| 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 PKM2 variant. Herein, we describe a step-by-step protocol of the ultraviolet (UV) light cross-linking and immunoprecipitation (CLIP) method to determine the direct binding of an RBP to specific regions ...
[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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| Determination of Cellular Phosphatidylinositol-3-phosphate (PI3P) Levels Using a Fluorescently Labelled Selective PI3P Binding Domain (PX)
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Author:
Date:
2016-08-20
[Abstract] The lipid Phosphatidylinositol-3-phosphate [PtdIns3P or PI(3)P] plays many membrane trafficking roles and is primarily produced by the Class III PI3K, VPS34. Determining the level of cellular PI(3)P however can be complex. Extraction of cellular lipids by methanol/chloroform can struggle to separate and identify distinct phospholipid species. Alternately mass spectrometry may be utilised but this requires significant set up of specialised equipment and time to utilise.
Use of a PI(3)P-binding-specific recombinant protein domain is a quick method for ascertaining cellular PI(3)P levels and can also allow visualisation of sub-cellular localisation. The PX domain of p40phox (herein referred to as PX) is very specific for PI(3)P over other phospholipid species (Kanai et al., ...
[摘要] 脂质磷脂酰肌醇-3-磷酸[PtdIns3P或PI（3）P]扮演许多膜贩运角色，主要由III类PI3K，VPS34产生。然而，确定细胞PI（3）P的水平可以是复杂的。通过甲醇/氯仿提取细胞脂质可能难以分离和鉴定不同的磷脂种类。或者可以使用质谱法，但这需要大量设置专门的设备和时间来利用。
使用PI（3）P结合特异性重组蛋白结构域是用于确定细胞PI（3）P水平的快速方法，并且还可以允许亚细胞定位的可视化。 p40phox的PX结构域（本文称为PX）对PI（3）P比其它磷脂种类非常特异（Kanai等人，2001）。然而，在细胞中直接表达PX可能是有问题的，因为它将以显性负性方式作用，以比内源蛋白更大的亲和力结合和螯合PI（3）P，从而干扰细胞途径和PI（3）P水平。因此，使用荧光标记的PX后细胞固定更合适，因为它能突出显示PI（3）富集结构，没有扰乱系统的风险。
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| Determination of VPS34/PIK3C3 Activity in vitro Utilising 32P-γATP
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Author:
Date:
2016-08-20
[Abstract] VPS34 is the only class III phosphatidylinositol-3-kinase (PI3K) in mammalian cells and produces the vast majority of cellular phosphatidylinositol-3-phosphate [PI(3)P]. PI(3)P is a key signalling lipid that plays many membrane trafficking roles in processes such as endocytosis and autophagy. VPS34 is a key cellular regulator, loss of function can have catastrophic effects and is embryonic lethal (Zhou et al., 2011). The levels of cellular PI(3)P can be determined by fluorescent staining techniques and can be used to monitor effects upon VPS34 activity, however it is important to verify that any changes are mediated by VPS34, particularly as alternate pathways of PI(3)P production are possible such as via class II PI3Ks (Devereaux et al., 2013). Assaying VPS34 activity ...
[摘要] VPS34是哺乳动物细胞中唯一的III类磷脂酰肌醇-3-激酶（PI3K），并且产生绝大多数细胞磷脂酰肌醇-3-磷酸[PI（3）P]。 PI（3）P是一个关键的信号脂质，在诸如内吞作用和自噬的过程中起许多膜运输的作用。 VPS34是关键的细胞调节剂，功能丧失可具有灾难性作用并且是胚胎致死的（Zhou等人，2011）。 细胞PI（3）P的水平可以通过荧光染色技术确定，并且可以用于监测对VPS34活性的影响，然而重要的是验证任何变化由VPS34介导，特别是作为PI（3）的替代途径， P生产是可能的，例如通过II类PI3K（Devereaux等人，2013）。 直接在体外测定VPS34活性可以是描绘特定刺激的作用的关键阶段。
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