Multiple Distinct Splicing Enhancers in the Protein-Coding Sequences of a Constitutively Spliced Pre-mRNA

Abstract

We have identified multiple distinct splicing enhancer elements within protein-coding sequences of the constitutively spliced human β-globin pre-mRNA. Each of these highly conserved sequences is sufficient to activate the splicing of a heterologous enhancer-dependent pre-mRNA. One of these enhancers is activated by and binds to the SR protein SC35, whereas at least two others are activated by the SR protein SF2/ASF. A single base mutation within another enhancer element inactivates the enhancer but does not change the encoded amino acid. Thus, overlapping protein coding and RNA recognition elements may be coselected during evolution. These studies provide the first direct evidence that SR protein-specific splicing enhancers are located within the coding regions of constitutively spliced pre-mRNAs. We propose that these enhancers function as multisite splicing enhancers to specify 3′ splice-site selection.

ACKNOWLEDGMENTS

We thank Brenton Graveley, Klemens Hertel, Bhavin Parekh, Christopher Sears, Jinghua Yang, and other members of Maniatis lab; and Kevin Jarrell (Boston University School of Medicine), Kristen W. Lynch (University of California, San Francisco), Robin Reed (Harvard Medical School), and Ming Tian (Harvard Medical School) for helpful discussions, encouragement, and critical comments on the manuscript. We are grateful to Jim Bruzik (Case Western Reserve University) for his S100 extract preparation protocol; Renate Gattoni and James Stévenin (CNRS, Strasbourg, France), Adrian Krainer (Cold Spring Harbor Laboratory), and Mark Roth (Fred Hutchinson Cancer Research Center) for monoclonal antibodies/hybridomas; Michael Zhang (Cold Spring Harbor Laboratory) for communicating unpublished data; and Dave Smith (Harvard University Biological Laboratories Imaging Center) for help with figure preparation.

This work was supported by National Institutes of Health grant GM42231 to T.M.