Formation of Pseudouridine in U5 Small Nuclear RNA

Abstract

The formation of pseudouridine (psi) on U5 small nuclear RNA (U5 snRNA) was studied using an in vitro modification system. Labeled U5 RNA, synthesized in vitro and therefore unmodified, was incubated in reactions containing S100 and/or nuclear extracts (NE) from HeLa cells, and the levels of psi were determined. There are three psi residues found in human U5 RNA, at positions 43, 46, and 53. Incubation of unmodified U5 RNA in reactions containing either S100 or NE supports psi formation at positions 43 and 46, which are found in a loop in the predicted secondary structure of U5 RNA. However, psi formation at position 53, which is found in a stem, is dependent on the presence of NE during the incubation. The order of extract addition does not have a significant effect on the formation of psi at position 53 as long as NE is present. The most efficient psi formation was observed with a combination of S100 and NE which allowed for efficient small nuclear ribonucleoprotein particle (snRNP) assembly and psi formation. When 9S and 20S U5 snRNPs were isolated by velocity sedimentation gradient centrifugation after incubation in the combined extracts, there was little difference in the psi levels at any of the positions for the two distinct particles. Mutations in the U5 RNA sequence do affect psi formation. U5 RNAs that have mutated Sm binding sites or are truncated prior to the Sm binding site have very low levels of psi formation at positions 43 and 46 and no detectable psi formation at position 53. A deletion of five nucleotides from 39 to 43 abolishes psi formation at positions 43 and 46, but the modification of position 53 is unaffected.

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

“…However, the observation that Pus1p catalyzes only one of the ⌿ residues detected in the S. cerevisiae UsnRNAs is in agreement with the previous observation for vertebrate UsnRNAs showing the involvement of several pseudouridine synthases (73)(74)(75). Other putative pseudouridine synthase genes are present in the S. cerevisiae genome, and we have initiated systematic gene disruption experiments in order to identify the enzymes responsible for the formation of the five other ⌿ residues that were detected in this study.…”

“…Using in vitro-transcribed snRNAs and nuclear or S100 extracts from HeLa cells, the existence of multiple RNApseudouridine synthase activities that specifically recognize U1, U2, and U5 snRNAs was demonstrated (73)(74)(75)77). However, to date, none of the implicated RNA-pseudouridine synthases has been identified, nor was their precise specificity elucidated.…”

Immunopurified Small Nucleolar Ribonucleoprotein Particles Pseudouridylate rRNA Independently of Their Association with Phosphorylated Nopp140

“…Several methods have been developed to detect the pseudouridylation of RNA, in particular, the release of 3 H from position 5 of the pyridine ring (6), the strong stops generated during reverse transcription of RNA after specific derivatization of ⌿s (2), and the separation of ⌿ from uridine by TLC after digestion of the RNA to single nucleotides (33). This last method has been used extensively to study in vitro the pseudouridylation of 32 P-labeled spliceosomal small nuclear RNAs (snRNAs) (for a review, see reference 39), which appears to depend on at least partial RNP assembly (38,40). We adapted this assay, which also supports snoRNAguided pseudouridylation of snRNA (19), for the pseudouridylation of rRNA.…”

Recognition of the spliceosomal branch site RNA helix on the basis of surface and electrostatic features

“…Pseudouridine, a post-transcriptional modification of the canonical uridine (U) residue, is prevalent in structural RNAs such as tRNA, rRNA and snRNA of all eukaryotes. In particular, ψ residues in certain conserved locations in U2 snRNA, including the branch site pairing region, have been found to be important for spliceosome assembly and function (9–11). Structural models reveal that the conserved ψ induces a dramatically different structure from that seen in its unmodified counterpart (8).…”