裂殖酵母Dis312的结构与功能研究

发布时间：2018-08-14 09:34

【摘要】：RNA外切核酸酶是修饰RNA的一个大的核酸酶家族,在许多生命活动过程中发挥着重要作用。一方面RNA外切核酸酶可以通过降解多余的RNA来调节RNA的丰度,并由此来监视RNA的代谢。同时,一些RNA前体分子也需要通RNA外切核酸酶的剪切来形成成熟体。Dis312是存在于高等真核生物中的RNA外切核酸酶,在RNA的降解过程中发挥着重要的作用,它从3’往5’逐个地降解单核苷酸。Dis312位于细胞质中,在基因上与一些在细胞质中参与RNA降解通路的分子有联系。例如,在小鼠胚胎干细胞中Dis312通过降解尿苷化的pre-let-7来阻断let-7 microRNAs的表达。人源Dis312的突变可导致Perlman综合征,引起过增长并可引发Wilm's肿瘤。另有研究表明Dis312选择性降解尿苷化的RNA分子,这种活性可被腺苷化的RNA分子所抑制。随着最近几年Dis312功能和催化性质不断地被报道,很多研究组也在进行Dis312结构生物学的研究。在2014年小鼠的Dis312-RNA复合物结构已经被解出(PDB序列号4pmw),该结构和之前解出的Rrp44-RNA复合物结构(PDB序列号2nvu)以及RNase Ⅱ-RNA复合物结构(PDB序列号2ix1)都具有相似的构象：三个OB折叠的结构域(CSD1,CSD2和S1结构域)位于负责催化的RNB结构域的一侧,形成了RNA分子通道的入口,RNA底物从这个入口进入RNB结构域的内部,从而到达催化RNA降解的活性位点。但是在这三个RNA复合物的结构中,RNA通道还是有一些明显的差异。例如,酿酒酵母Rrp44的三个OB折叠结构域所形成的通道相比于Dis312显得更窄,这是因为CSDs的柔性loop在RNB以及S1结构域的距离上更为紧密,呈现一种关闭的状态；而在小鼠的Dis312-RNA复合物结构中,三个OB折叠的结构域的取向形成一个特别的漏斗状通道,使得RNA通道显得更加笔直和开放。小鼠的Dis312-RNA复合物结构的解析首次阐述了Dis312这种外切酶和RNA底物的作用方式,很好地解释了Dis312选择特异性结合尿苷酸链的原因。但是对于Dis312的催化机理仍然还有很多问题有待挖掘。例如Dis312不结合RNA时的构象,Dis312结合RNA的整体动态过程等。为了继续揭示Dis312的催化机制,本人解析了2.3A的裂殖酵母Dis312的不含RNA底物的结构,该结构中CSDs的取向和小鼠Dis312-RNA复合物结构中CSDs的取向出现了很大差别,位于RNB结构域的侧面而远离RNB结构域上方的RNA通道入口。从不含RNA的Dis312结构和Rrp44、RNAseⅡ两个同源物的RNA复合物结构的比较中也出现了类似差别。对这种构象上差别的可能原因分析结果表明,序列差异和晶体堆积对构象差异的影响不大,而RNA的结合状态的不同是不结合RNA的Dis312结构和复合物结构出现构象差别的主要原因。荧光偏振实验结果也显示RNB和S1参与最初整个酶对RNA分子的结合；同时CSDs也参与结合的整个过程。结合结构比对和突变体结合实验的结果可以推测,由于RNA的结合,Dis312可能会发生一个明显的构象变化。目前Dis312-RNA的结构只有小鼠的被解析出来,该工作解析出了首个不含RNA的Dis312晶体结构。此外,该工作也同步解析裂殖酵母Dis312-RNA的结构,来探究Dis312在结合RNA前后构象的变化。由于收到的复合物衍射数据分辨率不高,最终解析出的复合物结构中只确定了四分之三的肽链的走向(不含CSDs),但是通过检验发现,在已有的复合物结构模型中,RNB和S1结构域的电子密度和模型吻合度良好,这说明这两结构域的主链走向和空间大致位置是基本准确的。现有的复合物模型和电子密度提供的信息,以及解析出来的裂殖酵母Dis312结构模型一致表明,Dis312在结合RNA之前呈现一种较为开放的状态,CSDs偏向RNB结构域的侧面而相聚S1结构域较远,结合RNA之后,CSDs会发生一定角度的位置变化,和S1结构域一起形成钳状的结构来包裹住RNA分子,从而大大提高Dis312对底物的亲和力。
[Abstract]:RNA exonucleases, a large family of nucleases that modify RNA, play an important role in many life processes. On the one hand, RNA exonucleases can regulate RNA abundance by degrading excess RNA and thereby monitor RNA metabolism. At the same time, some RNA precursors also require cleavage of RNA exonucleases to form. Mature bodies. Dis312 is an RNA exonuclease present in higher eukaryotes and plays an important role in RNA degradation. It degrades mononucleotides one by one from 3'to 5'. Dis312 is located in the cytoplasm and is genetically associated with some molecules involved in RNA degradation pathways in the cytoplasm. For example, mouse embryonic stem cells. Dis312 inhibits the expression of let-7 microRNAs by degrading ureaylated pre-let-7. Human Dis312 mutations can lead to Perlman syndrome, overgrowth and Wilm's tumors. Other studies have shown that Dis312 selectively degrades ureaylated RNA molecules, which can be inhibited by adenylated RNA molecules. Functional and catalytic properties are continually reported, and many research groups are also working on the structural biology of Dis312. In 2014, the structure of the mouse Dis312-RNA complex (PDB sequence number 4pmw), the structure of the Rrp44-RNA complex (PDB sequence number 2nvu) and the structure of the RNase II-RNA complex (PDB sequence number 2ix1) have been released. All have similar conformations: three OB-folded domains (CSD1, CSD2, and S1) are located on one side of the catalytic RNB domain, forming the entrance of the RNA molecular channel from which the RNA substrate enters the interior of the RNB domain and reaches the active site for catalytic RNA degradation. For example, the channels formed by the three OB folding domains of Saccharomyces cerevisiae Rrp44 are narrower than those of Dis312, because the flexible loop of CSDs is more closely spaced between the RNB and S1 domains, presenting a closed state; and in the mouse's Dis312-RNA complex, the three OBs fold. The orientation of the domain forms a peculiar funnel-shaped channel that makes the RNA channel more straight and open. The structure of the mouse Dis312-RNA complex first elucidates the way Dis312 acts as an exonuclease and RNA substrate, which explains why Dis312 selectively binds to the uridine chain. In order to further reveal the catalytic mechanism of Dis312, the structure of 2.3A fission yeast Dis312 without RNA substrates, the orientation of CSDs and the CSDs in mouse Dis312-RNA complex structure were analyzed. The alignment varies greatly from side to side of the RNB domain and away from the entrance of the RNA channel above the RNB domain. Similar differences also occur in the comparison of the RNA complexes of two homologues, namely, DIS 312 and Rrp44, and RNAse II, which do not contain RNA. The results of fluorescence polarization experiments also showed that RNB and S1 were involved in the binding of the whole enzyme to RNA molecules, and CSDs were also involved in the binding process. The results of the mutant binding experiments suggest that a significant conformational change may occur in Dis312 due to RNA binding. At present, only mice have been able to identify the structure of Dis312-RNA. This work has identified the first crystal structure of Dis312 that does not contain RNA. In addition, the structure of Dis312-RNA in Schizosaccharomyces cerevisiae has been analyzed synchronously to explore Di. The conformational changes of S312 before and after binding RNA. Due to the low resolution of the complex diffraction data, only three-fourths of the peptide chains (excluding CSDs) were determined in the final structure of the complex. However, it was found that the electron density of RNB and S1 domains in the existing complex structure models was in good agreement with the model. The information provided by the existing complex model and electron density, as well as the structure model of the cleavage yeast Dis312, showed that Dis312 exhibited a relatively open state before binding RNA, and CSDs were inclined to the side of the RNB domain and aggregated S1. After binding to RNA, CSDs change their position at a certain angle and form clamp-like structures with S1 domain to encapsulate RNA molecules, thus greatly enhancing the affinity of Dis312 to substrate.
【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q617

【相似文献】