Poster abstracts
Poster number 58 submitted by Jillian Houtz
Identifying mitochondrial DNA-associated trans-acting factors required for mtRNA editing in Physarum polycephalum
Jillian Houtz (Center for RNA Molecular Biology, CWRU), Jonatha Gott (Center for RNA Molecular Biology, CWRU)
Abstract:
Proper gene expression in the mitochondria of the acellular slime mold Physarum polycephalum requires extensive RNA editing. RNA editing refers to a change in nucleotide sequence from that encoded in DNA and includes both nucleobase substitutions or conversions as well as nucleotide insertions and deletions. While both types of RNA editing occur in P. polycephalum mitochondria, the overwhelming majority (94%) of editing events comprise single cytosine (C) insertions. While it is known that this insertional editing is co-transcriptional (i.e., nucleotides are added at the 3' end of the nascent RNA during transcription), it is unclear both how editing sites are recognized as well as how the insertion of non-templated nucleotides is achieved. Preliminary data implicate at least one trans-acting protein factor is required for insertional editing. As efforts to uncouple editing from transcription have thus far been unsuccessful, it remains unclear whether this trans-acting factor associates with the mitochondrial DNA (mtDNA) or with both the mtDNA and mitochondrial RNA polymerase (mtRNAP). While our initial efforts to enrich for proteins associated with active transcription complexes via affinity selection using a nascent RNA handle were unsuccessful, we have more recently focused on enrichment of mtDNA template-associated proteins.
Previous work in our lab has yielded a mitochondrial lysate fractionation method which reduces the protein complement of the lysate by greater than 90%. The excluded mtDNA and associated protein factors support both transcription (and thus we refer to it as a mitochondrial Ttranscription Eelongation Complex, mtTEC) as well as mtRNA editing (to approximately 50% efficiency compared to intact mitochondria). The DNA in mtTEC preparations also serves as a substrate for restriction digestion, and our current aim is to enrich for protein factors associated with a discreet region of the mitochondrial genome that is actively transcribed and whose transcript is also edited. Restriction-digested mtTEC fragments are then substrates for affinity selection once filled-in or tailed with biotinylated deoxynucleotide triphosphates (dNTPs) and immobilized on streptavidin-coated magnetic beads.
Keywords: Physarum polycephalum, RNA Editing, mitochondria