关于RNA定位的信息对于理解生理和病理过程至关重要,如基因表达,细胞重编程,宿主-病原体相互作用,以及在无膜或膜结合的细胞器和细胞外囊泡水平上涉及RNA交易的信号通路。在许多情况下,评估RNA定位的拓扑结构非常重要,即,将包封在感兴趣的细胞器内的转录物与仅附着在其表面上的转录物区分开。这允许确定哪些RNA可以,原则上,参与局部分子相互作用,并被膜或其他物理屏障阻止相互作用。最广泛使用的询问RNA定位拓扑的技术是基于用RNA酶处理分离的细胞器,随后通过northern印迹鉴定存活的转录本。qRT-PCR,或RNA-seq。然而,这种方法会产生不连贯的结果和许多假阳性。这里,我们描述了与深度测序(CoLoC-seq)耦合的受控污染水平,一种克服这些陷阱的更精细的亚细胞转录组学方法。CoLoC-seq通过纯化感兴趣的细胞器开始。然后将它们保持完整或裂解,并进行RNase浓度梯度以产生独特的RNA降解动力学谱,可以通过Northern印迹或RNA-seq进行监测。通过简单的数学建模,CoLoC-seq将真正的膜包封转录物与任何丰度的可降解和不可降解污染物区分开。该方法已在HEK293细胞的线粒体中实施,它优于其他亚细胞转录组学方法。它适用于其他膜结合的细胞器,例如,质体,囊泡系统的单膜细胞器,细胞外囊泡,或病毒颗粒。主要特征•在人线粒体上测试;可能适用于细胞培养,非模式生物,细胞外囊泡,包膜病毒,组织;不需要遗传操作或高纯度的细胞器。•在人类细胞的情况下,起始材料的所需量是~2,500cm2的80%汇合细胞(或~3X108HEK293细胞)。•CoLoC-seq实现了一种特殊的RNA-seq策略来选择性地捕获完整的转录本,这需要产生5'-羟基和2'/3'-磷酸末端的RNase(例如,RNaseA,RNaseI)。•依赖于具有可定制指数函数的非线性回归软件。
Information on RNA localisation is essential for understanding physiological and pathological processes, such as gene expression, cell reprogramming, host-pathogen interactions, and signalling pathways involving RNA transactions at the level of membrane-less or membrane-bounded organelles and extracellular vesicles. In many cases, it is important to assess the topology of RNA localisation, i.e., to distinguish the transcripts encapsulated within an organelle of interest from those merely attached to its surface. This allows establishing which RNAs can, in principle, engage in local molecular interactions and which are prevented from interacting by membranes or other physical barriers. The most widely used techniques interrogating RNA localisation topology are based on the treatment of isolated organelles with RNases with subsequent identification of the surviving transcripts by northern blotting, qRT-PCR, or RNA-seq. However, this approach produces incoherent results and many false positives. Here, we describe Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq), a more refined subcellular transcriptomics approach that overcomes these pitfalls. CoLoC-seq starts by the purification of organelles of interest. They are then either left intact or lysed and subjected to a gradient of RNase concentrations to produce unique RNA degradation dynamics profiles, which can be monitored by northern blotting or RNA-seq. Through straightforward mathematical modelling, CoLoC-seq distinguishes true membrane-enveloped transcripts from degradable and non-degradable contaminants of any abundance. The method has been implemented in the mitochondria of HEK293 cells, where it outperformed alternative subcellular transcriptomics approaches. It is applicable to other membrane-bounded organelles, e.g., plastids, single-membrane organelles of the vesicular system, extracellular vesicles, or viral particles. Key features • Tested on human mitochondria; potentially applicable to cell cultures, non-model organisms, extracellular vesicles, enveloped viruses, tissues; does not require genetic manipulations or highly pure organelles. • In the case of human cells, the required amount of starting material is ~2,500 cm2 of 80% confluent cells (or ~3 × 108 HEK293 cells). • CoLoC-seq implements a special RNA-seq strategy to selectively capture intact transcripts, which requires RNases generating 5\'-hydroxyl and 2\'/3\'-phosphate termini (e.g., RNase A, RNase I). • Relies on nonlinear regression software with customisable exponential functions.