Cell cycle arrest promotes trans-hammerhead ribozyme action in yeast

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Yeast Genetics

Background:

• Hammerhead ribozymes are catalytic RNA molecules with self-cleaving activity.
• Targeting specific mRNA for degradation is a key strategy in gene regulation and therapy.
• Investigating ribozyme function in vivo is crucial for their therapeutic potential.

Purpose of the Study:

• To assess the in vivo activity of a hammerhead ribozyme designed to cleave yeast ADE1 mRNA.
• To investigate factors influencing ribozyme efficacy within a living yeast system.
• To explore the potential of ribozymes as gene-silencing agents in eukaryotes.

Main Methods:

• Expression of a hammerhead ribozyme in yeast under a galactose-inducible promoter.
• In vitro cleavage assays using RNA from induced yeast cultures.
• In vivo assessment of ADE1 mRNA levels under various stress conditions (alpha-factor, lithium acetate, nitrogen-free medium).
• Analysis of ribozyme variants with altered flanking recognition arm lengths.

Main Results:

• The expressed hammerhead ribozyme showed in vitro activity against ADE1 mRNA.
• No in vivo cleavage activity was observed under standard galactose induction.
• Significant reduction (50%) in ADE1 mRNA levels occurred upon alpha-factor treatment, lithium acetate, or nitrogen-free medium.
• Control experiments with disabled ribozymes showed no mRNA reduction.
• Increased flanking arm length of the ribozyme diminished its inhibitory effect.

Conclusions:

• Hammerhead ribozymes can exhibit trans-cleavage activity against target mRNA in vivo in yeast.
• Cellular stress conditions can modulate or enable ribozyme function.
• Ribozyme design, particularly the length of recognition arms, influences its in vivo efficacy.