RNA sequence and two-dimensional structure features required for efficient substrate modification by the Saccharomyces cerevisiae RNA:Ψ-synthase Pus7p

Abstract

The RNA:pseudouridine (Ψ) synthase Pus7p of Saccharomyces cerevisiae is a multisite-specific enzyme that is able to modify U13 in several yeast tRNAs, U35 in the pre-tRNATyr (GΨA), U35 in U2 small nuclear RNA, and U50 in 5 S rRNA. Pus7p belongs to the universally conserved TruD-like family of RNA:Ψ-synthases found in bacteria, archaea, and eukarya. Although several RNA substrates for yeast Pus7p have been identified, specificity of their recognition and modification has not been studied. However, conservation of a 7-nt-long sequence, including the modified U residue, in all natural Pus7p substrates suggested the importance of these nucleotides for Pus7p recognition and/or catalysis. Using site-directed mutagenesis, we designed a set of RNA variants derived from the yeast tRNAAsp(GUC), pre-tRNATyr(GΨA), and U2 small nuclear RNA and tested their ability to be modified by Pus7p in vitro. We demonstrated that the highly conserved U-2 and A+1 residues (nucleotide numbers refer to target U0) are crucial identity elements for efficient modification by Pus7p. Nucleotide substitutions at other surrounding positions (-4, -3, +2, +3) have only a moderate effect. Surprisingly, the identity of the nucleotide immediately 5′ to the target U0 residue (position -1) is not important for efficient modification. Alteration of tRNA three-dimensional structure had no detectable effect on Pus7p activity at position 13. However, our results suggest that the presence of at least one stem-loop structure including or close to the target U nucleotide is required for Pus7p-catalyzed modification. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.