Imaging mRNA and protein interactions within neurons
Imaging mRNA and protein interactions within neurons
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DOI：
10.1073/pnas.1621440114
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发表时间：
2017-03-07
影响因子：
11.1
通讯作者：
Singer, Robert H.
中科院分区：
文献类型：
--
作者：
Eliscovich, Carolina;Shenoy, Shailesh M.;Singer, Robert H.
RNA-蛋白质相互作用对于适当的基因表达调控是必不可少的，特别是在具有独特空间限制的神经元中。目前，这些相互作用的生物化学定义，但需要一种方法来评估它们的形态学范围内定量。使用宽场显微镜的双色标签的共定位是推断这些相互作用的方法。然而，由于物镜透镜中的色差，这种方法缺乏确定两个分子是物理接触还是仅仅偶然地在附近的分辨率。在这里，我们开发了一种强大的超级配准方法，将整个图像场的色差校正到10 nm以内，这能够确定两个分子是物理相互作用还是只是随机接近。我们将这种方法应用于单分子FISH结合免疫荧光（smFISH-IF）成像，并确定神经元内mRNA和结合蛋白之间的关联是显著的还是偶然的。我们评估了几种从RNA下拉试验中鉴定的mRNA结合蛋白，以确定其中哪些表现出真正的相互作用。令人惊讶的是，许多已知的mRNA结合蛋白不结合mRNA原位，表明外源相互作用是显着的使用现有技术。这种方法提供了一种能力，以评估与分子相互作用的规模兼容的双色配准。
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-fieldmicroscopy 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.