Pseudouridine Mapping in the Saccharomyces cerevisiae Spliceosomal U Small Nuclear RNAs (snRNAs) Reveals that Pseudouridine Synthase Pus1p Exhibits a Dual Substrate Specificity for U2 snRNA and tRNA

Abstract

Pseudouridine (⌿) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few ⌿ residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-pseudouridine synthase Cbf5p were tested for UsnRNA ⌿ content, only the loss of the Pus1p activity was found to affect ⌿ formation in spliceosomal UsnRNAs. Indeed, ⌿ 44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze ⌿ 44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNApseudouridine synthase Cbf5p had no effect on UsnRNA ⌿ content, formation of ⌿ residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.Introns are universally present in the nuclear genes transcribed by RNA polymerase II. Introns with GU and AG terminal dinucleotides and some introns with AU and AC terminal dinucleotides are removed by spliceosomal complexes containing the U1, U2, U4, U5, and U6 small nuclear RNAs (UsnRNAs) (for reviews, see references 54 and 59), the remaining part of introns with AU and AC terminal dinucleotides being excised by complexes containing the U11, U12, U4atac, U5, and U6atac UsnRNAs (32,79,106,105). In yeast cells, only introns with GU and AG borders have been detected, and their excision is catalyzed by ribonucleoprotein complexes containing UsnRNAs homologous to the vertebrate U1, U2, U4, U5, and U6 snRNAs (for a review, see reference 31). However, compared to their counterparts in other eukaryotes, the Saccharomyces cerevisiae spliceosomal UsnRNAs differ by their larger size. For example, U2 snRNA is 1,175 nucleotides (nt) long in S. cerevisiae versus 187 nt in humans, and U1 snRNA is 568 nt long in S. cerevisiae versus 164 nt in humans (for a review, see reference 31).In spite of this difference, the splicing machineries for the elimination of the GU-AG type of introns, in both vertebrates and S. cerevisiae, share several common properties. In particular, UsnRNPs are assembled in the same sequential order (17, 27; for a review, see reference 59), and the same kinds of bi-and multimolecular RNA-RNA interactions are implicated. The picture that now emerges from a large body of experiments in several laboratories is rather complex. First, upon U1 snRNP association, the 5Ј extremity of U1 forms a base-pair interaction with the intron 5Ј extremity (62,94,95,96,123). Then, the U2 snRNP is associated and a base-pair interaction is formed between U2 and the intron branch-point sequence (71,72,116,124). A U4/U6 RNA duplex is presen...

Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg2+ Binding Site is Located Close to the Stacked Branch Adenosine

“…[9] In all eukaryotic spliceosome systems investigated to date, a pseudouridine (Ψ) residue opposite of the branchpoint adenosine has been identified. [36,37] NMR structural analyses have shown that this Ψ is responsible for an extrahelical positioning of the branch adenosine in the isolated branch helix. [35,38] Although no Ψ has been identified in group II introns to date, the relationship with the spliceosome makes a bulged-out adenosine in context of the whole group II intron also tempting to propose.…”

The Specificities of Four Yeast Dihydrouridine Synthases for Cytoplasmic tRNAs

“…In yeast, the only other known members of this class are Trm7p, which catalyzes 2Ј-O-methylation at positions 32 and 34 (29), and Pus3p, which forms ⌿ 38 and ⌿ 39 (30). Other modification enzymes act either at multiple sites, like Pus1p (6,31,32) and Trm4 (33), or, more commonly, at single sites. This region specificity argues either for conformational flexibility between the protein binding site and the active site or a flexible region of the tRNA substrate.…”

Pseudouridines in spliceosomal snRNAs

“…Interestingly, early efforts showed that pseudouridylation in yeast was fundamentally different from that in higher eukaryotes. Specifically, using both in vivo and in vitro assays, the Branlant lab indicated that Pus1, a single-peptide enzyme known to pseudouridylate yeast tRNA at various positions, also catalyzes pseudouridylation of yeast U2 at position 44 (Massenet et al, 1999).…”

“…6). Taken together, pseudouridylation of yeast U2 snRNA (and perhaps the other yeast spliceosomal snRNAs as well) is catalyzed by two completely different mechanisms: the RNA-independent mechanism (Pus7 and Pus1), which modifies U2 at positions 35 and 44 (Massenet et al, 1999;Ma et al, 2003), and the RNA-dependent mechanism (snR81) that pseudouridylates U2 at position 42 (Ma et al, 2005).…”