An Evolution Experiment Testing the Drift Model of RNA Editing Evolution

Abstract

Enzymes that correct or alter messenger RNAs (mRNAs) have evolved independently multiple times. One proposed mechanism for the evolution of RNA editing is via genetic drift. In this model an editing activity emerges that is initially selectively neutral with no target, this allows mutations that require editing to correct to appear and persist. This creates conditions for RNA editing to be fixed in the population. This process is more likely to occur due to the accumulation of additional mutations during genetic drift. A previous mutation accumulation experiment demonstrated that pre-existing RNA polymerase slippage can correct frameshift mutations acquired through drift as. My project built upon this, aiming to test whether the same might occur for post-transcriptional RNA editing under drift. I introduced synthetic genes for RNA editing enzymes to 30 lines of Escherichia coli. These lines were evolved under high drift conditions for 50 days with constant overexpression of these RNA editing enzymes. Subsequently, the fitness impact of RNA editing on these evolved lines was assessed by inhibiting the expression of the RNA editing enzymes and measuring growth rates. Due to issues with the expression vector and evolved line strain this assay was inconclusive. These issues are detailed in this thesis, along with steps that could be taken to resolve them in future work. To assess rates of mutation and gain of mutations that may be potential editing targets, genomes for all evolved lines were sequenced and compared with ancestral strains. A significant number of mutations were observed consistent with mutation rates under high drift conditions. However, a search for specific mutations as likely RNA editing targets was largely unsuccessful. While these assays and sequence-based analyses were not successful, there may still be undetected editing in some strains. Further exploration through transcript sequencing is required. This evolution experiment generated many mutations under high drift conditions in the presence of overexpressed RNA editing enzymes. The strains generated during this evolution experiment are now ready for transcript sequencing and analysis to assess the possible impact of RNA editing upon the mutations gained and maintained throughout the experiment.