Poster abstracts
Poster number 53 submitted by Jillian Houtz
Identification of mitochondrial DNA-associated trans-acting factors potentially required for mtRNA editing in Physarum polycephalum
Jillian Houtz (Center for RNA Molecular Biology, CWRU), Jonatha Gott (Center for RNA Molecular Biology, CWRU)
Abstract:
Extensive RNA editing is required for proper gene expression in the mitochondria of the acellular slime mold Physarum polycephalum. The term RNA editing describes changes in nucleotide sequence from that encoded in DNA, comprising both nucleobase substitutions or conversions as well as nucleotide insertions and deletions; however, the majority (>94%) of editing events in P. polycephalum mtRNAs comprise single cytosine (C) insertions. While it is known that insertional editing in P. polycephalum is co-transcriptional (i.e., non-templated nucleotides are added at the 3' end of the nascent RNA during transcription), the mechanism of insertional editing remains unclear. Preliminary data from chimeric template experiments implicate a requirement for at least one template-associated trans-acting protein factor. Previous work from our lab has yielded a method for fractionation of mitochondrial lysates which reduces the protein complement of the native mtDNA template by greater than 90%. The resulting partially purified mtDNA-protein complex is a substrate for transcription - as well as mtRNA editing - and thus we refer to it as a mitochondrial Transcription Elongation Complex (mtTEC).
Following digestion of the DNA in mtTECs with restriction endonucleases, we have been able to enrich for protein factors associated with discreet regions of the mitochondrial genome that are both actively transcribed and whose transcripts are also extensively edited. When ligated to a short biotinylated DNA cassette, restriction-digested mtTEC DNA fragments can be affinity selected on streptavidin-coated magnetic beads. We have recently demonstrated that these immobilized fragments retain editing competency and thus any potential editing factors. The proteins associated with these editing-competent mtTEC fragments have been sequenced via mass spectrometry, narrowing down the number of potential candidates for factors required for nucleotide insertion to a manageable number. The subset of these proteins that have probable DNA or RNA binding domains will be the first targeted for silencing by RNAi to determine their role (if any) in co-transcriptional mtRNA editing in P. polycephalum.
Keywords: Physarum polycephalum, mitochondria, RNA editing