RNA结合蛋白与RNA互作鉴定技术

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,iCLIP),TRIBE (targets of RNA-binding protein identified by editing),RNA 标记,相互作用组捕获(interactome capture,IC) 和SerIC (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。     

Protein Interaction Between p53 and △113p53 Is Required for the Anti-Apoptotic Function of △113p53

Zebrafish △113p53, an N-terminal truncated p53 isoform, is a p53-target gene that antagonises p53-mediated apoptotic activity. Interestingly, △113p53 does not act on p53 in a dominant-negative manner, but rather interferes with the p53 function by differentially modulating p53-target gene expression to protect cells from apoptosis. Previous studies showed that over-expressed △113p53 and p53 proteins formed a complex. However, it is not known whether endogenous p53 and △113p53 proteins also interact with each other, and if this interaction is required for △113p53 to inhibit the apoptotic activity of full-length p53. In this study, we used two available zebrafish p53 antibodies to address these questions. One, Zfp53-N, only recognises full-length p53, whereas the other, Zfp53-A7C10, detects both full-length p53 and △113p53. Using Zfp53-N for immunoprecipitation and Zfp53-A7C 10 for detection, we demonstrated that endogenous △113p53 and full-length p53 induced by a DNA-damaging drug formed a complex in vivo. Furthermore, of the six △113p53 mutants we generated with different point mutations in the oligomerisation domain, two failed to interact with p53 and lost the ability to modulate p53-target gene expression and inhibit p53-induced cell apoptosis. However, those △113p53 mutants that could interact with p53 retained the ability to antagonise the apoptotic activity of p53. Therefore, our data demonstrated that protein--protein interaction between △113p53 and p53 is essential for the anti-apoptotic function of △113p53. In addition, the two △113p53 mutants that failed to interact with p53 are also useful for the study of the mechanisms of other functions of △113p53.     

A Yeast BiFC-seq Method for Genome-wide Interactome Mapping

Genome-wide physical protein±protein interaction(PPI) mapping remains a major challenge for current technologies. Here, we reported a high-efficiency BiFC-seq method, yeastenhanced green fluorescent protein-based bimolecular fluorescence complementation(y EGFPBiFC) coupled with next-generation DNA sequencing, for interactome mapping. We first applied y EGFP-BiFC method to systematically investigate an intraviral network of the Ebola virus.Two-thirds(9/14) of known interactions of EBOV were recap...     

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.     

RBPBind: Quantitative Prediction of Protein-RNA Interactions

There are hundreds of RNA binding proteins in the human genome alone and their interactions with messenger and other RNAs in a cell regulate every step in an RNA's life cycle. To understand this interplay of proteins and RNA it is important to be able to know which protein binds which RNA how strongly and where. Here, we introduce RBPBind, a web-based tool for the quantitative prediction of the interaction of single-stranded RNA binding proteins with target RNAs that fully takes into account the effect of RNA secondary structure on binding affinity. Given a user-specified RNA and a protein selected from a set of several RNA-binding proteins, RBPBind computes their binding curve and effective binding constant. The server also computes the probability that, at a given protein concentration, a protein molecule will bind to any particular nucleotide along the RNA. The sequence specificity of the protein-RNA interaction is parameterized from public RNAcompete experiments and integrated into the recursions of the Vienna RNA package to simultaneously take into account protein binding and RNA secondary structure. We validate our approach by comparison to experimentally determined binding affinities of the HuR protein for several RNAs of different sequence contexts from the literature, showing that integration of raw sequence affinities into RNA secondary structure prediction significantly improves the agreement between computationally predicted and experimentally measured binding affinities. Our resource thus provides a quick and easy way to obtain reliable predicted binding affinities and locations for single-stranded RNA binding proteins based on RNA sequence alone.     

Molecular Insights into the Coding Region Determinant-binding Protein-RNA Interaction through Site-directed Mutagenesis in the Heterogeneous Nuclear Ribonucleoprotein-K-homology Domains

The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function.     

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。     

Genome-wide Identification and Quantitative Analysis of Cleaved tRNA Fragments Induced by Cellular Stress

Certain stress conditions can induce cleavage of tRNAs around the anticodon loop via the use of the ribonuclease angiogenin. The cellular factors that regulate tRNA cleavage are not well known. In this study we used normal and eIF2α phosphorylation-deficient mouse embryonic fibroblasts and applied a microarray-based methodology to identify and compare tRNA cleavage patterns in response to hypertonic stress, oxidative stress (arsenite), and treatment with recombinant angiogenin. In all three scenarios mouse embryonic fibroblasts deficient in eIF2α phosphorylation showed a higher accumulation of tRNA fragments including those derived from initiator-tRNA^Met. We have shown that tRNA cleavage is regulated by the availability of angiogenin, its substrate (tRNA), the levels of the angiogenin inhibitor RNH1, and the rates of protein synthesis. These conclusions are supported by the following findings: (i) exogenous treatment with angiogenin or knockdown of RNH1 increased tRNA cleavage; (ii) tRNA fragment accumulation was higher during oxidative stress than hypertonic stress, in agreement with a dramatic decrease of RNH1 levels during oxidative stress; and (iii) a positive correlation was observed between angiogenin-mediated tRNA cleavage and global protein synthesis rates. Identification of the stress-specific tRNA cleavage mechanisms and patterns will provide insights into the role of tRNA fragments in signaling pathways and stress-related disorders.     

The next wave of interactomics: Mapping the SLiM-based interactions of the intrinsically disordered proteome

Short linear motifs (SLiMs) are a unique and ubiquitous class of protein interaction modules that perform key regulatory functions and drive dynamic complex formation. For decades, interactions mediated by SLiMs have accumulated through detailed low-throughput experiments. Recent methodological advances have opened this previously underexplored area of the human interactome to high-throughput protein–protein interaction discovery. In this article, we discuss that SLiM-based interactions represent a significant blind spot in the current interactomics data, introduce the key methods that are illuminating the elusive SLiM-mediated interactome of the human cell on a large scale, and discuss the implications for the field.     

Identification of a conserved interface between PUF and CPEB proteins

Members of the PUF (Pumilio and FBF) and CPEB (cytoplasmic polyadenylation element-binding) protein families collaborate to regulate mRNA expression throughout eukaryotes. Here, we focus on the physical interactions between members of these two families, concentrating on Caenorhabditis elegans FBF-2 and CPB-1. To localize the site of interaction on FBF-2, we identified conserved amino acids within C. elegans PUF proteins. Deletion of an extended loop containing several conserved residues abolished binding to CPB-1. We analyzed alanine substitutions at 13 individual amino acids in FBF-2, each identified via its conservation. Multiple single point mutations disrupted binding to CPB-1 but not to RNA. Position Tyr-479 was particularly critical as multiple substitutions to other amino acids at this position did not restore binding. The complex of FBF-2 and CPB-1 repressed translation of an mRNA containing an FBF binding element. Repression required both proteins and was disrupted by FBF-2 alleles that failed to bind CPB-1 or RNA. The equivalent loop in human PUM2 is required for binding to human CPEB3 in vitro, although the primary sequences of the human and C. elegans PUF proteins have diverged in that region. Our findings define a key region in PUF/CPEB interactions and imply a conserved platform through which PUF proteins interact with their protein partners.