Proximity of the U12 snRNA with both the 5′ Splice Site and the Branch Point during Early Stages of Spliceosome Assembly

Abstract

ABSTRACT U12 snRNA is required for branch point recognition in the U12-dependent spliceosome. Using site-specific cross-linking, we have captured an unexpected interaction between the 5′ end of the U12 snRNA and the −2 position upstream of the 5′ splice site of P120 and SCN4a splicing substrates. The U12 snRNA nucleotides that contact the 5′ exon are the same ones that form the catalytically important helix Ib with U6atac snRNA in the spliceosome catalytic core. However, the U12/5′ exon interaction is transient, occurring prior to the entry of the U4atac/U6atac.U5 tri-snRNP to the spliceosome. This suggests that the helix Ib region of U12 snRNA is positioned near the 5′ splice site early during spliceosome assembly and only later interacts with U6atac to form helix Ib. We also provide evidence that U12 snRNA can simultaneously interact with 5′ exon sequences near 5′ splice site and the branch point sequence, suggesting that the 5′ splice site and branch point sequences are separated by <40 to 50 Å in the complex A of the U12-dependent spliceosome. Thus, no major rearrangements are subsequently needed to position these sites for the first step of catalysis.