U2 snRNA sequences that bind U2-specific proteins are dispensable for the function of U2 snRNP in splicing

Previously we showed that microinjection of purified U2 snRNA from HeLa cells into Xenopus laevis oocytes, depleted of their endogenous U2 snRNPs by oligonucleotide-targeted degradation, led to assembly of hybrid snRNPs that were fully functional for splicing of SV40 late pre-mRNA. We have extended these results by examining features of U2 RNA that are required for its role in splicing. Injection of Xenopus U2 snRNA transcribed in vitro by T7 RNA polymerase, differing in sequence from authentic U2 by only one nucleotide, although capable of efficient assembly into snRNP-like particles, did not complement U2-predepleted oocytes for splicing. However, when injected into pretargeted oocytes, a plasmid containing Xenopus U2 snRNA sequences resulted in synthesis of U2 snRNA that was assembled into snRNPs capable of mediating splicing of SV40 late pre-mRNA. This allowed us to test several U2 RNA mutants for their function in splicing. Mutants with sequences deleted within U2 stem-loops I and II, although efficiently assembled into snRNP-like particles upon injection, failed to restore splicing. Interestingly, however, injection of a mutant that lacks the binding site for the U2-specific proteins A' and B", restored pre-mRNA splicing. These data suggest that the direct binding of U2-specific proteins with snRNA is not essential for the function of U2 snRNPs in splicing of pre-mRNA.