Functional characterization of trypanosoma mRNA capping enzymes and archaeal RNA ligase
MetadataShow full item record
The dissertation consists of three chapters. In chapter 1, I present the characterization of the Trypanosoma brucei mRNA capping enzyme apparatus. The 5’ end of kinetoplastid mRNA possesses a hypermethylated cap 4 structure, which is derived from the standard m 7 GpppN (cap 0) by addition of seven methylations within the first four nucleotides on the spliced leader (SL) RNA. T. brucei encodes two sets of cap 0 forming enzymes: TbCe1 guanylyltransferase and TbCmt1 (guanine N-7) methyltransferase, and the novel bifunctional TbCgm1. Recombinant TbCgm1 transfers GMP to SL RNA via a covalent enzyme-GMP intermediate, and methylates the guanine N-7 position of the GpppN-terminated RNA to form cap 0 structure. Silencing of TbCGM1 by RNA interference (RNAi) increased the abundance of uncapped hypometylated SL RNA. In contrast, depletion of TbCe1 or TbCmt1 did not affect SL RNA capping or parasite growth. We conclude that TbCgm1 specifically caps SL RNA, and cap 0 is a prerequisite for subsequent methylation events leading to the formation of mature cap 4. TbCgm1 specifically localizes in the nucleus, whereas TbCe1 is expressed predominantly in the cytoplasm. Our findings represent the first instance in which two sets of RNA capping enzymes coexist within the same organism, each with a genetically distinct function in a cell. In chapter 2, the identification and characterization of the T. brucei enzyme that is responsible for mRNA decapping (TbDcp2) is discussed. Recombinant TbDcp2 hydrolyzes m 7 Gpp from m 7 GpppN-terminated RNA. Depletion of TbDcp2 by RNAi resulted in mild growth phenotype with stabilization of some mRNA populations. Unlike decapping enzymes from other organisms, TbDcp2 is also capable of hydrolyzing unmethylated capped termini (GpppN-RNA) in vitro . On the other hand, the activity was significantly reduced when the first nucleotide was 2’- O -methylated (cap 1), and methylation at second nucleotide (cap 2) may also have an additive effect in reducing the decapping efficiency. These unique substrate specificities suggest that TbDcp2 may act as a surveillance enzyme to preferentially degrade mRNA with premature cap structures. In chapter 3, I report the characterization of a novel homodimeric RNA ligase from a thermophilic Archaeon, Methanobacterium thermoautotrophicum (MthRnl). MthRnl catalyzes intramolecular ligation of a single-stranded RNA to form a covalently closed circular RNA molecule through ligase-adenylylate and RNA-adenylylate intermediates. Sedimentation analysis indicates that MthRnl is a homodimer in solution. The C-terminal 127-amino acid segment is required for dimerization, is itself capable of oligomerization, and acts in trans to inhibit the ligation activity of a native MthRnl. In an attempt to define the in vivo role of Archaeal RNA ligase, we generated a ligase knockout strain in Thermococcus kodakarensis . Analysis of extracted RNA from the knockout cells suggests that the MthRnl and its homologues participate in tRNA and rRNA biogenesis.