RNA结合蛋白与RNA相互作用鉴定技术

RNA结合蛋白(RNA binding proteins,RBPs)通过与RNA相互作用,广泛参与到RNA的剪切、转运、编辑、胞内定位及翻译调控等过程中。RNA领域尤其是非编码RNA(non-coding RNA,ncRNA)研究的快速发展,催生了多种RBPs-RNAs相互作用鉴定技术。这些技术反之又推动了RNA领域的研究进程。本文对紫外交联免疫沉淀(ultraviolet crosslinking and immunoprecipitation,CLIP),CLIP cDNA文库高通量测序(high-throughput sequencing of CLIP cDNA library,HITS-CLIP),光活化核苷增强的CLIP(photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation,PAR-CLIP),单核苷酸分离CLIP(individual nucleotide resolution CLIP,i CLIP),TRIBE(targets of RNA-binding protein identified by editing),RNA标记,相互作用组捕获(interactome capture,IC)和Ser IC(serial RNA interactome capture)等RBPs-RNAs相互作用鉴定技术的基本原理和优缺点以及应用进行综述。     

蛋白质-RNA相互作用鉴定技术研究进展

RNA结合蛋白(RNA binding protein, RBP)是基因表达调控的关键因子，参与包括蛋白质复合物的协调与稳定、RNA的加工与成熟以及mRNA的转运、稳定、翻译和降解等重要的细胞生物学过程。而RBP和RNA之间的相互作用可以在它们各自的生物学过程中起到重要作用。因此，快速、准确检测RBP-RNA相互作用的技术对研究RBP和RNA的功能至关重要。对近些年发展起来的RNA纯化的染色质分离（chromatin isolation by RNA purification，ChIRP）、RNA靶标的捕获杂交分析（capture hybridization analysis of RNA targets，CHART）、三分子荧光互补技术（trimolecular fluorescence complementation，TriFC）、RNA免疫共沉淀(RNA immunoprecipitation, RIP)、紫外交联免疫沉淀(UV-crosslinking and immunoprecipitation，CLIP)、RNA Pull-down和RNA电泳迁移分析等主要RBP-RNA相互作用鉴定技术的基本原理和优缺点以及应用进行了综述，旨在为新型技术的发现提供新的思路。     

Genome-wide Mapping of Cellular Protein–RNA Interactions Enabled by Chemical Crosslinking

RNA–protein interactions influence many biological processes. Identifying the binding sites of RNA-binding proteins（RBPs） remains one of the most fundamental and important challenges to the studies of such interactions. Capturing RNA and RBPs via chemical crosslinking allows stringent purification procedures that significantly remove the non-specific RNA and protein interactions. Two major types of chemical crosslinking strategies have been developed to date, i.e., UV-enabled crosslinking and enzymatic mechanism-based covalent capture. In this review, we compare such strategies and their current applications, with an emphasis on the technologies themselves rather than the biology that has been revealed. We hope such methods could benefit broader audience and also urge for the development of new methods to study RNA RBP interactions.     

紫外交联免疫沉淀技术原理及其应用

紫外交联免疫沉淀（UV cross-linking immunoprecipitation,CLIP）技术最初建立于2003年。通过紫外交联、免疫沉淀、逆转录及后续的高通量测序等步骤,可在全转录组范围鉴定特定RNA结合蛋白（RNA-binding proteins,RBP）的靶标RNA序列和结合位点。在近20年的应用过程中,该技术被不断改进和完善,可操作性、实验结果的准确性都有所提升,技术的应用范围也有所拓展。本文对CLIP技术的基本原理、实验方法、实际应用进行介绍,着重比较几种主流CLIP技术的异同,并对如何选择具体的技术路线提出建议。     

GoldCLIP: Gel-omitted Ligation-dependent CLIP

Protein–RNA interaction networks are essential to understand gene regulation control.Identifying binding sites of RNA-binding proteins(RBPs) by the UV-crosslinking and immunoprecipitation(CLIP) represents one of the most powerful methods to map protein–RNA interactions in vivo. However, the traditional CLIP protocol is technically challenging, which requires radioactive labeling and suffers from material loss during PAGE-membrane transfer procedures. Here we introduce a super-efficient CLIP method(Gold CLIP) that omits all gel purification steps. This nonisotopic method allows us to perform highly reproducible CLIP experiments with polypyrimidine tract-binding protein(PTB), a classical RBP in human cell lines. In principle, our method guarantees sequencing library constructions, providing the protein of interest can be successfully crosslinked to RNAs in living cells. Gold CLIP is readily applicable to diverse proteins to uncover their endogenous RNA targets.     

RNA结合蛋白在多能干细胞命运转变中的研究进展

RNA结合蛋白(RNA binding proteins,RBPs)是一类通过其RNA结合结构域与RNA相互作用的蛋白质,在细胞内发挥着非常重要的作用。RBPs参与从RNA代谢(包括RNA的可变剪接、稳定性、翻译)到表观遗传修饰等多种调控途径。已有大量文献报道转录因子、表观遗传修饰和细胞外信号通路参与调控干细胞的多能性维持、分化和体细胞重编程,但对于RBPs在细胞命运转变中作用的研究报道甚少。该文主要综述了RBPs通过调控RNA的可变剪接、mRNA稳定性、翻译水平、microRNA代谢及组蛋白修饰进而调控干细胞多能性维持和体细胞重编程。     

紫外交联合并串联亲和纯化高效鉴定蛋白复合物组分的RNA结合活性

RNA结合蛋白在RNA的生成与代谢中发挥着重要作用.我们在近年报道的PAR-CLIP(photoactivatableribonucleoside-enhanced crosslinking and immunoprecipitation)技术的基础上建立了一套快速、有效鉴定RNA结合蛋白的实验方法:以串联亲和纯化替代一步免疫沉淀获得高纯度蛋白-RNA复合物;将Sypro Ruby蛋白染色与RNA放射自显影相结合判断复合物中哪种或哪些组分为RNA结合蛋白,该方法命名为紫外交联合并的串联亲和纯化(cross-linkingand tandem affinity purification,CLiTAP).运用该方法对布氏锥虫的三种锌指蛋白ZC3H7、ZC3H34和ZC3H5进行分析,发现ZC3H7作为帽结合蛋白复合物的核心组分具有很强的RNA结合能力;ZC3H34结合RNA能力较弱,但其互作蛋白具有强的RNA结合活性;相比之下,ZC3H5及其复合物组分皆无RNA结合活性.这些结果表明,CLiTAP与蛋白质鉴定方法相结合,能够有效鉴定靶蛋白复合物中的RNA结合蛋白种类,也为进一步定位RNA结合位点、研究RNA结合蛋白的结构及作用机制奠定了基础.     

RNA结合蛋白与巨噬细胞炎症因子表达

RNA结合蛋白(RNA-binding proteins, RBPs)是转录后基因表达的关键调控因子,参与剪接、出核、翻译和稳定性等RNA代谢调控。RBPs表达或功能异常可导致炎症性疾病、代谢性疾病以及神经系统疾病等多种疾病的发生发展。炎症是机体对外界刺激及损伤的防御性免疫反应。巨噬细胞作为机体重要的免疫细胞,通过快速响应刺激并且释放大量炎症因子,进而调控炎症反应。巨噬细胞中炎症因子的表达受到转录以及转录后水平的调控。其中,RBPs参与大量RNA的转录后调控过程。研究发现,一方面,RBPs直接结合炎症因子mRNA中的顺式作用元件,参与其mRNA稳定性和翻译等过程,例如TTP(tristetraprolin);另一方面,某些RBPs通过参与炎症信号通路中一些关键基因mRNA的稳定性、翻译或选择性剪接调控,进而间接影响炎症因子表达及分泌。例如,剪接因子3A亚基1(splicing factor 3A subunit 1, SF3A1)。本文主要总结RBPs在mRNA稳定性、翻译和选择性剪接不同转录后水平调控巨噬细胞炎症因子表达的作用机制。这些RBPs从不同的层面直接或者间接参与调控炎症因子...     

RNA结合蛋白Sam68及其功能

细胞通过基因表达调控来应对外界刺激,其中影响mRNA稳定性及翻译效率的转录后调控发挥重要作用。RNA结合蛋白(RNA binding proteins, RBPs)是介导转录后调控的重要分子,Sam68（SRC associated in mitosis of 68 kD）是集信号转导特性与RNA激活功能于一身的RNA结合蛋白,参与转录、可变剪接及核输出等mRNA 的代谢过程,且Sam68可通过信号通路参与细胞应答、细胞周期调控和疾病发生等。最新研究表明,Sam68可通过非编码RNAs(noncoding RNA, ncRNAs)参与表观遗传、转录与转录后调控。本文在介绍Sam68结构和转录后修饰的基础上,着重讨论Sam68在信号转导、可变剪接、ncRNAs代谢、疾病发生等方面的最新研究进展。     

Genome-wide identification of protein binding sites on RNAs in mammalian cells

RNA-binding proteins (RBPs) are proteins that bind to the RNA and participate in forming ribonucleoprotein complexes. They have crucial roles in various biological processes such as RNA splicing, editing, transport, maintenance, degradation, intracellular localization and translation. The RBPs bind RNA with different RNA-sequence specificities and affinities, thus, identification of protein binding sites on RNAs (R-PBSs) will deeper our understanding of RNA-protein interactions. Currently, high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP, also known as CLIP-Seq) is one of the most powerful methods to map RNA-protein binding sites or RNA modification sites. However, this method is only used for identification of single known RBPs and antibodies for RBPs are required. Here we developed a novel method, called capture of protein binding sites on RNAs (RPBS-Cap) to identify genome-wide protein binding sites on RNAs without using antibodies. Double click strategy is used for the RPBS-Cap assay. Proteins and RNAs are UV-crosslinked in vivo first, then the proteins are crosslinked to the magnetic beads. The RNA elements associated with proteins are captured, reverse transcribed and sequenced. Our approach has potential applications for studying genome-wide RNA-protein interactions.     

TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites

TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5′ ends of pan-edited RNAs than at their 3′ ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3′ to 5′ progression of editing. Detailed analyses of edited RNAs from wild-type and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3′ ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA–RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3′ to 5′ progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs.