Abstract
Intronic G triplets are frequently located adjacent to 5′ splice sites in vertebrate pre-mRNAs and have been correlated with splicing efficiency and specificity via a mechanism that activates upstream 5′ splice sites in exons containing duplicated sites (26). Using an intron dependent upon G triplets for maximal activity and 5′ splice site specificity, we determined that these elements bind U1 snRNPs via base pairing with U1 RNA. This interaction is novel in that it uses nucleotides 8 to 10 of U1 RNA and is independent of nucleotides 1 to 7. In vivo functionality of base pairing was documented by restoring activity and specificity to mutated G triplets through compensating U1 RNA mutations. We suggest that the G-rich region near vertebrate 5′ splice sites promotes accurate splice site recognition by recruiting the U1 snRNP.
ACKNOWLEDGMENTS
We thank the members of the Berget laboratory, especially Leslie Landree and Hua Lou, for valuable discussions throughout the course of this project. We recognize Valerija Vitkauskas, an undergraduate summer student, for performing several transfection experiments, and we acknowledge the dedicated technical assistance of Wade Wilson. We thank James Manley (Columbia University) for providing us with ASF-SF2 and SC35 expression plasmids, and we acknowledge Alan Weiner (University of Washington) for providing the wild-type U1 snRNA gene used in these studies.
This work was supported by grant RO1 GM38526 to S.M.B.