rRNA fragmentation induced by a yeast killer toxin.

Area of Science:

• Microbiology
• Molecular Biology
• Yeast Genetics

Background:

• Virus-like dsDNA elements (VLEs) in yeast encode killer toxins targeting tRNA.
• PaT and zymocin are known VLE-encoded toxins with anticodon nuclease (ACNase) activities.

Purpose of the Study:

• To characterize a novel VLE-encoded toxin, PiT, from Pichia inositovora.
• To identify the cytotoxic subunit of PiT and elucidate its RNA target and mechanism of action.

Main Methods:

• Conditional expression of PiOrf4 in Saccharomyces cerevisiae.
• Assessing toxicity rescue by manipulating tRNA modification and expression.
• Purification and characterization of rRNA cleavage products using linker ligation and reverse transcription.
• Site-directed mutagenesis of putative catalytic residues in PiOrf4.

Main Results:

• PiOrf4, the cytotoxic subunit of PiT, was identified.
• PiOrf4 toxicity was not rescued by altering tRNA modifications or expression.
• PiOrf4 induced specific fragmentation of 25S and 18S rRNA.
• Cleavage occurred at nucleotides 131-132 of 18S rRNA.
• Mutations in conserved catalytic residues (Glu9, His214) abolished PiOrf4 toxicity and rRNA fragmentation.

Conclusions:

• PiOrf4 represents a novel class of yeast killer toxins targeting rRNA, distinct from tRNA-targeting toxins.
• PiOrf4 possesses RNase activity, potentially sharing conserved catalytic modules with ACNase toxins.
• This discovery expands the known mechanisms of VLE-mediated cytotoxicity in yeast.