Pseudouridine Modification of U5 RNA in Ribonucleoprotein Particles Assembled In Vitro

Abstract

The formation of pseudouridine (Ψ) in U5 RNA during ribonucleoprotein (RNP) assembly was investigated by using HeLa cell extracts. In vitro transcribed, unmodified U5 RNA assembled into an RNP particle with the same buoyant density and sedimentation velocity as did U5 small nuclear RNP from extracts. The greatest amount of Ψ modification was detected when a combination of S100 and nuclear extracts was used for assembly. Ψ formation was inhibited when ATP and creatine phosphate or MgCl₂ were not included in the assembly reaction, paralleling the inhibition of RNP particle formation. A time course of assembly and Ψ formation showed that Ψ modification lags behind RNP assembly and that at very early time points, Sin-reactive U5 small nuclear RNPs are not modified. Two of three Ψ modifications normally found in U5 RNA were present in RNA incubated in the extracts. Mutations in the form of deletions and truncations were made in the U5 sequence, and the effect of these mutations on Ψ formation was investigated. A mutation in the area of stem-loop I which contains the Ψ moieties or in the Sm binding sequence affected Ψ formation.

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ERRATUM: Pseudouridine Modification of U5 RNA in Ribonucleoprotein Particles Assembled In Vitro