Live Cell Imaging of Endogenous mRNA Using RNA-Based Fluorescence “Turn-On” Probe

Messenger RNA (mRNA) plays a critical role in cellular growth and development. However, there have been limited methods available to visualize endogenous mRNA in living cells with ease. We have designed RNA-based fluorescence “turn-on” probes that target mRNA by fusing an unstable form of Spinach with target-complementary sequences. These probes have been demonstrated to be selective, stable, and capable of targeting various mRNAs for live <i>E. coli</i> imaging

2D ¹⁵N-HSQC spectra of monomeric CM2 show a calcium sensitive interaction of calmodulin.

<p>(A) 2D ¹⁵N-HSQC spectra of monomeric CM2 alone (gray) overlaid with the spectra after the addition of 18μM CaM with 2mM CaCl₂ (pink) shows significant shifts. While 18μM CaM in the presence of 2mM EGTA (purple) shows more minimal shifts. Inset: Shifts on leucine 1131 and leucine 1137 are apparent. (B) Quantitation of shifts from a CaM titration (2mM CaCl₂) where several shifts are greater than 0.01ppm (dashed line) on addition of 5.3μM CaM. Residues that align with the predicted CaM binding site in mammalian CDK5RAP2 are shown in red. The star indicated residue N1135 that is not visible in the HSQC spectra while the gray shading indicates residues that are not modeled in the 5MWE crystal structure. (C) Mapping of chemical shift changes (blue-red) onto the 5MWE crystal structure (CNNLZ is shown in gray) reveals shifts along the length of the helices including the region where dimer contacts are made.</p

Conserved domain, CM2, of centrosomin characterized by SEC-MALS, SIM, and yeast-two-hybrid experiments.

<p>(A) Schematic of Centrosomin with coiled-coil prediction shown in blue. CM1 and CM2 are highlighted in red. Alignment of <i>D</i>. <i>Melanogaster’s</i> CM2 with the zebra fish, mouse and human (CDK5RAP2) homologues, with residue numbering according to <i>D</i>. <i>Melanogaster</i> residues. Higher conservation can be seen starting at residue E1090 in line with the start of the predicted coiled coil region. The predicted calmodulin binding site in human CDK5RAP2 is shown in red. (B) Molecular weights (left axis) measured with SEC-MALS are consistent with aa1064-1148 forming a dimer of CM2 (red) with a predicted monomer weight of 11kDa and a calculated weight of 22.4kDa. The construct aa1090-1148 exists as monomer (blue) with a predicted monomer weight of 7kDa and a calculated weight of 7.7kDa. (C) SEC-MALS of a more minimal dimer construct aa1074-1148 (blue) that has a calculated molecular weight of 19.4kDa and predicted monomer weight of 9kDa. A construct with the final 18 residues truncated (Δ1130–1148) maintains a dimeric oligomer state (red) with a calculated molecular weight of 21.6kDa and predicted monomer weight of 8.8kDA. (D) Z-projections of the aligned and averaged PLP and GFP distributions for both the monomer and dimer GFP-CM2 fusion constructs in interphase S2 cells. Scale bar represents 100nm. (E) Z-projections of the monomer and dimer GFP-CM2 fusion constructs in mitotic S2 cells. Scale bar represents 100nm (F) Radial averages of the data represented in figures D and E with vertical bars indicating the average and horizontal bars representing the standard deviation of the Gaussian fit. (G) Schematic summaries of yeast-two-hybrid experiments showing the CM2 construct (aa1087-1148) interacting with a minimal domain of PLP(aa583-740) and the middle domain of CNN (aa454-556). Yeast plates of the interaction test are shown with red boxes indicating plating on selection media.</p

2D ¹⁵N-HSQC spectra of assigned monomeric CM2 shows interaction along three patches of residues.

<p>(A) Overlays of 2D ¹⁵N-HSQC spectra and assignments of CM2 monomer (gray) and dimer (pink) showing the peaks corresponding to residues F1105-L1148 have identical chemical shifts. All backbone monomer peaks are assigned. Though N1135 does appear in backbone tracing experiments, it does not appear in HSQC spectra. (B) Mapping of the combined chemical shift differences (blue to red) between the monomer and dimer HSQC spectra onto the crystal structure of CM2 (pdb:5MWE). Peaks that were not present in the dimer HSQC spectra were arbitrarily colored as a combined chemical shift difference of 0.1ppm for visualization purposes. (C) Overlay of monomer CM2 (gray) with 200μM PLPMD (pink) and 150μM of CNNLZ (purple) illustrates chemical shift changes, particularly for L1131 and L1137 (inset). (D) Quantitation of the combined chemical shift changes of CNNLZ (purple) compared to monomeric CM2 alone reveals three regions with greatest shift changes. Dashed line indicates a combined chemical shift change of 0.0025ppm. The star indicates residue N1135 that is not visible in the HSQC spectra. Gray shading indicates the residues that are not modeled in the crystal structure (pdb:5MWE). (E) Quantitation of chemical shift changes of PLPMD (pink) compared to monomeric CM2. (F) Combined chemical shift changes of CM2 in the presence of CNNLZ mapped onto the crystal structure of CM2 with CNNLZ (pdb:5MWE). CM2 is colored blue to red in proportion to chemical shift changes. CNNLZ is shown in gray. Inset shows the interface between CM2 and CNNLZ and the proximity of I1130 to L539 and L1137 to L532.</p