Yeast tRNA precursor mutated at a splice junction is correctly processed in vivo

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Transfer RNA (tRNA) genes are crucial for protein synthesis.
• Post-transcriptional modifications, including splicing, are essential for tRNA maturation.
• Ochre suppressors, like SUP4 in yeast, are specialized tRNAs that insert tyrosine and can be used to study tRNA processing.

Purpose of the Study:

• To investigate the molecular basis of altered tRNA expression in yeast mutants.
• To identify mutations affecting the processing of a tyrosine-inserting ochre suppressor tRNA (tRNATyr).
• To elucidate the role of splice junction sequences in tRNA precursor maturation.

Main Methods:

• Yeast genetics: Selection and screening for mutants with altered SUP4 suppressor activity.
• In vivo analysis: Assessing tRNA gene product production.
• RNA analysis: RNA sequencing to identify mutations and analyze precursor and mature tRNA levels.
• Biochemical assays: Evaluating tRNA splicing and processing fidelity.

Main Results:

• A yeast mutant with reduced SUP4 suppressor activity exhibited decreased mature tRNATyr levels.
• Accumulation of a precursor tRNA containing an intervening sequence was observed in the mutant.
• RNA sequence analysis revealed an A-to-G transition at the 5' splice junction of the tRNATyr precursor.
• Despite the mutation, the intervening sequence was accurately excised, and mature tRNA was produced.

Conclusions:

• The specific nucleotide sequence at the tRNA splice junction influences the efficiency of precursor processing.
• The identified mutation affects the rate of intervening sequence removal but not the accuracy of splicing.
• This study highlights the importance of splice site sequences in regulating RNA processing pathways in yeast.