RNA editing in African trypanosomes requires a 3' nucleotidyl phosphatase - the biochemical consequences of the exoUase activity of TbMP42.
Technische Universität, Darmstadt
[Ph.D. Thesis], (2008)
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|Item Type:||Ph.D. Thesis|
|Title:||RNA editing in African trypanosomes requires a 3' nucleotidyl phosphatase - the biochemical consequences of the exoUase activity of TbMP42|
Mitochondrial transcripts in African trypanosomes are post-transcriptionally modified by a process known as RNA editing. The reaction transforms cryptic pre-mRNAs into functional mRNAs and is characterized by the insertion and deletion of exclusively uridylyl residues. RNA editing is catalyzed by subcellular machines, so called editosomes. Editosomes are protein complexes consisting of ~20 polypeptides. In addition, the process relies on small, non-coding RNAs termed guide (g) RNAs. gRNAs are trans-acting molecular components, which specify the insertion and deletion events via basepairing. TbMP42 (Trypansoma brucei mitochondrial protein, MW: 42kDa) is an integral protein of the editosomal machinery. Chapter one and two of the thesis describe the endo/exoribonuclease activity of TbMP42 and characterize the reaction pathway that leads to pre-mRNA substrate cleavage. Experiments using recombinant (r) protein show that TbMP42 is a topology-dependent ribonuclease that is sensitive to base-stacking. The protein has a preference for single stranded uridylyl residues and the ribonuclease activity depends on Zn2+-ions and Zn2+-coordinating amino acids within the oligonucleotide/oligosaccharide binding site (OB-fold) at the C-terminus of the protein. RNAi-mediated gene silencing of TbMP42 is lethal for T. brucei and results in reduced endo/exoribonuclease and RNA editing activities in vitro. However, all three activities can be restored by the addition of rTbMP42. Chapter three addresses the biochemical consequences of the in vitro exoribonuclease activity of TbMP42 and its implication for the editing cycle. The data suggest that RNA editing includes an additional enzymatic reaction step that is conducted by a 3’-specific nucleotidyl phosphatase. Evidence is presented that the nucleotidyl phosphatase activity is part of the editosome and that it is mediated by two editosomal protein components: TbMP99 and TbMP100. Both polypeptides have endo-exo-phosphatase (EEP) domains and the simultaneous knockdown of the two proteins results in a reduction of the phosphatase activity in vitro. A final chapter deals with the identification of novel gRNAs, including gRNAs that potentially direct “alternative” editing events. “Alternative” RNA editing is a source for the generation of protein diversity within T. brucei mitochondria and thus, provides an additional regulatory layer for the editing machinery.
|Place of Publication:||Darmstadt|
|Classification DDC:||500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie|
|Divisions:||10 Department of Biology|
|Date Deposited:||17 Oct 2008 09:23|
|Last Modified:||07 Dec 2012 11:54|
|Referees:||Göringer, Prof. Dr. H. Ulrich and Thiel, Prof. Dr. Gerhard|
|Refereed:||1 July 2008|