【作者】 张文娟；

【导师】 滕脉坤； 牛立文；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 第一部分：前体RNA (pre-mRNA)的剪切在基因表达过程中是一个关键的调节步骤,细胞可以在不同水平上对它进行组织特异性或者发育阶段特异性的调节。几乎所有的RNAPⅡ转录出的pre-mRNA都能进行可变剪切。可变剪切可以增加从基因组转录出的mRNA的多样性,从一个基因的pre-mRNA产生多种mRNA,并且翻译出不同的蛋白变体(iso forms),行使不同的功能。因此,可变剪切具有普遍的生物学意义,而可变剪切调控的异常将会导致疾病。hnRNP L是一种重要的可变剪切调控的蛋白,能够调节很多基因的pre-mRNA的可变剪切。此外,它还在染色质的修饰,转录调控,mRNA出核与蛋白质翻译以及mRNA的稳定性调节过程中起到重要作用。在本文的工作中,我们首先对hnRNP L各蛋白区段结合RNA的能力进行了测定和分析,发现hnRNP L的多个RRM结构域协同结合RNA。RRM1需要N端的58个氨基酸的加入成RRMN1时才能微弱地结合RNA,RRM2结合RNA能力也较弱,但RRMN12结合能力明显增强。RRM34能作为一个功能单位较强地结合RNA,并且RRM34结合能力比RRMN12更强。我们利用X-射线晶体学方法解析了hnRNP L RRM1和RRM34的结构。在结构分析的基础上,通过定点突变和SPR实验,鉴定出它们主要是通过β折叠面与RNA结合；通过基于结构的序列比对,对hnRNP L选择结合CA重复序列的分子机制进行了初步探讨。通过基于FRET-based EMSA,稳态FRET, Cross-linking和DLS实验,证明hnRNP L RRM34能同时结合在具有合适间隔的两个RNA结合位点上,促进RNA成环。应用hnRNP L在细胞内的天然底物RNA进行EMSA和ITC实验,验证了hnRNP L介导的RNA成环也能够发生在细胞内。最后,我们提出两种hnRNP L在可变剪切调控中的发挥激活或者抑制作用的作用模型,为解释hnRNP L在可变剪切调控中的位置依赖性双重功能提供了结构基础,并且预示了hnRNP L可能通过RNA成环,不需要直接的蛋白-蛋白相互作用就可以募集蛋白质因子的功能。第二部分：染色质是一种高度包装和精密调控的核糖核蛋白复合物,它通过一种稳定而有序调节的方式保存细胞的遗传信息。因此,与DNA相关的基本生物过程,例如转录,复制和损伤修复等,需要一些蛋白因子的协助,克服核小体的阻碍作用,才能使DNA信息得以表达。FACT复合物是一种组蛋白伴侣,能够在以染色质为模板的相关的转录,复制和损伤修复等过程中起重要作用,既能通过破坏组蛋白-组蛋白和组蛋白-DNA之间的作用来解构核小体也能帮助组蛋白重组装到DNA上。人源FACT复合物由Spt16和SSRP1两个亚基组成。其中的SSRP1蛋白包含有三个确定的结构域：N端/二聚结构域(NTD/DD),中间结构域(MD)和HMG结构域。我们解析了人源SSRP1蛋白的中间结构域(SSRP1-M)的1.93A的晶体结构,该结构域采取了两个串联的紧密结合的PH结构域的折叠方式。通过结构比对,发现SSRP1-M蛋白具有独特的p-α-β的超二级结构花样插入在p桶与C端a螺旋中间,并且在不同物种中非常保守。进一步分析发现SSRP1-M表面两侧各有一个横跨两个PH结构域的正电荷富集区,并且通过EMSA实验证明SSRP1-M确实能结合DNA,且这种结合没有序列选择性。通过突变实验表明,SSRP1-M的这两个正电荷富集区参与结合DNA。此外,我们初步的pull down结果表明SSRP1-M并不参与组蛋白的结合,这与其在酵母中的同源蛋白Pob3-M表现出不同。这些研究结果,为进一步了解SSRP1蛋白在核小体解离和重组装过程中发挥的具体作用提供了结构生物学和生物化学的分子基础。更多还原

【Abstract】 Part I:Pre-mRNA splicing is a crucial mechanisom for gene expression. It is regulated at different levels in a tissue-or developmental stage-specific manner. Almost all polymerase II transcripts undergo alternative pre-mRNA splicing. The overall function of alternative splicing is to increase the diversity of mRNA expressed from the genome, generating different splicing isoforms from one pre-mRNA to fulfill various functions. The aberrant regulation of alternative splicing leads to human diseases. hnRNP L is an important splicing regulator involved in alternative-splicing regulation of many genes. Besides, it plays roles in chromatin modification, transcriptional regulation, mRNA export of intronless genes, IRES-dependent translation and mRNA stability regulation.hnRNP L contains four RNA recognition motifs (RRMs) that bind with CA repeats or CA-rich elements. In our study, results of surface plasmon resonance (SPR) spectroscopy assays revealed that all the four RRM domains contribute to RNA-binding. Compared to RRM12, RRM34shoulders the major responsibility for RNA binding of hnRNP-L. Then, we elucidated the crystal structures of RRM1and RRM34of hnRNP-L at2.0A and1.8A, respectively. These RRMs all adopt the typical β1α1β2β3α2β4topology except the presence of an unusual fifth P-strand in RRM3. RRM3and RRM4interact intimately with each other mainly through helical surfaces, leading the two β-sheets to point to the opposite directions. Structure-based mutations and SPR assays results suggested that all the antiparallel β-sheets of RRMs are accessible for RNA binding. FRET-based gel shift assays (FRET-EMSA) and steady-state FRET assays (ss-FRET), together with cross-linking and dynamic light scattering (DLS) assays, demonstrated that hnRNP L RRM34facilitates RNA looping when binding to two appropriately separated binding sites within the same target pre-mRNA. EMSA and ITC binding studies with in vivo target RNA suggested that hnRNP L-mediated RNA looping may occur in vivo. Our study provides a mechanistic explanation for the dual functions of hnRNP L in alternative-splicing regulation either as an activator or repressor. Our results also enlighten the possible ability of hnRNP L in recruiting additional factors through RNA looping without direct protein-protein interactions. Part II:Chromatin is a kind of densely packed and tightly regulated nucleoprotein complex that stores the cellular genetic material in a stable yet readily accessible form. To overcome the inhibitory effects of nucleosomes on the accessibility of DNA during basic chromatin-templated progresses such as transcription, DNA replication and repair, it needs assistance from many factors to alter the chromatin structure. FACT (FAcilitates Chromatin Transcription) complex is an important histone chaperone, which reorganizes nucleosome without hydrolyzing ATP and translocating histone octamers relative to DNA. It can not only disrupt core histone-histone and histone-DNA interactions, but also possess the ability to deposit H2A-H2B dimer and (H3-H4)2tetramer onto DNA. Human FACT complex is composed of Spt16and SSRP1. SSRP1protein contains three well-defined domains:the N-terminal/dimerization Domain (NTD/DD), middle domain (MD), and HMG-1domain (HMG).We determined the crystal structure of the middle domain of SSRP1(SSRP1-M) at a resolution of1.93A. The SSRP1-M structure adopts a compact double PH domain architecture. In structural comparison to typical PH domains, PHI domain contains extra two antiparallel strands linked by a helix, which are inserted between the last strand and the C-terminal helix. The residues constituting such unique super secondary structure are conserved through different species. The analysis of electrostatic potential surface of the structure of SSRP1-M showed that one positively charged ridge region exists on each side of surface, suggesting its DNA binding ability. The results of EMSA assays and mutagenesis assays revealed that SSRP1-M binds nonspecificly with DNA and the two positively charged regions are involved in DNA binding. Besides, our pull-down experiments showed that SSRP1-M doesn’t interact with histone, differently from its homologous protein in yeast Pob3-M. Our study provides structural and biochemical insights for SSRP1’s detailed roles in the reorganization and reassembly of nucleosomes.更多还原