Evaluating group I intron catalytic efficiency in mammalian cells

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Biochemistry

Background:

• Group I ribozymes from Tetrahymena thermophila show potential for trans-splicing reactions to repair mutant RNAs.
• Therapeutic applications require efficient ribozyme function within human cells, but intracellular expression effects are not well understood.

Purpose of the Study:

• To evaluate the self-splicing efficiency of group I introns expressed via RNA polymerase II in human cells.
• To directly measure ribozyme catalysis in a therapeutically relevant context.
• To investigate the influence of flanking sequences on ribozyme activity within the cellular environment.

Main Methods:

• Transfection of human cell lines with intron-containing expression cassettes.
• Analysis of RNA transcripts for intron removal to assess self-splicing efficiency.
• In vitro folding and self-splicing assays to correlate with in vivo activity.

Main Results:

• Self-splicing efficiency ranged from 0% to 50% depending on the specific construct.
• Self-splicing activity is supported in the mammalian cellular environment.
• Flanking sequences significantly influenced the extent of self-splicing, likely by affecting intron folding into an active conformation.

Conclusions:

• The sequence context surrounding group I introns is critical for their catalytic efficiency in therapeutic RNA repair.
• In vitro folding assays correlate well with in vivo self-splicing activity, providing a predictive model.
• The described self-splicing system aids in evaluating and enhancing in vivo ribozyme activity for RNA repair applications.