| Preparation of Sequencing RNA Libraries through Chemical Cross-linking Coupled to Affinity Purification (cCLAP) in Saccharomyces cerevisiae
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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 ...
[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）。 ...
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| Analysis of in vivo Interaction between RNA Binding Proteins and Their RNA Targets by UV Cross-linking and Immunoprecipitation (CLIP) Method
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Author:
Date:
2017-05-20
[Abstract] RNA metabolism is tightly controlled across different tissues and developmental stages, and its dysregulation is one of the molecular hallmarks of cancer. Through direct binding to specific sequence element(s), RNA binding proteins (RBPs) play a pivotal role in co- and post-transcriptional RNA regulatory events. We have recently demonstrated that, in pancreatic cancer cells, acquisition of a drug resistant (DR)-phenotype relied on upregulation of the polypyrimidine tract binding protein (PTBP1), which in turn is recruited to the pyruvate kinase pre-mRNA and favors splicing of the oncogenic ...
[摘要] RNA代谢在不同的组织和发育阶段被严格控制，其失调是癌症的分子特征之一。 通过直接结合特定的序列元件，RNA结合蛋白（RBP）在共转录和转录后调控事件中起关键作用。 我们最近证实，在胰腺癌细胞中，获得耐药（DR） - 表型取决于多聚嘧啶区结合蛋白（PTBP1）的上调，其又被引入丙酮酸激酶前mRNA并有利于剪接 致癌性PKM2变体。 在这里，我们描述了紫外（UV）光交联和免疫沉淀（CLIP）方法的逐步方案，以确定RBP与贴壁人细胞系中其目标RNA的特定区域的直接结合。
背景 在细胞核中转录时，新生的RNA立即与被称为RNA结合蛋白（RBP）的反式因子立即组装。这些因子直接与RNA分子中特定的顺式调控序列相互作用，从而形成核糖核蛋白（RNP）复合物（Dreyfuss et al。，2002; ...
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| Micrococcal Nuclease (MNase) Assay of Arabidopsis thaliana Nuclei
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Author:
Date:
2013-04-05
[Abstract] Micrococcal nuclease (MNase) is able to produce double-strand breaks within nucleosome linker regions. The efficiency of MNase digestion depends on the degree of chromatin compaction, being more easily digested the regions of less compacted chromatin. The MNase protocol described here can be used to asses changes in the chromatin structure of nuclei extracted from Arabidopsis seedlings.
[摘要] 微球菌核酸酶（MNase）能够在核小体接头区域内产生双链断裂。 MNase消化的效率取决于染色质压实的程度，更容易消化较少压实的染色质的区域。 这里描述的MNase方案可以用于评估从拟南芥幼苗提取的核的染色质结构的变化。
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