Arabidopsis RNA immunoprecipitation

RNA-binding proteins are key regulators of plant gene expression. Consistent with this, the Arabidopsis genome encodes many RNA-binding proteins that are genetically required for normal development and for responding to environmental changes. However, the direct RNA targets and RNA processing events that these RNA-binding proteins control are poorly understood. In order to facilitate the functional characterization of RNA-binding proteins, we have applied the RNA immunoprecipitation assay to Arabidopsis. Working with the U2B"–U2 snRNA interaction as a model experimental system, we show that treatment of intact plants with formaldehyde allows immunocapture of U2 snRNA using antibodies that recognize U2B" fused to the generic GFP tag. When coupled with recent developments in whole-genome tiling arrays and high-throughput next-generation sequencing, this combination of procedures and technology has the potential to allow systematic functional analysis of plant RNA-binding proteins.     

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。     

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...     

Proteins that interact with bacterial small RNA regulators

Small regulatory RNAs have been identified in a wide range of organisms, where they modify mRNA stability, translation or protein function. Small RNA regulators (sRNAs) either pair with mRNA targets or modify protein activities. Here we discuss current knowledge of the various proteins that interact with RNA regulators and review the physiologic implications of sRNA-protein complexes in DNA, RNA and protein metabolism, as well as in RNA and protein quality control in prokaryotes. Proteins that interact with the sRNAs can possess catalytic activity, induce conformational changes of the sRNA, or be sequestered by the sRNA to prevent the action of the protein.     

Crystal Structure of the Escherichia coli DExH-Box NTPase HrpB

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A Nucleolar PUF RNA-binding Protein with Specificity for a Unique RNA Sequence

PUF proteins are a conserved group of sequence specific RNA-binding proteins that bind to RNA in a modular fashion. The RNA-binding domain of PUF proteins typically consists of eight clustered Puf repeats. Plant genomes code for large families of PUF proteins that show significant variability in their predicted Puf repeat number, organization, and amino acid sequence. Here we sought to determine whether the observed variability in the RNA-binding domains of four plant PUFs results in a preference for nonclassical PUF RNA target sequences. We report the identification of a novel RNA binding sequence for a nucleolar Arabidopsis PUF protein that contains an atypical RNA-binding domain. The Arabidopsis PUM23 (APUM23) binding sequence was 10 nucleotides in length, contained a centrally located UUGA core element, and had a preferred cytosine at nucleotide position 8. These RNA sequence characteristics differ from those of other PUF proteins, because all natural PUFs studied to date bind to RNAs that contain a conserved UGU sequence at their 5′ end and lack specificity for cytosine. Gel mobility shift assays validated the identity of the APUM23 binding sequence and supported the location of 3 of the 10 predicted Puf repeats in APUM23, including the cytosine-binding repeat. The preferred 10-nucleotide sequence bound by APUM23 is present within the 18S rRNA sequence, supporting the known role of APUM23 in 18S rRNA maturation. This work also reveals that APUM23, an ortholog of yeast Nop9, could provide an advanced structural backbone for Puf repeat engineering and target-specific regulation of cellular RNAs.     

RNA-binding proteins in neurological diseases

Emerging studies support that RNA-binding proteins(RBPs)play critical roles in human biology and pathogenesis.RBPs are essential players in RNA processing and metabolism,including pre-mRNA splicing,polyadenylation,transport,surveillance,mRNA localization,mRNA stability control,translational control and editing of various types of RNAs.Aberrant expression of and mutations in RBP genes affect various steps of RNA processing,altering target gene function.RBPs have been associated with various diseases,including neurological diseases.Here,we mainly focus on selected RNA-binding proteins including Nova-1/Nova-2,HuR/HuB/HuC/HuD,TDP-43,Fus,Rbfox1/Rbfox2,QKI and FMRP,discussing their function and roles in human diseases.     

PPR蛋白调控植物细胞器RNA加工的分子功能

35肽重复（pentatricopeptide repeat, PPR）蛋白是2000年发现的一类由多个重复单位串联而成的核编码蛋白质。PPR蛋白广泛存在于真核生物中,在陆生植物中尤为普遍。PPR蛋白大多定位于线粒体或叶绿体。多项研究表明,PPR蛋 白为序列特异性RNA结合蛋白质,在细胞器RNA编辑、剪接、稳定、切割及翻译等转录后加工过程发挥重要作用。PPR蛋白功能缺陷导致植物生长发育异常,甚至胚胎致死。本文主要就PPR蛋白功能及作用机制进行综述,并对尚待解决的问题及研究前景加以探讨,以期为PPR蛋白的深入研究提供思路。     

miR-21: a small multi-faceted RNA

More than 1000 microRNAs (miRNAs) are expressed in human cells, some tissue or cell type specific, others considered as house-keeping molecules. Functions and direct mRNA targets for some miRNAs have been relatively well studied over the last years. Every miRNA potentially regulates the expression of numerous protein-coding genes (tens to hundreds), but it has become increasingly clear that not all miRNAs are equally important; diverse high-throughput screenings of various systems have identified a limited number of key functional miRNAs over and over again. Particular miRNAs emerge as principal regulators that control major cell functions in various physiological and pathophysiological settings. Since its identification 3 years ago as the miRNA most commonly and strongly up-regulated in human brain tumour glioblastoma [ 1 ], miR-21 has attracted the attention of researchers in various fields, such as development, oncology, stem cell biology and aging, becoming one of the most studied miRNAs, along with let-7, miR-17–92 cluster ('oncomir-1'), miR-155 and a few others. However, an miR-21 knockout mouse has not yet been generated, and the data about miR-21 functions in normal cells are still very limited. In this review, we summarise the current knowledge of miR-21 functions in human disease, with an emphasis on its regulation, oncogenic role, targets in human cancers, potential as a disease biomarker and novel therapeutic target in oncology.