| Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae
|
|
Author:
Date:
2018-10-05
[Abstract] Ribonucleoprotein particles (mRNPs) are complexes consisting of mRNAs and RNA-binding proteins (RBPs) which control mRNA transcription localization, turnover, and translation. Some mRNAs within the mRNPs have been shown to undergo degradation or storage. Those transcripts can lack general mRNA elements, like the poly(A) tail or 5’ cap structure, which prevent their identification through the application of widely-used approaches like oligo(dT) purification. Here, we describe a modified cross-linking affinity purification protocol (cCLAP) based on existing cross-linking and immunoprecipitation (CLIP) methods to isolate mRNAs which could be deadenylated, decapped and/or partially degraded in mRNPs, opening the possibility to detect different types of non-coding RNAs (ncRNAs). Once isolated, ...
[摘要] 核糖核蛋白颗粒（mRNP）是由mRNA和RNA结合蛋白（RBP）组成的复合物，其控制mRNA转录定位，转换和翻译。已显示mRNP内的一些mRNA经历降解或储存。那些转录物可能缺乏一般的mRNA元件，如poly（A）尾或5'帽结构，这通过应用广泛使用的方法如oligo（dT）纯化来阻止它们的鉴定。在这里，我们描述了基于现有的交联和免疫沉淀（CLIP）方法的修饰的交联亲和纯化方案（cCLAP），以分离mRNP中可被去腺苷酸化，去除和/或部分降解的mRNA，从而开启了检测不同的可能性。非编码RNA（ncRNA）的类型。分离后，将RNA进行衔接子连接，然后进行下一代测序（NGS）。由于快速有效的交联和淬灭步骤，该方案也适用于瞬时诱导的mRNP颗粒。实例包括由外在应激物触发的处理体（PB）或应力颗粒（SG）。其重现性和广泛应用使该方案成为研究特定RNP的RNA组成的有用且有力的工具。
【背景】mRNP内转录物的表征对于理解细胞转录和转录后过程至关重要。通过交联和免疫沉淀，然后通过RNA-Seq从mRNP颗粒中分离RNA已经成为鉴定mRNA靶标的常用方法（Tagwerker et al。，2006; Hafner et al。，2010; Kishore et al。，2011）。 ...
|
|
|
| An in vitro Transcription/translation System for Detection of Protein Interaction
|
|
Author:
Date:
2016-05-05
[Abstract] Studying protein-protein interaction is crucial to understand the fundamental processes of molecular biology. High-throughput screening, such as immunoprecipitation followed by proteomic analysis, allows for the identification of numerous candidate partners that might interact with a selected protein. However, experimental validation of protein-protein interaction requires conventional cloning and recombinant protein expression/purification, which are complicated and labor-intensive techniques. Here, we demonstrate an efficient experimental pipeline for verifying protein-protein interactions between a bait protein using the example of Odontoglossum ringspot virus (ORSV) capsid protein (CP) and the host CP-binding protein. These candidate CP-binding proteins were identified ...
[摘要] 研究蛋白质 - 蛋白质相互作用对于理解分子生物学的基本过程是至关重要的。高通量筛选，如免疫沉淀，然后蛋白质组分析，允许鉴定可能与选择的蛋白质相互作用的许多候选伙伴。然而，蛋白质 - 蛋白质相互作用的实验验证需要常规克隆和重组蛋白表达/纯化，这是复杂和劳动密集型技术。在这里，我们演示了一个有效的实验管道，用于验证使用Odontoglossum环斑病毒（ORSV）衣壳蛋白（CP）和宿主CP结合蛋白的例子的诱饵蛋白之间的蛋白质 - 蛋白质相互作用。这些候选CP结合蛋白通过高通量蛋白质组学和转录组学方法进行鉴定。使用TOPO克隆策略，将每个候选基因克隆到表达载体中，用于在体外转录/翻译系统的单个步骤中表达His标记的重组蛋白。这种表达的His标记的候选物可以在共免疫沉淀（co-IP）测定中用作CP诱饵蛋白的猎物，以验证它们的物理相互作用。不需要传统的蛋白质表达和纯化，该管道简化了验证过程，并为高通量蛋白质 - 蛋白质相互作用研究提供了解决方案。
|
|
|