Poster abstracts
Poster number 137 submitted by Gabriel Silveira dAlmeida
Overcoming a “molecular ruler” mechanism: the unusual heterotrimeric tRNA splicing endonuclease of Trypanosoma brucei
Gabriel Silveira dAlmeida (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University), Mary Anne Rubio (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University), Christopher Trotta (PTC Therapeutics Inc), Arthur Gnzl (Arthur Gnzl), Juan Alfonzo (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University)
Abstract:
In all three major lines of descent (Bacteria, Archaea and Eukarya) introns interrupt tRNA sequences rendering them non-functional for protein synthesis; intron removal is therefore essential. In eukaryotes, intron cleavage is catalyzed by an evolutionarily conserved heterotetrameric tRNA splicing endonuclease composed of four subunits: Sen 54, 34, 15 and 2. Bioinformatic analysis using previously published eukaryotic Sen sequences led us to the identification of only one homolog of the tRNA splicing endonuclease in Trypanosoma brucei, suggesting that either the other subunits are missing or, as a whole, the enzyme is very divergent in these organisms. Here we present evidence for a highly divergent and unique enzyme composed only of three sub-units; homologs of Sen 34, 15 and 2, while lacking the larger Sen54 subunit. By performing tandem-affinity chromatography followed by mass spectrometry analysis we purified and identified TbSen subunits from a T. brucei S100 fraction. Gel filtration chromatography and in vitro activity assays revealed that the active enzyme had a size within the range of 58 to 72 kDa, consistent with that of a heterotrimer. Furthermore, in contrast with most eukaryotes, immunofluorescence localization assays showed that the enzyme was cytoplasmic. This observation is also in stark contrast with the yeast system where the enzyme localizes to the surface of the mitochondria. Our results demonstrate that TbSen diverges from previously described eukaryotic enzymes, both in structure and localization. Interestingly, in most eukaryotes, Sen54 serves as a “molecular ruler” that carefully measures the distance between the splice sites and the backbone of the folded tRNA, aiding in substrate identification and catalytic site positioning. Our finding of heterotrimeric endonuclease then obviates the need for a Sen54 sub-unit and may remove the substrate recognition restriction set forth by the “molecular ruler” mechanism. These observations have direct implications for both the evolution of the enzyme in trypanosomes and its potential for targeting of additional substrates while not just being limited to tRNAs.
Keywords: trypanosoma, tRNA, splicing