长链非编码RNA在癌症和心血管疾病中的作用

长链非编码RNA（long non-coding RNA, lneRNA）与癌症和心血管疾病的发生发展密切相关。lncRNA可通过与转录因子相互作用,或参与染色质的表观遗传学修饰,从转录水平调控疾病相关基因的转录效率;或通过调节靶mRNA的稳定性,从转录后水平调控疾病相关基因的翻译效率。本文就lncRNA的来源、作用机制及其在癌症和心血管疾病中的作用的研究进展做一综述。     

miRNA，siRNA，saRNA与基因表达调控

近年来的研究发现,生物体内存在着大量的非编码RNA（non．codingRNAs,ncRNA）,它们在染色质修饰、基因转录、RNA剪接和mRNA翻译等多种水平上参与了基因表达的调控。ncRNA中的小分子RNA如miRNA能够识别特定的目标mRNA,通过与mRNAs3’非翻译区结合,影响mRNA转录及蛋白质翻译;siRNA是RNA干扰的引发物,能够导致与dsRNA同源的mRNA降解,进而抑制相应基因表达;saRNA是目前最新发现的一种靶向目的基因启动子区的在转录水平激活目的基因表达的dsRNA。miRNA、siRNA和saRNA在生成机制、作用途径等方面关系密切,既区别又相互联系,小分子RNA的研究将是今后分子生物学的研究热点之一。     

拟南芥EBP1蛋白与RNA相互作用的初步研究

基因表达调控是现代分子生物学研究的热点话题,随着研究手段的不断成熟,其RNA转录后调控已成为备受关注的领域。RNA结合蛋白(RBPs)是转录后调控的关键因子,参与RNA可变剪切、RNA稳定、翻译等多个过程的调控。ErbB3绑定蛋白(EBP1)是结构功能高度保守的RNA结合蛋白,可与rRNA、tRNA、mRNA等多种RNA结合,参与调控核糖体生物生成、蛋白质翻译多个过程,但目前已报道的靶RNA甚少,对靶RNA的作用机制目前仍不清楚。通过生物信息学分析发现拟南芥EBP1结合RNA的保守结构域,并在体外可直接结合"GUCUCUCACUGCGACGGCUU"序列;通过RNA免疫共沉淀实验找到了3个靶mRNA(AT1G24792、AT3G25211、AT3G24320);结合核糖体提取及虫草素处理实验发现EBP1可显著调控特定靶mRNA的稳定性及其翻译速率。研究结果不仅证实拟南芥EBP1具有结合RNA的功能,还显示其参与调控靶RNA的转录后事件,为进一步研究EBP1在转录后调控的生物学机制奠定基础。     

狼疮La蛋白的分子生物学研究进展

狼疮La蛋白,又叫La核糖核蛋白、La自身抗原或干燥综合征B型抗原,是原发性干燥综合征的特异性自身抗原之一。La蛋白拥有多种结构和运输元件,可定位于不同的亚细胞部位与RNA相互作用。通常I丑蛋白主要存在于细胞核内,作为RNA聚合酶Ⅲ的转录因子,与RNA聚合酶Ⅲ转录新生产物结合,调节其转录终止,在转录后发挥分子伴侣作用,稳定RNA前体并促进其正确折叠,帮助其进行加工处理。La蛋白还可以与一类拥有内部核糖体进入位点的细胞内mRNA和病毒RNA结合,调节这类RNA的表达翻译;也可在细胞凋亡时被颗粒酶B酶解,产生特异性酶解片段,诱导特异性抗La自身抗体和干燥综合征的生成。我们就I丑蛋白的分子生物学方面的近期研究进展进行综述。     

PCBP1在基因表达过程中的功能和作用机理

Poly（C）-binding protein 1 （PCBP 1）是一种RNA结合蛋白,其蛋白质相对分子质量约为38000。PCBP1含三个KH（hnRNP K homology）结构域,这些结构域对其结合RNA有重要作用。PCBP1的功能主要是参与基因转录及转录后调节,如前体mRNA的剪切、mRNA稳定性、mRNA翻译过程的沉默或增强等。本文主要对PCBP1参与的信号通路以及与人类疾病的关系进行综述,试图阐明PCBP1的生物学功能和作用机理。     

植物microRNAs研究进展

植物microRNAs(miRNAs)是一类与RNA诱导沉默复合体相关的约由22个核苷酸组成的单链小RNA分子, 其主要功能是, 通过特异性剪切靶mRNA或阻遏靶mRNA的正常翻译在转录后水平调控基因的负表达。植物miRNAs的靶标主要是参与调控植物生长发育和防御应答的转录因子家族。文章主要综述miRNAs在植物体内的生物发生、作用机制及其调控作用研究新进展。     

mRNA翻译起始区二级结构改变对干扰素基因在大肠杆菌中翻译的影响

为研究mRNA翻译起始区结构与基因表达的关系,利用密码子的简并性,在不改变表达产物氨基酸序列的前提下定点突变α8干扰素及αA干扰素衍生物基因的5′端若干位点,使其与表达载体重组后转录形成的mRNA翻译起始区结构发生改变。SDS-PAGE及活性测定证实这些改变提高了外源基因的表达水平。RNA斑点印迹表明突变前后基因转录水平差别不大,表达水平的提高主要由于翻译效率的提高。mRNA翻译起始区二级结构预测提示其生成自由能(ΔG)的变化可能与表达水平的提高有关。     

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。     

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的表达,有些相互协同,有些相互拮抗,是宏观的、整体的对机体炎症反应的调控。深入探讨RBPs调控巨噬细胞炎症因子以及炎症反应的作用机制,对于从不同角度认识、预防以及治疗炎症性相关疾病,具有重要意义。     

细菌RNA结合蛋白Hfq的DNA压缩和复制作用研究进展

RNA结合蛋白(RNA-Binding Protein)Hfq是一种重要的细菌转录后调节因子,之前对Hfq的研究大多集中在该蛋白对小分子非编码RNA (Small Non-Coding RNA,sRNA)和mRNA的作用上。Hfq最典型的功能是促进sRNA与其靶标mRNA碱基配对,在转录后介导对RNA的稳定性和翻译的调控。此外,Hfq也能与多种蛋白质直接或间接相互作用。然而,近年来的研究表明,除了RNA和蛋白质,Hfq还可以与DNA相互作用,在DNA压缩(DNA Compaction)和DNA复制(DNA Replication)等多种DNA代谢过程中发挥直接或间接的调控作用。额外的靶标和功能的鉴定将进一步夯实Hfq作为细菌中多种代谢途径核心调控因子的重要地位,也表明该蛋白的功能并不局限于其在RNA和蛋白质代谢中的作用。本文总结了Hfq在DNA代谢调控中的近几年最新研究进展,并展望了其前景。     

Translational control and target recognition by Escherichia coli small RNAs in vivo

Small non-coding RNAs (sRNAs) are an emerging class of regulators of bacterial gene expression. Most of the regulatory Escherichia coli sRNAs known to date modulate translation of trans-encoded target mRNAs. We studied the specificity of sRNA target interactions using gene fusions to green fluorescent protein (GFP) as a novel reporter of translational control by bacterial sRNAs in vivo. Target sequences were selected from both monocistronic and polycistronic mRNAs. Upon expression of the cognate sRNA (DsrA, GcvB, MicA, MicC, MicF, RprA, RyhB, SgrS and Spot42), we observed highly specific translation repression/activation of target fusions under various growth conditions. Target regulation was also tested in mutants that lacked Hfq or RNase III, or which expressed a truncated RNase E (rne701). We found that translational regulation by these sRNAs was largely independent of full-length RNase E, e.g. despite the fact that ompA fusion mRNA decay could no longer be promoted by MicA. This is the first study in which multiple well-defined E.coli sRNA target pairs have been studied in a uniform manner in vivo. We expect our GFP fusion approach to be applicable to sRNA targets of other bacteria, and also demonstrate that Vibrio RyhB sRNA represses a Vibrio sodB fusion when co-expressed in E.coli.     

The phosphorylation state of eucaryotic initiation factor 2 alters translational efficiency of specific mRNAs.

Phosphorylation of the alpha subunit of the eucaryotic translation initiation factor (eIF-2 alpha) by the double-stranded RNA-activated inhibitor (DAI) kinase correlates with inhibition of translation initiation. The importance of eIF-2 alpha phosphorylation in regulating translation was studied by expression of specific mutants of eIF-2 alpha in COS-1 cells. DNA transfection of certain plasmids could activate DAI kinase and result in poor translation of plasmid-derived mRNAs. In these cases, translation of the plasmid-derived mRNAs was improved by the presence of DAI kinase inhibitors or by the presence of a nonphosphorylatable mutant (serine to alanine) of eIF-2 alpha. The improved translation mediated by expression of the nonphosphorylatable eIF-2 alpha mutant was specific to plasmid-derived mRNA and did not affect global mRNA translation. Expression of a serine-to-aspartic acid mutant eIF-2 alpha, created to mimic the phosphorylated serine, inhibited translation of the mRNAs derived from the transfected plasmid. These results substantiate the hypothesis that DAI kinase activation reduces translation initiation through phosphorylation of eIF-2 alpha and reinforce the importance of phosphorylation of eIF-2 alpha as a way to control initiation of translation in intact cells.     

Competition among Hfq-binding small RNAs in Escherichia coli

A major class of small bacterial RNAs (sRNAs) regulate translation and mRNA stability by pairing with target mRNAs, dependent upon the RNA chaperone Hfq. Hfq, related to the Lsm/Sm families of splicing proteins, binds the sRNAs and stabilizes them in vivo and stimulates pairing with mRNAs in vitro. Although Hfq is abundant, the sRNAs, when induced, are similarly abundant. Therefore, Hfq may be limiting for sRNA function. We find that, when overexpressed, a number of sRNAs competed with endogenous sRNAs for binding to Hfq. This correlated with lower accumulation of the sRNAs (presumably a reflection of the loss of Hfq binding), and lower activity of the sRNAs in regulating gene expression. Hfq was limiting for both positive and negative regulation by the sRNAs. In addition, deletion of the gene for an expressed and particularly effective competitor sRNA improved the regulation of genes by other sRNAs, suggesting that Hfq is limiting during normal growth conditions. These results support the existence of a hierarchy of sRNA competition for Hfq, modulating the function of some sRNAs.     

Mutations in Interaction Surfaces Differentially Impact E. coli Hfq Association with Small RNAs and Their mRNA Targets

The RNA chaperone protein Hfq is required for the function of all small RNAs (sRNAs) that regulate mRNA stability or translation by limited base pairing in Escherichia coli. While there have been numerous in vitro studies to characterize Hfq activity and the importance of specific residues, there has been only limited characterization of Hfq mutants in vivo. Here, we use a set of reporters as well as co-immunoprecipitation to examine 14 Hfq mutants expressed from the E. coli chromosome. The majority of the proximal face residues, as expected, were important for the function of sRNAs. The failure of sRNAs to regulate target mRNAs in these mutants can be explained by reduced sRNA accumulation. Two of the proximal mutants, D9A and F39A, acted differently from the others in that they had mixed effects on different sRNA/mRNA pairs and, in the case of F39A, showed differential sRNA accumulation. Mutations of charged residues at the rim of Hfq interfered with positive regulation and gave mixed effects for negative regulation. Some, but not all, sRNAs accumulated to lower levels in rim mutants, suggesting qualitative differences in how individual sRNAs are affected by Hfq. The distal face mutants were expected to disrupt binding of ARN motifs found in mRNAs. They were more defective for positive regulation than negative regulation at low mRNA expression, but the defects could be suppressed by higher levels of mRNA expression. We discuss the implications of these observations for Hfq binding to RNA and mechanisms of action.     

Alternative Hfq‐sRNA interaction modes dictate alternative mRNA recognition

Many bacteria use small RNAs (sRNAs) and the RNA chaperone Hfq to regulate mRNA stability and translation. Hfq, a ring‐shaped homohexamer, has multiple faces that can bind both sRNAs and their mRNA targets. We find that Hfq has at least two distinct ways in which it interacts with sRNAs; these different binding properties have strong effects on the stability of the sRNA in vivo and the sequence requirements of regulated mRNAs. Class I sRNAs depend on proximal and rim Hfq sites for stability and turn over rapidly. Class II sRNAs are more stable and depend on the proximal and distal Hfq sites for stabilization. Using deletions and chimeras, we find that while Class I sRNAs regulate mRNA targets with previously defined ARN repeats, Class II sRNAs regulate mRNAs carrying UA‐rich rim‐binding sites. We discuss how these different binding modes may correlate with different roles in the cell, with Class I sRNAs acting as emergency responders and Class II sRNAs acting as silencers.