Bioorthogonal mRNA labeling at the poly(A) tail for imaging localization and dynamics in live zebrafish embryos† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc05981d

Live imaging of mRNA in cells and organisms is important for understanding the dynamic aspects underlying its function.


Cell culture and confocal imaging
HeLa cells (Sigma Aldrich) were cultured in growth media comprising MEM Earle's media (Merck) supplemented with L-glutamine (2 mM), nonessential amino acids (1%), penicillin and streptomycin (1%), and fetal calf serum (FCS, 10%) and under standard conditions (5% CO 2 , 37°C). One day before transfection, 2 × 10 5 cells were seeded in media (1 mL) on 15 mm cover slips in a 12-well cell culture plate. Cells were transfected using Metafectene® Pro (3 µL) in MEM Earle's media (47 µL) and egfp/mcherry mRNAs (1 µg) in MEM Earl's media (50 µL) for 6 h at 37°C in a total volume of 1 mL per chamber. Then, media with transfection reagent was replaced with fresh growth media. After 24 h, cells were washed twice in 1 x PBS, fixed in 4% paraformaldehyde, washed again twice in 1 x PBS, stained with DAPI (10 µg/µL) for 30 s, washed twice with 1 x PBS and once quickly with water before the coverslips were quickly inverted and mounted on Aqua-Poly/Mount-coated microscope glass slides. Images were taken in using a Leica A TCS SP8 confocal laser scanning microscope with a 63x water-immersion objective and the settings tabulated in Table S2.

Cell culture and western blot analysis
For western blot analysis, HeLa cells were cultured and transfected as described in the imaging experiments. Twenty-four hours after transfection, cells were lyzed using the protocol of the manufacturer for CelLytic™ M (Sigma Aldrich). To determine the protein concentration of cell lysate, the Bradford assay was applied by diluting cell lysate (1:50) along with BSA calibration standards (0-10 µg/µL). Samples (50 µL) were then incubated (15 min, rt, exclusion of light) with 1× Roti®-Quant (Roth) staining solution (200 µL) and then, the extinction at 595 nm was determined. Proteins were separated via tris-glycine-PAGE (10% PA, 120 V, 1.5 h, rt, 50 µg of protein) and then the proteins were transferred onto a nitrocellulose membrane Roti®-NC (Roth) in semi-dry transfer buffer with 80 mA for 75 min at rt. After ascertaining transfer efficiency using Ponceaus S-staining, the membrane was cut into suitable pieces for subsequent antibody treatment and washed with 1× PBS + 0.01% Tween (PBST). The membrane was blocked in blocking buffer (3% BSA) for 1 h at rt, followed by incubation with the anti-GFP or anti-α-tubulin (loading control) primary antibodies overnight at 4°C. Then the membrane was washed three times with PBST each for 5 min at rt. The membrane pieces were then incubated with HRP-conjugated secondary antibodies for 1 h at rt and washed three times with PBST afterwards. For chemiluminescence detection the EZ-ECL Chemiluminescence detection kit (Biological Industries) was used and results were analysed with a Chemo Star Advanced Fluorescence & ECL Imager (Intas). Primary antibodies for GFP (B-2) and α-tubulin were purchased from Santa Cruz Biotechnology and Sigma Aldrich. Secondary antibody polyclonal rabbit anti-Mouse/HRP was from Dako Diagnostics.

Zebrafish work
Wildtype zebrafish (Danio rerio) of the AB genetic background were used in all experiments. For the experiment presented in Figure 2B, the fish carried a transgene that directs the expression of GFP on the membrane of PGCs (tg(kop:egfp-f'-nos3'UTR-cry:dsRed)). [2] The fish were handled according to the regulations of the state of North Rhine-Westphalia, supervised by the veterinarian office of the city of Muenster.

Construction of plasmids, mRNA synthesis and labeling
For PGC-specific expression of cytoplasmic GFP (gfp-nos, internal database no. 355) and for marking germ granules with GFP-tagged Vasa protein (vasa-gfp, internal database no. 291), previously published constructs were used. 70,27 To distinguish between labelled injected mRNA and endogenous mRNA, a nos 3'UTR-containing construct was designed (STOP mcherry-nos, internal database no. D396). This construct contains an mcherry coding sequence, which can be detected in the RNAscope procedure using the mcherry probe. In this construct a single base pairinsertion was introduced following the start codon, leading to a stop codon after 24 nucleotides and no mCherry protein translation. This allowed detection of the red SRB signal of the click-labelled mRNA, with no spectral overlap with mCherry protein. For visualising mRNA via the PP7 detection system, a nos 3'UTR-containing construct was designed. In this construct 24 PP7-recognition loop sequences were inserted in the 3'UTR (STOP nanos-nos 24xpPP7, internal database no. D016). The PP7 coat protein (PCP) was fused to a nuclear localization sequence (NLS) to direct unbound coat protein to the nucleus, and was tagged with a YFP for visualization of the protein (nls-ha-tdpcp-yfp-glob, internal database no. C987). BFP-tagged lifeact expressed in the germ cells using the nos 3'UTR (lifeact-tagbfp-nos, internal database no. E058) labelled actin rich structures in the PGCs with this fluorescent protein.
Capped sense mRNAs were synthesized using the mMessage mMachine kit (Ambion) according to the protocol of the manufacturer. Then 4000 ng of each mRNA was subjected to enzymatic addition of 2'-N 3 -2'-dATP as described above. The click reactions were performed with azidomodified mRNA (1000 ng) and DBCO-conjugated SRB (7.5 µM) for 60 min at 37°C followed by isopropanol precipitation overnight and washing with 70% ethanol. Then, the labeled mRNAs were analyzed on a 7.5% denaturing PA gel.

Microinjection into zebrafish embryos
To visualize the distribution of injected mRNAs in figures 3 and 4, 1 nL containing 80 pg or 200 pg of gfp-nos (internal database no. 355) or of STOP mcherry-nos (internal database no. D396) mRNAs either non-labeled or labelled with SRB were used. To visualize germ granules with GFPtagged Vasa protein, 1 nL containing 80 pg of vasa-gfp (internal database no. 291) mRNA was injected. For detection of nos 3' UTR-containing mRNA using the click-labelling approach and the PP7 detection system in figure 5, 1 nl containing 80 pg of STOP mcherry-nos mRNA labeled with SRB were co-injected with an equimolar amount of STOP nanos-nos 24xPP7 mRNA (internal database no. D016). In addition, 8 pg of nls-ha-tdpcpyfp-glob (internal database no. C987) mRNA were co-injected to express the YFP-tagged PCP protein. To visualize PGCs in figure 5, 80 pg of lifeact-tagBFP-nos (internal database no. E058) were co-injected. mRNAs were injected into the yolk of 1 cell-stage zebrafish embryos using glass capillaries and the PV830 Pneumatic PicoPump microinjector (WPI).

Microscopy and image analysis in zebrafish embryos
Confocal images were acquired on an LSM 710 microscope (Zeiss) equipped with 405, 488, 561 and 633 nm lasers and a 63x water-immersion objective (Zeiss) controlled by the ZEN software (Zeiss, version 2010B SP1, 6.0). Time-lapses were acquired on a Yokogawa CSU-X1 Spinning Disk microscope equipped with a 63x water-immersion objective (Zeiss) and connected to a Piezo stage and a Hamamatsu Orca Flash 4.0 V3 camera. The microscopy setup was controlled by the VisiView software (Visitron, version 4.0.0.14). For the time-lapse movies, images were acquired at 3.5 seconds intervals over a period of 206.5 seconds using an exposure of 200 ms. For each time point 18 different focal planes with a z-distance of 0.5 µm were acquired. Images were processed using the Fiji software (National Institutes of Health, version 2.0.0-rc-43/1.51a). The 509 nm emission channels showing GFP-positive PGCs in figure 2 were processed with a background subtraction and gaussian filter to reduce signal noise. Images were deconvolved with Huygens Professional version 19.04 (Scientific Volume Imaging, The Netherlands, http://svi.nl), using the CMLE algorithm, with a maximum of 40 iterations.

Figure S8
HPLC and LC-QQQ analysis of SPAAC reaction of RNAs modified at the 3′ end with 2′-azido-ddATP. RNAs were modified as indicated, then digested and dephosphorylated to single nucleosides for analysis. A) HPLC analysis at 260 nm shows the four nucleosides and azido-A (arrow) in the case of enzymatic modifications (red trace). After the click reaction, no azido-A is detectable, indicating complete conversion (blue trace), similar to unmodified RNA (black trace). B) EIC for azido-A (M+H = 293.10), confirming the mass of the peak. C) EIC of the mass expected for the click product of azido-A with biotin (M+H= 1042.45), showing that the expected product is formed. D) EIC for azido-A after the click reaction showing that no azido-A can be detected after the click reaction and also confirming complete conversion during the SPAAC reaction.