The emergence of slippage-type editing in an evolution experiment

Type of content
Theses / Dissertations
Publisher's DOI/URI
Thesis discipline
Cellular and Molecular Biology
Degree name
Doctor of Philosophy
Publisher
University of Canterbury
Journal Title
Journal ISSN
Volume Title
Language
English
Date
2016
Authors
Lai, Alicia Sook Wei
Abstract

RNA editing, the correction of genomic errors by altering the sequence of messenger RNA, has evolved multiple times independently, but it remains unclear exactly how such a complex yet low-fidelity process has evolved. A model for the emergence of RNA editing proposed that RNA editing activity pre-exists, but there is no substrate for editing activity to act upon. Subsequently, mutation creates editable nucleotide sites, which may be fixed by genetic drift making RNA editing indispensable for expression of functional genes. We sought to test this model by asking whether editing-type processes can evolve under experimental conditions designed to maximize the effect of genetic drift.

Previous work in our group has shown that, in the bacterial endosymbiont Buchnera, RNA polymerase slips at long poly(A/T) tracts, leading to stochastic incorporation or removal of As or Us in the nascent messenger RNA. This results in a heterogeneous population of mRNAs. Slippage-type editing was shown to correct errors in genes which had acquired natural frameshift mutations, but this may in turn reduce expression efficiency of genes with intact reading frames. It would appear that these editing processes in Buchnera may not always be beneficial thus raising a question: if editing-type processes do not offer any inherent advantage, how did this process emerge and why has it persisted after millions of years?

In a mutation accumulation (MA) experiment, we subjected Escherichia coli populations to daily single-cell bottlenecks, mimicking the genetic background of Buchnera. After approximately 4,000 generations, a general loss of fitness was observed while one of the lineages succumbed to mutational meltdown. Genome sequencing revealed the accumulation of mutations and the emergence of 22 frameshift mutations that require slippage-type editing for the production of functional proteins. We then introduced one of the frameshift mutations into wild-type E. coli and demonstrated that RNA polymerase slippage results in a loss of fitness. Further to this, we also conducted delta-bitscore (DBS) analyses to identify functional perturbation following mutation accumulation and we showed loss of fitness may be attributed to the loss of protein function in the bottlenecked lineage that succumbed to mutational meltdown.

We subsequently assessed the impact of RNA polymerase slippage on gene expression and protein production utilising GFP reporter systems. We present data showing that slippage-type editing rescues frameshift mutations but that protein production is reduced. Our results support the hypothesis that, under conditions favouring genetic drift, editing-type processes may readily emerge. To our knowledge, this is the first experimental demonstration of the evolutionary drivers for the emergence of RNA editing.

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All Rights Reserved