Abstract
The 2'-OH group of the branch point adenosine is a key moiety to initiate pre-mRNA splicing. We use RNA-guided RNA modification to target the pre-mRNA branch point adenosine for 2'-O-methylation, with the aim of blocking pre-mRNA splicing in vertebrate cells. We show that, under certain conditions, injection of a branch point-specific artificial box C/D RNA into Xenopus oocytes effectively 2'-O-methylates adenovirus pre-mRNA at the target nucleotide. However, 2'-O-methylation at the authentic branch point activates a host of cryptic branch points, thus allowing splicing to continue. These cryptic sites are mapped, and mutated. Upon injection, pre-mRNA free of cryptic branch points fails to splice when the branch point-specific box C/D RNA is present. However, 2'-O-methylation at the branch point does not prevent pre-mRNA from being assembled into pre-catalytic spliceosome-like complexes prior to the first chemical step of splicing. Our results demonstrate that RNA-guided pre-mRNA modification can occur in the nucleoplasm of vertebrate cells, thus offering a powerful tool for molecular biology research.
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