Repression of Prespliceosome Complex Formation at Two Distinct Steps by Fox-1/Fox-2 Proteins

Abstract

Precise and robust regulation of alternative splicing provides cells with an essential means of gene expression control. However, the mechanisms that ensure the tight control of tissue-specific alternative splicing are not well understood. It has been demonstrated that robust regulation often results from the contributions of multiple factors to one particular splicing pathway. We report here a novel strategy used by a single splicing regulator that blocks the formation of two distinct prespliceosome complexes to achieve efficient regulation. Fox-1/Fox-2 proteins, potent regulators of alternative splicing in the heart, skeletal muscle, and brain, repress calcitonin-specific splicing of the calcitonin/CGRP pre-mRNA. Using biochemical analysis, we found that Fox-1/Fox-2 proteins block prespliceosome complex formation at two distinct steps through binding to two functionally important UGCAUG elements. First, Fox-1/Fox-2 proteins bind to the intronic site to inhibit SF1-dependent E′ complex formation. Second, these proteins bind to the exonic site to block the transition of E′ complex that escaped the control of the intronic site to E complex. These studies provide evidence for the first example of regulated E′ complex formation. The two-step repression of presplicing complexes by a single regulator provides a powerful and accurate regulatory strategy.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/ .

ACKNOWLEDGMENTS

We thank the following individuals for providing antibodies and plasmids: Angela Krämer at the University of Geneva (anti-SF1 antibody), Stefan Stamm at the University of Erlangen (anti-Tra2β antibody), Douglas Black at the University of California at Los Angeles (MS2-MBP plasmid and anti-Fox-1 antibody), and James Patton at Vanderbilt University (His-SRp55-expressing and His-SRp35-expressing viruses). We thank Helen Salz, Jo Ann Wise, and Melissa Hinman for critical reading and editing of the manuscript.

This study was supported by an NIH grant (NS-049103-01) to H.L. H.-L.Z. was supported by a postdoctoral fellowship from the American Heart Association.