Poster abstracts
Poster number 57 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 (School of Medicine, University of Connecticut), Juan Alfonzo (Department of Microbiology and OSU Center for RNA Biology, The Ohio State University)
Abstract:
Introns interrupt tRNA sequences in all major lines of descent (Bacteria, Archaea and Eukarya), rendering them nonfunctional for protein synthesis. Intron removal is therefore, essential. In all known eukaryotes, intron cleavage, the first step in the tRNA splicing pathway, is catalyzed by a conserved heterotetrameric tRNA splicing endonuclease (Sen) composed of four subunits: Sen54, 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 (Sen34), suggesting that either the other subunits are missing or, as a whole, the enzyme is highly divergent in these organisms. In this work, we present evidence for a divergent and unique enzyme composed of three subunits: homologs of Sen34, 15 and 2. 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, immunofluorescence localization assays showed that the enzyme was cytoplasmic, in stark contrast to the nuclear localization of Sen in most eukaryotes. The results presented here demonstrate that TbSen greatly diverges from previously described eukaryotic enzymes in both 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 a heterotrimeric endonuclease then obviates the need for a Sen54 subunit 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: tRNA, splicing, trypanosoma