The Human HDV-like CPEB3 Ribozyme Is Intrinsically Fast-Reacting

摘要

Self-cleaving RNAs have recently been identified in mammalian genomes. A small ribozymenrelated in structure to the hepatitis delta virus (HDV) ribozyme occurs in a number of mammals, includingnchimpanzees and humans, within an intron of the CPEB3 gene. The catalytic mechanisms for the CPEB3 andnHDV ribozymes appear to be similar, generating cleavage products with 50n-hydroxyl and 20n,30n-cyclicnphosphate termini; nonetheless, the cleavage rate reported for the CPEB3 ribozyme is more than 6000-foldnslower than for the fastest HDV ribozyme. Herein, we use full-length RNA and cotranscriptional self-ncleavage assays to compare reaction rates among human CPEB3, chimp CPEB3, and HDV ribozymes. Ourndata reveal that a single base change of the upstreamflanking sequence, which sequesters an intrinsically weaknP1.1 pairing in a misfold, increases the rate of the wild-type human CPEB3 ribozyme by ∼250-fold; thus, thenhuman ribozyme is intrinsically fast-reacting. Secondary structure determination and native gel analysesnreveal that the cleaved population of the CPEB3 ribozyme has a single, secondary structure that closelynresembles the HDV ribozyme. In contrast, the precleavage population of the CPEB3 ribozyme appears tonhave a more diverse secondary structure, possibly reflecting misfolding with the upstream sequence andndynamics intrinsic to the ribozyme. Prior identification of expressed sequence tags (ESTs) in human cellsnindicated that cleavage activity of the human ribozyme is tissue-specific. It is therefore possible that cellularnfactors interact with regions upstream of the CPEB3 ribozyme to unmask its high intrinsic reactivity.