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The editosome is protein complex that plays an essential role in RNA editing. The editosome is known to initiate reactions involving the addition and removal of uridylates, a unique feature that only occurs among trypanosomes. The process of editing uridylates involves trimming the nascent mRNA strand and generating an edited or mature mRNA sequence. The mechanism of action of the editosome involves several steps which are catalyzed by particular enzymes associated with RNA editing.
In order for an editosome to approach a specific sequence site along the nascent mRNA strand, short-sequence RNA guides that originate from the mitochondria assist in the identification of sequences along the strand that require editing (Correll et al., 1996). These guide RNAs trigger the cleavage of the nascent mRNA through its endonucleolytic action, resulting in the 3’ and 5’ open ends of the nascent mRNA. These blunt ends act as sites for the attachment or removal of uridylates through the action of the editosome. In the case of removal of uridylates, the enzyme exo-uridylase attaches to the site for subsequent deletion by the editosome. When uridylates are needed to be attached to the blunt end, the enzyme uridylyl transferase identifies the site of attachment and triggers the editosome to bring in the uridylate component. As soon as the addition or removal of uridylate is completed, the edited mRNA strand is checked for any gaps that were incurred during RNA editing. Similar to DNA replication, the enzyme RNA ligase is responsible for sealing the gaps that were accumulated during the RNA editing process.
Each guide RNA has the inherent feature of carrying a specific sequence that is used for identifying sites within the nascent mRNA strand that are targeted for editing. Molecular biology analysis has revealed that there is one editing sites in every stretch of 25 to 35 nucleotides along the nascent mRNA strand. It has also been determined that majority of mRNAs need several guide RNA molecular to complete the entire RNA editing process. The high number of guide RNA’s are essential in order to complete the entire stretch of the produced nascent mRNA strand.
Several attempts in the study of the editosome have generated essential information on this muti-protein complex. Cell biology techniques have revealed that the multi-protein complex sedimented together with the 20S and 40S ribosomal portions. This sedimentation coefficient is also similar to that of the guide RNA molecules and the RNA ligases that were earlier isolated and studied. Protein separation through an electric gradient using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has shown that the editosome is composed of at least 7, and at most 20, proteins (Panigrahi et al., 2001). The variation in the number of proteins that are collected for analysis largely depended on the protocol employed for protein isolation.
Two genes have been identified to be responsible for generating two forms of RNA ligases. The REL1 RNA ligase, earlier identified as the TbMP52 protein, and the REL2 RNA ligase, or previously known as the TbMP48 protein, were characterized using mass spectroscopy. The REL1 gene was further investigated by mutagenesis studies and was proven to be necessary in order for RNA editing to occur (Schnaufer et al., 2001). More importantly, the mutagenesis studies also revealed that the REL1 gene was essential for the survival of trypanosomes. On the other hand, the REL2 gene was observed to be an accessory yet non-essential component of the editing and survival aspects of trypanosomes (Drozdz et al., 2002).
Other genes that have been determined to be responsible for producing other editosome proteins include TbMP18, TbMP42 and TbMP81. These additional proteins were also identified through mass spectroscopy and have been observed to carry sequences that have are evolutionarily conserved within the species. Among the most significant consensus sequences are the zinc finger domains, of which play essential roles in RNA editing. The function of these zinc finger domains was determined through gene inactivation studies and gene editing assays. Another protein that is strongly associated with RNA editing is the DEAD box helicase, which has been determined to recognize the DEAD box motif that is situation along the mRNA strand. Analysis of mutant trypanosome strains with deleted DEAD box motifs show that there was a significant reduction in the amount of editing of the RNA strand.
The catalytic core of the editosome of trypanosomes is currently described as to be composed of at least 16 proteins. Protein analysis has shown that at least five of these components are related through sequence similarities. Protein sequence analysis has revealed that most of the protein components of editosomes carry sequence motifs that are known to trigger interactions with other proteins or nucleic acids. In other cases, the motifs have also been determined to catalyze transferases to either detach or incorporate uridylates from one site to another.
In-depth analysis of the proteins has strongly associated these molecules as major components of the editosome. Through the employment of high stringency washes, the proteins were determined to withstand harsh environment and are thus stable to perform their roles in RNA editing amidst high concentrations of salts or detergents in their surrounding environment. In addition, the protein components of the editosome interact with each other, resulting in a more stable tertiary protein configuration for RNA editing. Chromatographic studies have revealed that the editosome can be dissociated into their individual components and each protein can be further studied in order to generate more details with regards to the structure and function of the poly-protein editosome complex.
Protein purification assays have also revealed that each protein component of the editosome serve as specific function in the entire process of RNA editing. For example, the protein TbMP57 serve as a provider of uridylate transferase activity in the trypanosome. In the case wherein two variants of the TbMP57 protein were present, each variant had a different molecular weight and thus can be differentiated through gel electrophoretic assays (Rusché et al., 2001). Each variant was then determined to act on a specific site of the RNA strand, one variant transferred uridylates at the 5’ end while the other variant focused on the attachment or detachment of uridylates at the 3’ end of the nascent mRNA strand.
On the other hand, the TbMP90 protein component of the editosome has been discovered to be active in conferring nuclease functions during RNA editing. The nuclease functions of this protein include cleavage of the double-stranded nucleic acid because the nascent mRNA strand is then hybridized to several guide RNA strands for stabilization of the nucleic acid during its temporary existence in the cytoplasmic environment of the trypanosome cell (Koller et al., 1997). It has been estimated that an entire round of RNA editing may involve as much as six nucleases to the nascent RNA strand in order for the process to be completed.
Corell, R.A., Read, L.K., Riley, G.R., Nellissery, J.K., Allen, T., Kable, M.L., Wachal, M.D., Seiwert, S., Myler, P.J., and Stuart, K.D. (1996). Complexes from Trypanosoma brucei that exhibit deletion editing and other editing-associated properties. Molecular Cellular Biology 16:1410–1418.
Drozdz, M., Palazzo, S.S., Salavati, R., O’Rear, J., Clayton, C., and Stuart K. 2002. TbMP81is required for RNA editing in Trypanosoma brucei. EMBO Journal 21:1791–1799.
Koller, J., Muller, U., Schmid, B., Missel, A., Kruft, V., Stuart, K., and Göringer, H.U. 1997. Trypanosoma brucei gBP21: An arginine rich mitochondrial protein that binds to guide RNA with high affinity. Journal of Biological Chemistry 272:3749–3757.
Panigrahi, A.K., Gygi, S., Ernst, N., Igo Jr., R.P., Palazzo, S.S., Schnaufer, A., Weston, D., Carmean, N., Salavati, R., Aebersold, R. (2001): Association of two novel proteins, TbMP52 and TbMP48, with the Trypanosoma brucei RNA Editing Complex. Molecular and Cellular Biology 21:380–389.
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Rusché, L.N., Huang, C.E., Piller, K.J., Hemann, M., Wirtz, E., and Sollner-Webb, B. (2001): The two RNA ligases of the Trypanosoma brucei RNA editing complex: Cloning the essential Band IV gene and identifying the Band V gene. Molecular and Cellular Biology 21:979–989.
Schnaufer, A., Panigrahi, A.K., Panicucci, B., Igo Jr., R.P., Salavati, R., and Stuart, K.D. (2001): An RNA ligase essential for RNA editing and survival of the bloodstream form of Trypanosoma brucei. Science 291:2159–2162.