RNA sequence and two-dimensional structure features required for efficient substrate modification by the...

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Biology

Background:

• Pus7p is a multisite-specific RNA:pseudouridine (Psi) synthase in yeast.
• It modifies uridines in various RNAs, including tRNA, pre-tRNA, U2 small nuclear RNA, and 5S rRNA.
• The recognition specificity of Pus7p for its diverse RNA substrates remains largely uncharacterized.

Purpose of the Study:

• To investigate the RNA sequence and structural determinants governing Pus7p recognition and modification.
• To identify key nucleotides and structural features essential for Pus7p activity.
• To elucidate the specificity of Pus7p in modifying different RNA molecules.

Main Methods:

• Site-directed mutagenesis was employed to create RNA variants of yeast tRNA(Asp)(GUC), pre-tRNA(Tyr)(GPsiA), and U2 small nuclear RNA.
• In vitro assays were performed to test the ability of these RNA variants to be modified by Pus7p.
• Analysis focused on the impact of nucleotide substitutions and structural alterations on Pus7p activity.

Main Results:

• The conserved U(-2) and A(+1) residues relative to the target uridine (U(0)) are critical for efficient Pus7p modification.
• Nucleotide substitutions at positions -4, -3, +2, and +3 had minor effects on modification efficiency.
• The nucleotide at position -1 (5' to the target U) was found to be non-essential for modification.
• Altering tRNA three-dimensional structure did not affect Pus7p activity at position 13.
• A stem-loop structure near the target uridine appears necessary for Pus7p-catalyzed modification.

Conclusions:

• Pus7p recognition is primarily determined by specific nucleotide identities at positions -2 and +1.
• Pus7p exhibits flexibility regarding nucleotides at flanking positions and overall tRNA structure.
• The presence of a local stem-loop structure is a significant requirement for Pus7p-mediated RNA modification.