Distinct roles for sequences upstream of and downstream from Physarum editing sites

Abstract

RNAs in the mitochondria of Physarum polycephalum contain nonencoded nucleotides that are added during RNA synthesis. Essentially all steady-state RNAs are accurately and fully edited, yet the signals guiding these precise nucleotide insertions are presently unknown. To localize the regions of the template that are required for editing, we constructed a series of chimeric templates that substitute varying amounts of DNA either upstream of or downstream from C insertion sites. Remarkably, all sequences necessary for C addition are contained within ;9 base pairs on either side of the insertion site. In addition, our data strongly suggest that sequences within this critical region affect different steps in the editing reaction. Template alterations upstream of an editing site influence nucleotide selection and/or insertion, while downstream changes affect editing site recognition and templated extension from the added, unpaired nucleotide. The data presented here provide the first evidence that individual regions of the DNA template play discrete mechanistic roles and represent a crucial initial step toward defining the source of the editing specificity in Physarum mitochondria. In addition, these findings have mechanistic implications regarding the potential involvement of the mitochondrial RNA polymerase in the editing reaction.

RNA editing of 10 Didymium iridis mitochondrial genes and comparison with the homologous genes in Physarum polycephalum

“…This is consistent with the experimental finding of Rhee et al (2009), which determined that information in the 9 bp upstream of and downstream from an insertion site are necessary for proper insertional editing.…”

“…Recently, the P. polycephalum mitochondrial RNA polymerase has been cloned (Miller et al 2006), and it has been found to add non-DNA-templated nucleotides to the 39 end of RNAs (Miller and Miller 2008). Using chimeric templates and in vitro mitochondrial transcription elongation complexes Rhee et al (2009) demonstrated the importance of 9 base pairs (bp) to either side of C insertion sites in directing this type of editing event.…”

RNA editing is absent in a single mitochondrial gene ofDidymium iridis

“…Recent work provides strong evidence for the importance of the 9 bp template sequence immediately upstream and downstream of insertion sites in directing proper editing (Rhee et al 2009). These results explained observations of a minimum distance of 9 bp between editing sites (Miller et al 1993).…”

When you can’t trust the DNA: RNA editing changes transcript sequences

“…The mitochondrial RNA polymerase has been cloned and shown to indeed add nonencoded nucleotides to the 3 0 end of nascent RNAs [178,179]. Investigating the mechanisms that guide and control the cotranscriptional insertions of nucleotides has revealed that approximately 9 bp flanking the insertion sides on both sides of the DNA template play important roles [180][181][182][183][184]. How many further biochemically distinct and independent activities of RNA editing (e.g., of a deamination type for the C-to-U conversions) actually exist in myxomycetes is currently an open question addressed with different in vitro systems [185].…”