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

ABSTRACT: 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 trans-splicing, 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.

SUBMITTER: Amini ZN

PROVIDER: S-EPMC4509937 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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

Amini Zhaleh N ZN Müller Ulrich F UF

RNA (New York, N.Y.) 20150623 8

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 ...[more]

PMID: 26106216

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Project description:Mobile bacterial group II introns are evolutionary ancestors of spliceosomal introns and retroelements in eukaryotes. They consist of an autocatalytic intron RNA (a "ribozyme") and an intron-encoded reverse transcriptase, which function together to promote intron integration into new DNA sites by a mechanism termed "retrohoming". Although mobile group II introns splice and retrohome efficiently in bacteria, all examined thus far function inefficiently in eukaryotes, where their ribozyme activity is limited by low Mg2+ concentrations, and intron-containing transcripts are subject to nonsense-mediated decay (NMD) and translational repression. Here, by using RNA polymerase II to express a humanized group II intron reverse transcriptase and T7 RNA polymerase to express intron transcripts resistant to NMD, we find that simply supplementing culture medium with Mg2+ induces the Lactococcus lactis Ll.LtrB intron to retrohome into plasmid and chromosomal sites, the latter at frequencies up to ~0.1%, in viable HEK-293 cells. Surprisingly, under these conditions, the Ll.LtrB intron reverse transcriptase is required for retrohoming but not for RNA splicing as in bacteria. By using a genetic assay for in vivo selections combined with deep sequencing, we identified intron RNA mutations that enhance retrohoming in human cells, but <4-fold and not without added Mg2+. Further, the selected mutations lie outside the ribozyme catalytic core, which appears not readily modified to function efficiently at low Mg2+ concentrations. Our results reveal differences between group II intron retrohoming in human cells and bacteria and suggest constraints on critical nucleotide residues of the ribozyme core that limit how much group II intron retrohoming in eukaryotes can be enhanced. These findings have implications for group II intron use for gene targeting in eukaryotes and suggest how differences in intracellular Mg2+ concentrations between bacteria and eukarya may have impacted the evolution of introns and gene expression mechanisms.

Dataset Information

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

Publications

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

Similar Datasets

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