λ N -GFP: an RNA reporter system for live-cell imaging

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1 λ N -GFP: an RNA reporter system for live-cell imaging Nathalie Daigle & Jan Ellenberg Supplementary Figures and Text: Supplementary Figure 1 localization in the cytoplasm. 4 λ N22-3 megfp-m9 serves as a reporter of mrna Supplementary Figure 2 Dynamics of SRP RNA assessed by FRAP experiments. Supplementary Methods Note: Supplementary Movies 1 and 2 are available on the Nature Methods website.

2 Supplementary figure 1. a. mrna: mrfp-4 boxb-zipcode Protein: mrfp Ratio mrna/protein 0:00 0:30 1:00 1:30 2:00 b. Relative fluorescence intensity 1,2 1 0,8 0,6 0,4 0,2 Protein mrfp mrna mrfp-4xboxb-zipcode Time (sec) mrna: mrfp-4 boxb-zipcode Protein: mrfp Pre-bleach Bleach 4 s 60 s 4 λn22-3 megfp-m9 serves as a reporter of mrna localization in the cytoplasm. (a) Cells transfected with pmrfp-4 boxb-zipcode were imaged over 2 hours. Selected time points show localization of the mrna (arrowheads) in lamellipodia as the cell moves. Upper panel shows mrna localization, middle panel the encoded mrfp and lower panel shows the pseudocolor ratio images displaying the specific mrna localization. Warm pixels correspond to areas where mrna concentration is high. Images are projections of 3 confocal slices of 2 µm optical thickness (FWHM). See Movie 1 for full video. (b) Dynamics of mrna molecules assessed by FRAP experiments. Plots represent normalized fluorescence displayed over time, n = 6. Images show a single confocal slice of 5 µm optical thickness of mrna (upper panel) and protein (lower panel). Scale bar 10µm.

3 Supplementary figure 2. Relative fluorescence intensity 1, , , , , Time (s) RNA 5xboxB-SRPRNA Protein SRP19-mRFP mrna: 5 boxb-srprna Protein: SRP19-mRFP Bleach 10 s 110 s Dynamics of SRP RNA assessed by FRAP experiments. Plot represent normalized fluorescence displayed over time for 5 box-srprna, the non-coding RNA that forms the structural backbone of the signal recognition particle (SRP), and for SRP19-mRFP, one of the protein components of the particle. Images show a single confocal section of 5 µm optical thickness (FWHM) of SRP RNA (upper panel) and SPR19 protein (lower panel). n = 5. Scale bar 10 µm.

4 Supplementary Methods Cell Culture NRK cells were cultured as described elsewhere 1. All experiments were performed in NRK cells stably expressing 4 λ N22-3 megfp-m9. The stable cell line was selected according to standard protocols and maintained in 0,5mg/ml G418. For imaging, growth medium was replaced by CO 2 -independent phenol red free medium (Invitrogen). Transfection was with FuGene 6 (Roche). To promote movement of 4 λ N22-3 megfp- M9 NRK cells transfected with pmrfp-4 boxb-β-actin-zipcode, 20% FCS was added back after 4 hours of serum (FCS) starvation. DNA constructs An array of four λ N22 (MDAQTRRRERRAEKQAQWKAAN), spaced by 21 amino acid linkers (PPLDGAGAGAGAGAGAGGLAT), was fused to a tandem of three megfp 2 to increase the signal 3, and followed by an M9 nuclear localization signal 4. psrprb-mrfp- 4 boxb and psrprb-mrfp-16 boxb were made the following way: mrfp 5 has been subcloned in place of CFP into psrprb-cfp 3. An oligonucleotide containing four boxb (GCCCTGAAAAAGGGC) spaced by four nucleotides and flanked by restriction sites was synthesized (Sigma) and subcloned downstream of mrfp stop codon. The total length of the stem loop tag was thus 80 nucleotides. Restriction sites 5 and 3 of this oligonucleotide were designed to subclone multiples of this array of four boxb motifs and used to generate a tag containing 16 boxb motifs, with a total length of 310 nucleotides. pmrfp-4/16 boxb-β-actin-zipcode were generated using the same oligonucleotide containing 4 boxb and the same strategy as for the previous construct. Chicken β-actin-zipcode was designed as construct G in 6. The plasmid p5 boxb- SRPRNA was constructed using the forty one nucleotides of the human SRP RNA promoter followed by 5 boxb, the SRP RNA sequence and PolIII terminator 7. EGFP was replaced by mrfp in psrp19-egfp 8 to generate psrp19-mrfp.

5 Imaging Imaging was performed on a customized ZEISS LSM510 Axiovert confocal microscope as described previously 9,10 using a 63x Plan-Apochromat 1.4 NA oil immersion objective lens (Carl Zeiss, Jena). Images were analyzed in the LSM software (Carl Zeiss, Jena) and ImageJ ( Figures were assembled in Adobe Photoshop and Adobe Illustrator (Adobe Systems, Inc.). Image ratios were calculated by dividing background subtracted mrna intensities by the corresponding background subtracted protein intensities. For display purposes, cell contours were defined by automated segmentation in the mrfp channel and applied to the ratio images. Ratio values at each pixel are displayed as warm colors for higher ratio and cold colors for lower ratio. For correlating post-mitotic mrna export and encoded protein translation, quantitation during the first wave of cytoplasmic mrna localization is displayed. Fluorescence intensities were background subtracted and normalized between 0 and 1 in the following manner. For mrna, average cytoplasmic background of untransfected cells and highest values in the first wave where normalized to 0 and 1. For protein, average cytoplasmic background of untransfected cells and highest values were set to 0 and 1. Data from n = 6 cells have been registered in time to the highest mrna value of the wave. Photobleaching was done on mrna and protein molecules using the 488 nm line of the argon laser and the 561 nm diode laser simultaneously. Images were taken with wide open pinhole, fluorescence in the bleached region of the cytoplasm was background subtracted and corrected for bleaching due to acquisition and loss of total fluorescence due to the photobleach pulse and normalized to 1 for the prebleach intensity.

6 References 1. Ellenberg, J. et al. Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis. J Cell Biol 138, (1997). 2. Snapp, E.L. et al. Formation of stacked ER cisternae by low affinity protein interactions. J Cell Biol 163, (2003). 3. Daigle, N. et al. Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells. J Cell Biol 154, (2001). 4. Siomi, H. & Dreyfuss, G. A nuclear localization domain in the hnrnp A1 protein. J Cell Biol 129, (1995). 5. Campbell, R.E. et al. A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 99, (2002). 6. Kislauskis, E.H., Zhu, X. & Singer, R.H. Sequences responsible for intracellular localization of beta-actin messenger RNA also affect cell phenotype. J Cell Biol 127, (1994). 7. Ullu, E. & Weiner, A.M. Human genes and pseudogenes for the 7SL RNA component of signal recognition particle. Embo J 3, (1984). 8. Politz, J.C. et al. Signal recognition particle components in the nucleolus. Proc Natl Acad Sci U S A 97, (2000). 9. Lenart, P. et al. A contractile nuclear actin network drives chromosome congression in oocytes. Nature 436, (2005). 10. Rabut, G. & Ellenberg, J. Automatic real-time three-dimensional cell tracking by fluorescence microscopy. J Microsc 216, (2004).