Pseudouridine synthase 3 from mouse modifies the anticodon loop of tRNA

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Modification

Background:

• Pseudouridines are essential RNA modifications impacting tRNA structure and function.
• Yeast pseudouridine synthase 3 (Pus3) is known to modify specific uridine positions in yeast tRNA.
• The homologous enzyme in mammals, mouse pseudouridine synthase 3 (mPus3p), had not been fully characterized.

Purpose of the Study:

• To clone and characterize the mouse pseudouridine synthase 3 (mPus3p) gene and protein.
• To investigate the enzymatic activity of mPus3p on tRNA substrates.
• To determine the specific positions modified by mPus3p in mammalian tRNA.

Main Methods:

• Cloning of the mPus3p cDNA.
• Northern blot analysis to detect mPus3p mRNA expression.
• In vitro translation of mPus3p.
• Enzymatic assays using in vitro translated mPus3p and yeast/human tRNA substrates.

Main Results:

• The mPus3p cDNA was cloned, encoding a 481-amino acid protein with 34% identity to yeast Pus3.
• Northern blot analysis revealed an 1.8 kb mRNA transcript in mouse tissues and cultured cells.
• In vitro assays demonstrated that mPus3p efficiently converts uridine to pseudouridine at position 39 in both yeast and human tRNA.
• A very slight modification at position 38 of human tRNA(Leu) was observed.

Conclusions:

• Mouse pseudouridine synthase 3 (mPus3p) is a functional enzyme capable of pseudouridylation.
• mPus3p primarily targets uridine at position 39 in tRNA, similar to its yeast counterpart.
• This study provides molecular insights into mammalian tRNA pseudouridylation, a critical post-transcriptional modification.