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Imaging mRNA and protein interactions within neurons
#MMPMID28223507
Eliscovich C
; Shenoy SM
; Singer RH
Proc Natl Acad Sci U S A
2017[Mar]; 114
(10
): E1875-E1884
PMID28223507
show ga
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.
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