Random removal of inserts from an RNA genome: selection against single-stranded RNA

We have monitored the evolution of insertions in two MS2 RNA regions of known secondary structure where coding pressure is negligible or absent. Base changes and shortening of the inserts proceed until the excessive nucleotides can be accommodated in the original structure. The stems of hairpins can be dramatically extended but the loops cannot, revealing natural selection against single-stranded RNA. The 3' end of the MS2 A-protein gene forms a small hairpin with an XbaI sequence in the loop. This site was used to insert XbaI fragments of various sizes. Phages produced by these MS2 cDNA clones were not wild type, nor had they retained the full insert. Instead, every revertant phage had trimmed the insert in a different way to leave a four- to seven-membered loop to the now extended stem. Similar results were obtained with inserts in the 5' untranslated region. The great number of different revertants obtained from a single starting mutant as well as sequence inspection of the crossover points suggest that the removal of redundant RNA occurs randomly. The only common feature among all revertants appears the potential to form a hairpin with a short loop, suggesting that single-stranded RNA negatively affects the viability of the phage. To test this hypothesis, we introduced XbaI fragments of 34 nucleotides that could form either a long stem with a small loop or a short stem with a large loop (26 nucleotides). The base-paired inserts were perfectly maintained for many generations, whereas the unpaired versions were quickly trimmed back to reduce the size of the loop. These data confirm that single-stranded RNA adversely affects phage fitness and is strongly selected against. The repair of the RNA genome that we describe here appears as the result of random recombination. Of the plethora of recombinants, only those able to adopt a base-paired structure survive. The frequency with which our inserts are removed seems higher than measured by others for small inserts in a reading frame in Q beta RNA. To account for this higher frequency, we suggest models in which the single-stranded nature of our inserts induces random recombination at the site of the insertion.