Bustos-Sanmamed Pilar, Laffont Carole, Frugier Florian, Lelandais-Brière Christine, Crespi Martin
Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.
Methods Mol Biol. 2013;959:317-22. doi: 10.1007/978-1-62703-221-6_21.
Proteins are distributed in different cellular compartments. Our group studies the role of non-coding RNAs and associated RNPs in the development and stress response in legumes. Ribonucleoproteins (RNPs) are RNA-protein complexes that play different roles in many cellular processes. Long and small non-coding RNAs determine the specificity of action of several RNPs as the RNA Induced Silencing Complex (RISC), or affect mRNA translation, splicing and stability by interacting with other RNPs such as P-bodies, spliceosome or polysomes. Together with small and long RNAs (Chapter 20), the precise localization of the associated RNPs or the translational products regulated by small RNAs (ie target proteins regulated by miRNAs, or translationally-regulated products) by immunocytochemistry could bring novel insights into these regulatory processes. The protocol described is currently used for detection of RNP associated proteins in nodules and roots of Medicago truncatula but could be extended to any other protein. The critical points, as the choice of the antibody and the fixation and permeabilization steps, that allow preservation of tissue and cell integrity and increase the accessibility to epitopes, will be discussed.
蛋白质分布于不同的细胞区室。我们的研究小组研究非编码RNA和相关核糖核蛋白在豆科植物发育和应激反应中的作用。核糖核蛋白（RNP）是RNA-蛋白质复合物，在许多细胞过程中发挥不同作用。长链和小非编码RNA决定了几种核糖核蛋白（如RNA诱导沉默复合体（RISC））作用的特异性，或通过与其他核糖核蛋白（如P小体、剪接体或多核糖体）相互作用来影响mRNA的翻译、剪接和稳定性。与小RNA和长链RNA一起（第20章），通过免疫细胞化学对相关核糖核蛋白或由小RNA调节的翻译产物（即由miRNA调节的靶蛋白或翻译调节产物）进行精确定位，可能会为这些调节过程带来新的见解。所描述的方案目前用于检测蒺藜苜蓿根瘤和根中与RNP相关的蛋白质，但也可扩展到任何其他蛋白质。我们将讨论一些关键点，如抗体的选择以及固定和通透步骤，这些步骤能够保持组织和细胞的完整性，并增加抗原表位的可及性。
