Structure of a pre-mRNA branch point/3' splice site region

We have analyzed the solution structure of RNA containing the branch point/3' splice site region of the first intervening sequence (IVS1) of human beta-globin pre-mRNA by nuclease mapping and NMR. Nuclease mapping indicates that there are two distinct structural domains: one contains the branch point region, and the other includes the 3' splice site and second exon sequences. In the branch point domain, the adenosine at which the RNA branch forms appears to be in the loop of a stem/loop structure. The branch point structure does not appear to interact with other parts of the RNA, since its unique nuclease digestion pattern is conserved among transcripts containing the entire intron or only the branch point region. This is confirmed by a comparison of the NMR spectra of two RNA transcripts; a distinct set of resonances appears in the spectra of the RNA containing only branch sequences or including 3' splice site/exon 2 sequences. NMR studies further show that the 3' splice site/exon 2 domain has a lower melting temperature than the branch point domain, suggesting that the two regions are distinct dynamically as well as structurally. Nuclease mapping studies of adenovirus major late IVS1 indicate that this RNA has structural features in common with the human beta-globin transcript.