RNA editing in kinetoplastids, several of which are global pathogens, is a unique, and essential, process in the mitochondria of these ancient eukaryotes. This process uses hundreds of so-called """"""""guide RNAs"""""""" to edit an incomplete """"""""pre-messenger RNA"""""""" by U-insertion and deletion at hundreds of specific editing sites. Many more U's are inserted than deleted. In some cases, """"""""pan-editing"""""""" occurs which can more than double the size of the pre-message. A central role in this U-insertion/deletion editing is played by the """"""""editosome"""""""". This is an assembly of approximately 20 nuclear-encoded proteins including six different RNA editing enzymes performing a precisely orchestrated sequence of RNA cleavage, insertion/deletion and religation reactions. ? ? Our research aims to unravel the functioning of the editosome at the atomic level by crystallographic methods to ultimately: ? (i) obtain a full understanding of its architecture; ? (ii) unravel the substrate specificity of each editosomal enzyme; ? (iii) elucidate key interactions of the guide RNA:pre-mRNA duplex with the editosomal proteins; ? (iv) discover the large conformational changes the protein and RNA molecules must undergo while the pre-message is growing by the action of the six different enzymes. ? ? Our proposal builds on recent successes including the crystal structure determinations of the RNA Editing Ligase 1 catalytic domain and that of the editing 3'-Terminal-Uridylylate Transferase. In the latter case the lone pair of an exquisitely positioned water molecule appears to be the key to U-specificity. A subcomplex of three different editosomal proteins has been obtained which appears to be a heterohexamer. These initial results provide an excellent platform from which to proceed with unraveling the many remaining mechanistic mysteries of this marvelous U-insertion/deletion machinery. ? ? Since several editing proteins are essential in pathogenic kinetoplastids, the structures we plan to determine are also promising starting points for the design of selective inhibitors of key pathogen proteins. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM077418-03
Application #
7426816
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Preusch, Peter C
Project Start
2006-05-01
Project End
2010-04-30
Budget Start
2008-05-01
Budget End
2009-04-30
Support Year
3
Fiscal Year
2008
Total Cost
$477,403
Indirect Cost
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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Park, Young-Jun; Budiarto, Tanya; Wu, Meiting et al. (2012) The structure of the C-terminal domain of the largest editosome interaction protein and its role in promoting RNA binding by RNA-editing ligase L2. Nucleic Acids Res 40:6966-77
Park, Young-Jun; Hol, Wim G J (2012) Explorations of linked editosome domains leading to the discovery of motifs defining conserved pockets in editosome OB-folds. J Struct Biol 180:362-73
Wu, Meiting; Park, Young-Jun; Pardon, Els et al. (2011) Structures of a key interaction protein from the Trypanosoma brucei editosome in complex with single domain antibodies. J Struct Biol 174:124-36
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Tarun Jr, Salvador Zipagan; Schnaufer, Achim; Ernst, Nancy Lewis et al. (2008) KREPA6 is an RNA-binding protein essential for editosome integrity and survival of Trypanosoma brucei. RNA 14:347-58