Abstract
The large subunit of the U2 auxiliary factor (U2AF) recognizes the polypyrimidine tract (Py-tract) located adjacent to the 3′ splice site to facilitate U2 snRNP recruitment. While U2AF is considered essential for pre-mRNA splicing, its requirement for splicing on a genome-wide level has not been analyzed. Using Solexa sequencing, we performed mRNA profiling for splicing in the Schizosaccharomyces pombe U2AF59 (prp2.1) temperature-sensitive mutant. Surprisingly, our analysis revealed that introns show a range of splicing defects in the mutant strain. While U2AF59 inactivation (nonpermissive) conditions inhibit splicing of some introns, others are spliced apparently normally. Bioinformatics analysis indicated that U2AF59-insensitive introns have stronger 5′ splice sites and higher A/U content. Most importantly, features that contribute to U2AF59 insensitivity of an intron unexpectedly reside in its 5′-most 30 nucleotides. These include the 5′ splice site, a guanosine at position 7, and the 5′ splice site-to-branch point sequence context. A differential requirement (similar to U2AF59) for introns may also apply to other general splicing factors (e.g., prp10). Our combined results indicate that U2AF insensitivity is a common phenomenon and that varied intron features support the existence of unrecognized aspects of spliceosome assembly.
ACKNOWLEDGMENTS
We thank Dick McIntosh and his laboratory for help with the yeast work, Judith Potashkin, and Tokio Tani for plasmids, strains, and reagents/antibodies, and Tom Cech, Tom Blumenthal, Mark Winey, and Rajesh Gaur for helpful discussions and critical reading of the manuscript.
This work was supported in part by the American Cancer Society and a Butcher award to R.S.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01000-10.