Imaging mRNA and protein interactions within neurons

Abstract

RNA–protein interactions are essential for proper gene expression regulation, particularly in neurons with unique spatial constraints. Currently, these interactions are defined biochemically, but a method is needed to evaluate them quantitatively within morphological context. Colocalization of two-color labels using wide-field microscopy is a method to infer these interactions. However, because of chromatic aberrations in the objective lens, this approach lacks the resolution to determine whether two molecules are physically in contact or simply nearby by chance. Here, we developed a robust super registration methodology that corrected the chromatic aberration across the entire image field to within 10 nm, which is capable of determining whether two molecules are physically interacting or simply in proximity by random chance. We applied this approach to image single-molecule FISH in combination with immunofluorescence (smFISH-IF) and determined whether the association between an mRNA and binding protein(s) within a neuron was significant or accidental. We evaluated several mRNA-binding proteins identified from RNA pulldown assays to determine which of these exhibit bona fide interactions. Surprisingly, many known mRNA-binding proteins did not bind the mRNA in situ, indicating that adventitious interactions are significant using existing technology. This method provides an ability to evaluate two-color registration compatible with the scale of molecular interactions.

β-actin mRNA interactome mapping by proximity biotinylation

“…While ORF probes were used to detect β-actin mRNA in WT MEFs, MBS probes against the MS2 loop sequences were used to detect the β-actin mRNA in β-actin-MBS MEFs. The association between β-actin mRNA and the proteins was determined by super registration microscopy (47). Briefly, we corrected the images for chromatic aberration and mechanical shifts in Cy3 and Cy5 channels by using broad spectra fluorescent microsphere beads (S1 Appendix fig.…”

“…For the live cell imaging we used a dual band GFP/mCherry filter set (F56-319, AHF). For imaging of fixed cells, the microscope set up is the same as in Eliscovich et al (47).…”

“…Imaging Analysis. Single molecule localization was determined by FISH-QUANT (63) software; and super registration analysis was performed as described in Eliscovich et al (47) with existing software packages and custom algorithm programs written in MATLAB (MathWorks). For polarization index calculation, after taking the max projections from all the z-stacks, polarization and dispersion index measurement were measured as mentioned before (9) with existing software package written in MATLAB (MathWorks).…”

On the interaction of ribosomal protein RPL22e with microtubules

“…However, RPL22e is not required for capdependent translation (Lavergne et al, 1988). In addition to rRNA, RPL22e binds to some mRNAs including its own paralogue RPL22le mRNA (O'Leary et al, 2013;Wiest and Kennedy, 2013), β-actin mRNA (Eliskovich et al, 2017), the telomerase RNA component (Le et al, 2000), and some viral mRNA (Wood et al, 2001;Fok et al, 2006). The dysfunction of both RPL22e and RpL22le genes is lethal to mouse embryos (O'Leary et al, 2013).…”

Single mRNP analysis by super-resolution microscopy and fluorescence correlation spectroscopy reveals that small mRNP granules represent mRNA singletons

“…Insulin-like growth factor 2 (IGF2) mRNA-binding protein 1 (IMP1, IGF2BP1) belongs to a conserved family of heterochronic mRNA-binding proteins (IMP1, IMP2, and IMP3) (Hansen et al, 2004;Nielsen et al, 2001;Nielsen et al, 1999;Yaniv and Yisraeli, 2002). Together with the cytoplasmic mRNA-binding protein Y-box binding protein 1 (YBX1), IMPs form typical cytoplasmic RNP granules (Eliscovich et al, 2017) (Figure 1).…”