摘要
大多数的pre-mRNA的3’-端成熟包括核酸内切酶剪切和在上游剪切产物末端添加多聚腺苷酸末尾并需要多个蛋白复合物的参与。参与真核生物pre-mRNA3’-端加工的蛋白几乎都已被发现,其中Cleavage factor I (CFI_m)是一个参与多聚腺苷酸位点剪切过程的重要蛋白复合物,包含一个25 kDa亚基(CFI_m25)和三个较大的亚基中的任意一个(CFI_m59, CFI_m68, CFI_m72)。CFI_m识别AAUAAA上游的UGUAA元件从而促进pre-mRNA的3’-端加工复合物组装和提高体外多聚腺苷酸位点的体外剪切速率和效率。CFI_m也可通过与PAP和hFip1的相互作用调控不依赖AAUAAA信号的多聚腺苷酸末尾合成。将体外表达的CFI_m25-CFI_m68复合物添加到纯化后的3’-端加工因子中,可在体外实验中恢复多聚腺苷酸位点剪切活性。通过基因敲除抑制CFI_m25的表达不影响HeLa细胞的存活,但增加上游剪切位点的使用频率,说明CFI_m25在多聚腺苷酸位点选择中发挥重要作用。因此研究CFI_m复合物的结构和功能对于揭示多聚腺苷酸尾巴合成这一生物现象具有非常重要的意义。本论文研究了CFI_m复合物的晶体结构,阐述了CFI_m复合物识别pre-mRNA的分子机制。
我们研究了CFI_m25和CFI_m68之间的相互作用,发现CFI_m68通过其氨基端的RRM结构域结合CFI_m25并解析了CFI_m25-CFI_m68RRM复合物的晶体结构。CFI_m68RRM通过一种新颖的RRM-蛋白相互作用模式与CFI_m25结合形成四聚体。突变实验揭示CFI_m68RRM与CFI_m25二体的两个分子均有相互作用,同时CFI_m25二聚化可稳定CFI_m68RRM的结合面,提示CFI_m25的二聚化具有关键的生物学意义。我们进一步解析了CFI_m25-CFI_m68RRM-RNA的三元复合物晶体结构。CFI_m复合物以四聚体形式结合两个UGUAA元件,CFI_m25亚基通过疏水作用,氢键作用和芳香族残基侧链与碱基环之间的共轭作用在其带正电的沟中特异性结合UGUAA元件。CFI_m25-CFI_m68RRM结合pre-mRNA的亲和力测定及突变实验发现CFI_m68RRM可结合UGUAA元件5’-末端序列并且复合物的形成可大大提高底物亲和力,说明CFI_m68也参与识别pre-mRNA,同时CFI_m25-CFI_m68复合物与pre-mRNA的结合具有协同性。这些研究揭示了CFI_m复合物识别pre-mRNA的分子机制。
本文还介绍了酵母蛋白Pub1的结构生物学研究。Pub1是一个分布于细胞核和细胞质的蛋白,含有三个RRM结构域(Pub1-RRM1,Pub1-RRM2和Pub1-RRM3),调控细胞mRNA降解。已有的研究发现大约10%的mRNA的降解受到Pub1的调控。它可结合并稳定含ARE和类似ARE元件的mRNA来抑制其降解,同时也可结合并稳定含STE元件的mRNA来抑制NMD通路的降解作用。Pub1可在体外结合多聚尿嘧啶。我们成功解析Pub1-RRM2和连续RRM结构域Pub1-RRM1-RRM2(Pub1-RRM12)的结构。Pub1-RRM1和Pub1-RRM2具有典型RRM结构域的结构。结构生物学研究发现Pub1-RRM12中两个RRM结构域通过一段linker相连,结构域之间没有相互作用。Pub1-RRM12的晶体结构显示,Pub1-RRM12是一个CV-N类型domain-swapped二聚体。Pub1-RRM12与晶胞内的对称分子相互作用稳定了两个RRM结构域的空间位置。进一步研究发现Pub1-RRM12在溶液中是以单体形式存在的。通过点突变实验,我们确定了Pub1-RRM1和Pub1-RRM2结合多聚尿嘧啶的结合面及关键氨基酸残基。利用SPR技术,我们测定了三个RRM结构域和Pub1-RRM12结合10个和15个碱基多聚尿嘧啶的亲和力。结果显示Pub1的单个RRM结构域以相似的亲和力结合这两条多聚尿嘧啶。但是Pub1-RRM12结合15个碱基多聚尿嘧啶的亲和力显著高于10个碱基。这些研究结果为了解Pub1蛋白提高了结构生物学和生物化学的基础。
The maturation of the 3’-ends of most mRNAs is catalyzed by multiple protein complexes, including the endo-nucleolytic cleavage of primary transcripts and addition of poly(A) tails to the upstream cleavage products. Nearly all the critical protein complexes involved in eukaryotic pre-mRNA 3’-end processing have been identified. Cleavage factor I (CFI_m), consisting of a 25 kDa subunit (CFI_m25) and one of the three larger subunits (CFI_m59, CFI_m68, CFI_m72), is required for the 3’-end cleavage. CFI_m binds to the UGUAA elements upstream of AAUAAA elements of the pre-mRNA substrates that facilitates pre-mRNA 3’-end processing complex assembly and enhance the rate and overall efficiency of poly(A) site cleavage in vitro. Sequence-specific binding of CFI_m to pre-mRNA directs A(A/U)UAAA-independent poly(A) addition through interacting with poly(A) polymerase and hFip1. When added to partially purified 3’-end processing factors, recombinant CFI_m25-CFI_m68 complex was sufficient to reconstitute poly(A) site cleavage activity in vitro. Repression of CFI_m25-CFI_m68 complex activity by knocking down CFI_m25 does not affect the HeLa cell viability, but increases upstream poly(A) site usage, suggesting CFI_m25 plays an important role in poly(A) site selection. Therefore, it is important to exploring the structure-function relationship of CFI_m. This thesis presents the structural basis for pre-mRNA recognition by CFI_m.
CFI_m68 interacts with CFI_m25 through its N-terminal RRM domain (CFI_m68RRM). We determined the crystal structure of CFI_m25-CFI_m68RRM complex, revealing that CFI_m68RRM interacts with CFI_m25 through a novel RRM-protein interaction mode to form a tetramer. Mutagenesis analysis and pull-down experiment showed that CFI_m25 dimerization is crucial for CFI_m complex assembly, suggesting CFI_m complex is possibly a tetramer in vivo. We also determined the crystal structure of CFI_m25-CFI_m68RRM-RNA complex. The CFI_m25-CFI_m68RRM tetramer binds two UGUAA elements in the positively charged cavities of the CFI_m25 dimer via hydrogen-bonds, hydrophobic contacts and base pair stacking. The kinetic analysis demonstrates that CFI_m complex assembly increases pre-mRNA binding affinity, and the subsequent mutagenesis analysis reveals the RNA binding surface of CFI_m68, suggesting CFI_m68 may bind the immediately flanking region at 5’-end of the UGUAA element.
This thesis also presents the structural investigation of yeast poly(U) binding protein (Pub1). Yeast poly(U)-binding protein (Pub1) is a major nuclear and cytoplasmic protein, containing three RNA recognition motif (RRM) domains (termed Pub1-RRM1, Pub1-RRM2 and Pub1-RRM3), which has been implicated as a regulator of cellular mRNA decay. Nearly 10% of all yeast mRNAs decay occurs in a Pub1-dependent manner. Pub1 binds to and stabilizes AU-rich element (ARE) and ARE-like sequence-containing transcripts by protecting them from degradation through the deadenylation-dependent pathway, and also binds to and stabilizes stabilizer element (STE)-containing transcripts by preventing their degradation via the nonsense-mediated decay (NMD) pathway. We determined the crystal structures of Pub1-RRM2 and the first two tandem RRM domains (Pub1-RRM12). Pub1-RRM1 and Pub1-RRM2 adopt the canonicalαβsandwich structures of RRM domains. Pub1-RRM12 forms a CV-N type domain-swapped dimmer by crystal packing. Size exclusion chromatography assay and analysitcal ultracentrifugation (AUC) showed Pub1-RRM12 is a monomer in solution. Mutagenesis analysis revealed five residues, located on the twoβ-sheets of Pub1-RRM1 and Pub1-RRM2, are critical for poly(U) binding. Kinetic analysis showed that all the three individual RRM domains can bind to a 10- or 15-base poly(U) segment with similar affinities, whereas Pub1-RRM12 binds to the 15-base poly(U) segment with the affinity approximately an order of magnitude higher than the 10-base poly(U) segment. Our studies provide structural and biochemical information for Pub1.
引文
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