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。     

RRM RNA结合蛋白的结构与功能

RRM RNA结合蛋白是一类含一个或数个RRM结构域及附属结构域的RNA结合蛋白,参与RNA前体的剪接、RNA的细胞定位、RNA的稳定性等多种转录后调控过程.在RRM基序中含有许多保守的氨基酸以保证对RNA的结合活性,但是这一家族的不同蛋白质却能特异地结合各种不同的RNA分子.RRM RNA结合蛋白与某些人类遗传性疾病及肿瘤相关.     

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。     

SR蛋白家族在RNA剪接中的调控作用

SR蛋白家族成员都具有一个富含丝氨酸/精氨酸(S/R)重复序列的RS结构域,在RNA剪接体的组装和选择性剪接的调控过程中具有重要的作用。绝大多数SR蛋白是生存的必需因子,通过其RS结构域和特有的其他结构域,实现与前体mRNA的特异性序列或其他剪接因子的相互作用,协同完成剪接位点的正确选择或促进剪接体的形成。深入研究SR蛋白家族在RNA选择性剪接中的调控机制,可以促进以疾病治疗或害虫防治为目的的应用研究。该文总结了SR蛋白家族在基础研究和应用方面的进展。     

动植物中RNA结合蛋白的研究进展

RNA结合蛋白(RNA-binding proteins)在转录后基因表达调节中起着重要的作用,它通过和RNA相互作用来调节细胞的功能。RNA结合蛋白参与RNA剪接、多聚腺苷化作用、序列编辑、RNA转运、维持RNA的稳定和降解、细胞内定位和翻译控制等RNA代谢的各个方面。主要介绍了RNA结合蛋白的结构、靶标RNA及RNA结合蛋白在动植物和疾病中的研究。     

白桦叶绿体RNA结合蛋白的蛋白质组学分析

在高等植物叶绿体中,RNA结合蛋白在转录后RNA处理、运输以及mRNA的稳定等方面发挥重要作用.本项研究使用多聚腺苷酸(polyA）吸附柱或单链DNA(ssDNA)吸附柱富集白桦叶绿体的polyA结合蛋白或RNA结合蛋白,并通过MALDI-TOF-MS以及ESI MS/MS进行鉴定,13个叶绿体蛋白质得到了鉴定.按照Swiss Prot数据库的注释,这些蛋白质的功能主要包括4个相关种类,分别为NAD结合蛋白、RNA结合蛋白、DNA结合蛋白和ATP结合蛋白.使用这些方法还鉴定出包括转录因子的4个高丰度蛋白.这些结果加深了对树木中叶绿体RNA结合蛋白的全面了解,可以将其应用于其他树木叶绿体中RNA 蛋白质的相互作用的研究.     

植物细胞器RNA编辑因子的功能及其作用机制 *

在植物线粒体和叶绿体转录本上,数百个胞嘧啶(C)位点经脱氨基反应变为尿嘧啶(U),这是一种在转录本水平上对遗传信息进行修饰或调控的机制.在植物细胞器中,RNA编辑过程需要不同家族的RNA编辑因子相互作用组装成复杂的编辑复合体,特异地识别编辑位点进行编辑.最初的研究发现,植物RNA编辑受到高特异性五环肽重复(pentatricopeptide repeat, PPR)蛋白的调控,目前在植物中发现400多种PPR家族蛋白,编辑作用复杂.之后对RNA编辑因子互作蛋白/多细胞器RNA编辑因子(RNA editing factor interacting proteins /multiple organellar RNA editing factors,RIP/MORF),细胞器RNA识别基序(organelle RNA recognition motif,ORRM),细胞器锌指蛋白(organelle zinc-finger,OZ)等的研究表明,这些非PPR蛋白组分可以与PPR蛋白形成编辑复合体,共同参与编辑,且RNA编辑复合体具有多样性.RNA编辑因子的缺失会引起植物的生长发育受阻,果实成熟延迟等,对RNA编辑因子的研究显得尤为重要.对植物中RNA编辑因子的功能及其作用机制研究进展进行综述,旨在为后续RNA编辑的研究提供一定的参考.     

Pre-mRNA选择性剪接的调控及选择性剪接数据库

Pre-mRNA选择性剪接是真核生物转录组和蛋白质组多样性的主要来源,也是细胞分化、发育等过程中重要的基因表达调控方式。约95%的人类多外显子基因存在RNA选择性剪接|很多人类基因疾病的发生与RNA剪接错误相关。随着共转录现象的发现,RNA选择性剪接调控机制研究也取得了很大进展。本文分别从序列层面和核小体定位、组蛋白修饰、DNA甲基化及非编码RNA等表观遗传层面,系统地阐述了RNA选择性剪接的调控机制。为便于搜索,本文介绍了近10年来RNA选择性剪接相关的数据库。     

表观遗传因素在RNA剪接过程中的作用

RNA剪接是真核生物基因表达过程中的重要环节,增加了蛋白质的多样性和基因表达的可调节性. 日益增多的研究表明,RNA剪接并不是独立的生物过程.RNA Ⅱ型聚合酶(RNA polymerase-Ⅱ, RNA Pol Ⅱ)、核小体定位和组蛋白修饰等因素都与RNA剪接过程密切相关.阐明RNA Pol Ⅱ、核小体定位和组蛋白修饰等因素在RNA剪接过程中的作用,将为剪接位点的准确识别和剪接调控机制的研究提供新思路.本文综述了RNA Pol Ⅱ、核小体定位和组蛋白修饰等因素对RNA剪接的影响以及它们在RNA剪接过程中的调控作用.     

表观遗传调控pre-mRNA的选择性剪接

选择性剪接是真核生物基因表达过程中的关键环节,是蛋白质多样性的主要来源,在生物的分化、发育及疾病的发生中扮演重要角色。传统的选择性剪接调控机制的研究多集中于RNA序列元件及与之相关的一些剪接因子,但近期的突破性研究指出表观遗传因素在选择性剪接的调控中发挥重要作用。DNA甲基化、染色质结构、组蛋白修饰相互影响并作用于pre-mRNA的选择性剪接,构成一个庞大、复杂的调控网络,表明表观遗传因素不仅决定着基因转录的起始,还影响其转录本剪接的结果。文章综述了近年来pre-mRNA选择性剪接的表观遗传调控的研究进展,探讨了DNA甲基化、染色质结构、组蛋白修饰在pre-mRNA选择性剪接中的可能作用,并展望了其对人类疾病研究所带来的深远影响。     

Biochemical and genetic analyses of the U5, U6, and U4/U6 x U5 small nuclear ribonucleoproteins from Saccharomyces cerevisiae.

We have purified the yeast U5 and U6 pre-mRNA splicing small nuclear ribonucleoproteins (snRNPs) by affinity chromatography and analyzed the associated polypeptides by mass spectrometry. The yeast U5 snRNP is composed of the two variants of U5 snRNA, six U5-specific proteins and the 7 proteins of the canonical Sm core. The U6 snRNP is composed of the U6 snRNA, Prp24, and the 7 Sm-Like (LSM) proteins. Surprisingly, the yeast DEAD-box helicase-like protein Prp28 is stably associated with the U5 snRNP, yet is absent from the purified U4/U6 x U5 snRNP. A novel yeast U5 and four novel yeast U4/U6 x U5 snRNP polypeptides were characterized by genetic and biochemical means to demonstrate their involvement in the pre-mRNA splicing reaction. We also show that, unlike the human tri-snRNP, the yeast tri-snRNP dissociated upon addition of ATP or dATP.     

Tim50a,a nuclear isoform of the mitochondrial Tim50, interacts with proteins involved in snRNP biogenesis

Background  

The Cajal body (CB) is a nuclear suborganelle involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs), which are vital for pre-mRNA splicing. Newly imported Sm-class snRNPs traffic through CBs, where the snRNA component of the snRNP is modified, and then target to other nuclear domains such as speckles and perichromatin fibrils. It is not known how nascent snRNPs localize to the CB and are released from this structure after modification. The marker protein for CBs, coilin, may play a role in snRNP biogenesis given that it can interact with snRNPs and SMN, the protein mutated in Spinal Muscular Atrophy. Loss of coilin function in mice leads to significant viability and fertility problems and altered CB formation.     

Direct interactions between pre-mRNA and six U2 small nuclear ribonucleoproteins during spliceosome assembly.

Highly purified mammalian spliceosomal complex B contains more than 30 specific protein components. We have carried out UV cross-linking studies to determine which of these components directly contacts pre-mRNA in purified prespliceosomal and spliceosomal complexes. We show that heterogeneous nuclear ribonucleoproteins cross-link in the nonspecific complex H but not in the B complex. U2AF65, which binds to the 3' splice site, is the only splicing factor that cross-links in purified prespliceosomal complex E. U2AF65 and the U1 small nuclear ribonucleoprotein particle (snRNP) are subsequently destabilized, and a set of six spliceosome-associated proteins (SAPs) cross-links to the pre-mRNA in the prespliceosomal complex A. These proteins require the 3' splice site for binding and cross-link to an RNA containing only the branch site and 3' splice site. Significantly, all six of these SAPs are specifically associated with U2 snRNP. These proteins and a U5 snRNP component cross-link in the fully assembled B complex. Previous work detected an ATP-dependent, U2 snRNP-associated factor that protects a 30- to 40-nucleotide region surrounding the branchpoint sequence from RNase digestion. Our data indicate that the six U2 snRNP-associated SAPs correspond to this branchpoint protection factor. Four of the snRNP proteins that are in intimate contact with the pre-mRNA are conserved between Saccharomyces cerevisiae and humans, consistent with the possibility that these factors play key roles in mediating snRNA-pre-mRNA interactions during the splicing reaction.     

A novel EB-1/AIDA-1 isoform,AIDA-1c,interacts with the Cajal body protein coilin

Background  

Cajal bodies (CBs) are nuclear suborganelles that play a role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), which are crucial for pre-mRNA splicing. Upon nuclear reentry, Sm-class snRNPs localize first to the CB, where the snRNA moiety of the snRNP is modified. It is not clear how snRNPs target to the CB and are released from this structure after their modification. Coilin, the CB marker protein, may participate in snRNP biogenesis given that it can interact with snRNPs and SMN. SMN is crucial for snRNP assembly and is the protein mutated in the neurodegenerative disease Spinal Muscular Atrophy. Coilin knockout mice display significant viability problems and altered CB formation. Thus characterization of the CB and its associated proteins will give insight into snRNP biogenesis and clarify the dynamic organization of the nucleus.     

DExD/H-box Prp5 protein is in the spliceosome during most of the splicing cycle

The DExD/H-box Prp5 protein (Prp5p) is an essential, RNA-dependent ATPase required for pre-spliceosome formation during nuclear pre-mRNA splicing. In order to understand how this protein functions, we used in vitro, biochemical assays to examine its association with the spliceosome from Saccharomyces cerevisiae. GST-Prp5p in splicing assays pulls down radiolabeled pre-mRNA as well as splicing intermediates and lariat product, but reduced amounts of spliced mRNA. It cosediments with active spliceosomes isolated by glycerol gradient centrifugation. In ATP-depleted extracts, GST-Prp5p associates with pre-mRNA even in the absence of spliceosomal snRNAs. Maximal selection in either the presence or absence of ATP requires a pre-mRNA with a functional intron. Prp5p is present in the commitment complex and functions in subsequent pre-spliceosome formation. Reduced Prp5p levels decrease levels of commitment, pre-spliceosomal and spliceosomal complexes. Thus Prp5p is most likely an integral component of the spliceosome, being among the first splicing factors associating with pre-mRNA and remaining until spliceosome disassembly. The results suggest a model in which Prp5p recruits the U2 snRNP to pre-mRNA in the commitment complex and then hydrolyzes ATP to promote stable association of U2 in the pre-spliceosome. They also suggest that Prp5p could have multiple ATP-independent and ATP-dependent functions at several stages of the splicing cycle.     

Structure and assembly of the SF3a splicing factor complex of U2 snRNP

SF3a is an evolutionarily conserved heterotrimeric complex essential for pre-mRNA splicing. It functions in spliceosome assembly within the mature U2 snRNP (small nuclear ribonucleoprotein particle), and its displacement from the spliceosome initiates the first step of the splicing reaction. We have identified a core domain of the yeast SF3a complex required for complex assembly and determined its crystal structure. The structure shows a bifurcated assembly of three subunits, Prp9, Prp11 and Prp21, with Prp9 interacting with Prp21 via a bidentate-binding mode, and Prp21 wrapping around Prp11. Structure-guided biochemical analysis also shows that Prp9 harbours a major binding site for stem-loop IIa of U2 snRNA. These findings provide mechanistic insights into the assembly of U2 snRNP.     

The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.