o C b Percent brightness at 42 o C c Spinach Spinach1.2 a G1A, C98T C3T, G96A G4A, C95T C5T, G94A G6A, C93T C8T, G91A C9T, G90A G10A G14A G19A G20A G22A G25A, C34T G26A, C33T C28T G29A G30A G31A G36A, C60T G37A, C59T G63A, C88T G66A G68A G71A, C82T G73A, C80T G75A G77A C83T G84A G6A, C93T, C9T, G90A, G37A, C59T, C3T, G96A, G6A, C93T, C9T, G90A, G37A, C59T, C9T, G90A, G37A, C59T, G6A, C93T, G37A, C59T, G6A, C93T, C9T, G90A,

13 G6A, C93T, C9T, G90A, G37A, C59T, C3T, G96A, G6A, C93T, C9T, G90A, C3T, G96A, G6A, C93T, G37A, C59T, C3T, G96A, C9T, G90A, G37A, C59T, C3T, G96A, G6A, C93T, C3T, G96A, C9T, G90A, C3T, G96A, G37A, C59T, C3T, G96A, a Mutations are numbered according to Spinach nucleotide position. All mutants listed above contain mutations A6C, T9C, A90G, G37C, T38C, A58G, and Δ40,41,56 relative to Spinach unless otherwise noted. b Brightness values are determined relative to Spinach. c Percent brightness values represent the percent of signal remaining at 42 o C for a given mutant. Mutations in red improved both brightness and thermostability and were included in subsequent mutagenesis. Mutations in green were screened by combinatorial mutagenesis. 13

14 SUPPLEMENTARY NOTES Supplementary Note 1. MS2-GFP based imaging approaches for studying toxic RNAs. One strategy for labeling RNA in living cells involves targeting GFP to RNAs of interest by expressing GFP fused to the MS2 coat protein and inserting multiple copies of the MS2 coat protein recognition sequence into the target RNA 3. Although MS2-GFP-based strategies for imaging RNAs in living cells have been used successfully, they are technically complex 3. Additionally, this approach requires RNAs to be bound to numerous MS2-GFP molecules that contain their own nuclear-targeting signals, which can affect the localization or trafficking behavior of an RNA of interest 4. The MS2-GFP system has been successfully used to study the dynamics of CUG-repeat containing RNAs 5. The principle advantages of Spinach2 technology are the small size of the single Spinach2 tag relative to the 24 MS2-binding RNA tags bound by multiple MS2-GFP molecules needed for imaging the CUG repeats, and the nominal background cellular fluorescence observed using Spinach2-DFHBI compared to the MS2-GFP system in which unbound MS2-GFP molecules in the nucleus and cytoplasm are fluorescent. Additionally, the Spinach2-tagged RNA is the only construct that is expressed in cells, which contrasts with the MS2 system which also requires optimizing the expression level of MS2-GFP so that it stoichiometrically matches the level of the MS2 RNA tags on the mrna. SUPPLEMENTARY REFERENCES 1. Paige, J.S., Wu, K.Y. & Jaffrey, S.R. RNA mimics of green fluorescent protein. Science 333, (2011). 2. Strack, R.L. et al. A noncytotoxic DsRed variant for whole-cell labeling. Nat Methods 5, (2008). 14

15 3. Park, H.Y., Buxbaum, A.R. & Singer, R.H. Single mrna tracking in live cells. Methods Enzymol 472, (2010). 4. Tyagi, S. Imaging intracellular RNA distribution and dynamics in living cells. Nat Methods 6, (2009). 5. Querido, E., Gallardo, F., Beaudoin, M., Ménard, C. & Chartrand, P. Stochastic and reversible aggregation of mrna with expanded CUG-triplet repeats. J Cell Sci 124, (2011). 15