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Increased efficiency of evolved group i intron spliceozymes by decreased side product formation

  • Author(s): Amini, ZN
  • Müller, UF
  • et al.
Abstract

© 2015 Amini and Müller. The group I intron ribozyme from Tetrahymena was recently reengineered into a trans-splicing variant that is able to remove 100- nt introns from pre-mRNA, analogous to the spliceosome. These spliceozymes were improved in this study by 10 rounds of evolution in Escherichia coli cells. One clone with increased activity in E. coli cells was analyzed in detail. Three of its 10 necessary mutations extended the substrate binding duplexes, which led to increased product formation and reduced cleavage at the 5′-splice site. One mutation in the conserved core of the spliceozyme led to a further reduction of cleavage at the 5′- splice site but an increase in cleavage side products at the 3′-splice site. The latter was partially reduced by six additional mutations. Together, the mutations increased product formation while reducing activity at the 5′-splice site and increasing activity at the 3′-splice site. These results show the adaptation of a ribozyme that evolved in nature for cis-splicing to transsplicing, and they highlight the interdependent function of nucleotides within group I intron ribozymes. Implications for the possible use of spliceozymes as tools in research and therapy, and as a model for the evolution of the spliceosome, are discussed.

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