Engineering degradation-resistant messenger RNAs in yeast

ABSTRACT Virally-derived RNA structures provide useful tools for engineering messenger RNA (mRNA) transcripts with unique properties. Here we show that 5’→3’ e x oribonuclease- r esistant RNA structures (xrRNAs) from Flaviviruses can be used to protect heterologously-expressed messenger RNAs (mRNAs) from 5’→3’ degradation in Saccharomyces cerevisiae (budding yeast). Installation of xrRNAs ahead of a downstream internal ribosome entry site (IRES) leads to the accumulation of partially-degraded mRNAs designed to undergo cap-independent translation. In a yeast strain favoring cap-independent translation, this strategy led to a 30-fold increase in the enzymatic activity of lysates obtained from cells expressing degradation-resistant reporters. Overall, these finding demonstrate the possibility of coupling multiple viral RNA structures together to enhance protein expression.