Structure-function analysis of Rny1 in tRNA cleavage and growth inhibition

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• T2 ribonucleases (RNase T2) are crucial enzymes in eukaryotes, impacting processes from plant self-incompatibility to human neurological development and tumor progression.
• RNase T2 functions can be catalytic or catalytic-independent, but the specific protein features governing these roles are not well understood.

Purpose of the Study:

• To investigate the structural requirements for both catalytic tRNA cleavage and catalytic-independent growth inhibition by Rny1, a T2 ribonuclease in Saccharomyces cerevisiae.
• To elucidate the roles of the C-terminal extension, catalytic core, and signal peptide in Rny1 function.

Main Methods:

• Analysis of Rny1 features in Saccharomyces cerevisiae to determine requirements for in vivo tRNA cleavage and growth inhibition.
• Biochemical flotation assays to investigate tRNA association with cellular compartments.

Main Results:

• Catalytic-independent growth inhibition by Rny1 is a combinatorial property influenced by its fungal-specific C-terminal extension, conserved catalytic core, and signal peptide.
• Catalytic functions of Rny1 are independent of the C-terminal extension but require the catalytic core and signal peptide.
• Biochemical assays indicate that Rny1-mediated tRNA cleavage may involve tRNA association with or uptake into membrane compartments.

Conclusions:

• The distinct functional domains of Rny1 contribute differentially to its catalytic and catalytic-independent activities.
• Understanding these features provides insight into the diverse roles of T2 ribonucleases in cellular processes.