A Novel SR-Related Protein Is Required for the Second Step of Pre-mRNA Splicing

Abstract

The SR family proteins and SR-related polypeptides are important regulators of pre-mRNA splicing. A novel SR-related protein of an apparent molecular mass of 53 kDa was isolated in a gene trap screen that identifies proteins which localize to the nuclear speckles. This novel protein possesses an arginine- and serine-rich domain and was termed SRrp53 (for SR-related protein of 53 kDa). In support for a role of this novel RS-containing protein in pre-mRNA splicing, we identified the mouse ortholog of the Saccharomyces cerevisiae U1 snRNP-specific protein Luc7p and the U2AF⁶⁵-related factor HCC1 as interacting proteins. In addition, SRrp53 is able to interact with some members of the SR family of proteins and with U2AF³⁵ in a yeast two-hybrid system and in cell extracts. We show that in HeLa nuclear extracts immunodepleted of SRrp53, the second step of pre-mRNA splicing is blocked, and recombinant SRrp53 is able to restore splicing activity. SRrp53 also regulates alternative splicing in a concentration-dependent manner. Taken together, these results suggest that SRrp53 is a novel SR-related protein that has a role both in constitutive and in alternative splicing.

ACKNOWLEDGMENTS

We thank Colin Semple and Philippe Gautier (MRC HGU) for help with bioinformatic analysis. We are grateful to Rachel Davies and Nick Hastie for the constructs used in two-hybrid analysis. We also thank Albrecht Bindereif for the hnRNP L expression vector. Finally, we are grateful to David S. Horowitz (Bethesda, Md.) for critical reading of the manuscript and Sonia Guil and Jeremy R. Sanford for discussions.

This work was supported by the Medical Research Council.