A comprehensive tRNA pseudouridine map uncovers targets dependent on human stand-alone pseudouridine synthases

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Pseudouridine (Ψ) is a prevalent RNA modification in human cells, crucial for RNA function.
• Pseudouridine synthases (PUS) catalyze Ψ formation, but their specific targets and biological roles are not fully understood.
• Understanding PUS-mediated Ψ modifications is key to deciphering RNA biology and function.

Purpose of the Study:

• To systematically identify targets of stand-alone pseudouridine synthases (PUS) in human cells.
• To map pseudouridine (Ψ) modifications across human tRNAs and their relationship with PUS enzymes.
• To investigate the functional roles of PUS enzymes in tRNA processing.

Main Methods:

• Systematic knockout and knockdown of nine stand-alone PUS enzymes in HCT116 cells.
• Mapping of pseudouridine (Ψ) profiles using 2-bromoacrylamide-assisted cyclization sequencing.
• Integration of new data with existing findings to create a comprehensive PUS-dependent Ψ modification map.

Main Results:

• Uncovered novel targets for RPUSD1, RPUSD2, PUS3, PUSL1, and PUS7L.
• Revealed redundant function of TRUB1 and PUS10 in catalyzing Ψ55 modification in cytosolic tRNAs.
• Demonstrated distinct stages of pre-tRNA processing for PUS-dependent Ψ modifications.
• Identified RPUSD3 and TRUB2 as lacking detectable enzymatic activity in human cells.

Conclusions:

• Established a comprehensive map of stand-alone PUS-dependent Ψ modifications in human tRNAs.
• Provided insights into the functional diversification of PUS enzymes.
• Highlighted the dynamic role of PUS enzymes in different stages of tRNA biogenesis.