Discovery and characterization of novel RNA repair systems

structural biology;Phosphatase;Kinase;Hen1;RNA ligase;ribotoxin;RNA damage;RNA repair

Wang, Pei

关键词: structural biology;    Phosphatase;    Kinase;    Hen1;    RNA ligase;    ribotoxin;    RNA damage;    RNA repair;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/78582/Wang_Pei1.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

RNA is one of the major macromolecules essential for all known forms of life. RNA in the cell is subject to many types of damage as DNA, and the ubiquitous RNA degradation by surveillance machinery is an important way to respond RNA damage. However, RNA repair is an alternative way for cells to deal with RNA damage, which may play an important role in maintenance of cellular RNAs and even for cell survival.RNA repair is the mechanism that rectifies the purposeful RNA damage during RNA processing or cellular stress. To counter the unexpected RNA breakage, RNA repair systems have evolved in some organisms to restore the normal structure and function of RNA. The first RNA repair system was found in T4phage, in which two proteins T4Pnkp and T4Rnl carried out RNA healing and sealing, respectively. The bacterial Pnkp/Hen1 complex has also been shown to repair ribotoxin-cleaved RNAs in vitro, but it was distinguished from T4 systems by performing 3’-terminal 2’-O-methylation during RNA repair, which prevents the repaired RNA from repeated cleavage at the same site. Bacterial Pnkp and Hen1 appear pair-wise in the same operon in approximately 5% of known bacterial species. Although the bacterial Pnkp has been shown to possess kinase, phosphatase for RNA healing and all the signature motifs of a RNA ligase, it alone is not able to carry out RNA repair. In our study, we crystallized an active RNA ligase consisting of the C-terminal half of Pnkp (Pnkp-C) and the N-terminal half of Hen1 (Hen1-N) from Clostridium thermocellum, and provided the molecular basis for the ligase activation of bacterial Pnkp by Hen1. We also carried out a detailed functional study to pinpoint the activation step during RNA ligation. Guided by the sequence and structure, we created a series of point mutants for this new ligase and carried out biochemical assays. These studies provide additional insight into the mechanism of RNA ligation by Pnkp/Hen1.Based on a comprehensive Blast search, a novel RNA repair system composed of three proteins (Pnkp1, Rnl and Hen1) has been found in 10 bacterial species. This new system possesses the features from both T4Pnkp/Rnl and bacterial Pnkp/Hen1 systems. Efficient in vitro RNA repair only occurred in the presence of all three proteins. We showed that these three proteins formed a heterohexamer in vitro that contains two copies of each active site (kinase, phosphatase, methyltransferase and ligase). We crystallized and solved the strucuture of the heterohexamer to gauge four different enzymatic activities. Based on the structural and biochemical studies, we propose a novel mechanism of processive RNA repair with efficient 2’-O-methylation. The study of bacterial Pnkp1/Rnl/Hen1 complex may shed light on the bacterial Pnkp/Hen1 system due to their similarities.Both structural and biochemical assays indicated bacterial RNA repair systems have a broad range of RNA substrates, but we still have little knowledge of their biological functions in vivo. We therefore introduced genes encoding toxins and RNA repair proteins into bacteria through tightly controlled plasmids, and tested the inhibition and recovery of cellular growth upon the induction of gene expression. This method helped to identify potential toxin genes predicted by sequence alignment, and also facilitated the in vivo study by mimicking the environment the bacteria might be exposed to. If the RNA repair system provides surviving advantage for bacteria to defend themselves under stress, targeting this system may have the potential to limit the growth of pathogenic bacteria possessing the RNA repair systems.In addition to bacterial RNA repair, I am also interested in important NTase fold protein, which constitutes a large and highly diverse superfamily of proteins. Almost all known members of NTase transfer NMP to a hydroxyl group of substrates including proteins, nucleic acids and small molecules. A newly identified subgroup of this superfamily, Mab21, is shown to be essential for development or immune response. Particularly, Mab21D1 (cGAS) is important for interferon response activation by sensing dsDNA in cytosol and generating a second messenger molecule cGAMP. I crystallized and solved the structure of a close member Mab21D2, which is highly conserved in vertebrates but with unknown function. Through structural comparison to other NTase fold members as well as preliminary biochemical assays, we propose that it might work differently than cGAS.