PRIMARY FACULTY

David Horowitz, Ph.D.
Associate Professor
Biochemistry
 
4301 Jones Bridge Road
Bethesda MD 20814
Office: 301-295-3589
Fax: 301-295-1996
dhorowitz@usuhs.mil

Biochemistry of pre-mRNA splicing
 
Work in my lab focuses on several proteins that are required for pre-mRNA splicing. Splicing of pre-mRNA occurs via a two-step pathway. In the first step, the pre-mRNA the lariat intermediate and exon1 are generated from the pre-mRNA. In the second step, the two intermediates are converted into the mRNA and released lariat intron. The splicing reactions take place in a large particle called the spliceosome, which consists of the pre-mRNA, five small nuclear ribonuclear RNAs (snRNAs), and a large number proteins. We purified a complex of three U4/U6 snRNP-specific proteins from human cells. The first of these proteins is the human homolog of the yeast (S. cerevisiae) splicing factor Prp4, and the second is the homolog of yeast Prp3. The third protein is a cyclophilin called USA-CyP. We are studying the functions of the these three proteins in splicing. Of particular interest is the cyclophilin. Cyclophilins have been studied intensively because of their medical importance as the targets of the immunosuppressive drug cyclosporin A, but the normal cellular functions of cyclophilins have been difficult to establish. We have found that USA-CyP forms stable complexes with two splicing factors, hPrp4 and hPrp18, and apparently enters the spliceosome separately within each of these complexes, as shown in the figure. USA-CyP functions in both steps of splicing, although it is not essential for splicing.

We are also studying the second-step splicing protein Prp18. Prp18 was initially characterized in yeast and was shown to be specifically required for the second step of splicing. Subsequently, the human homolog, hPrp18, was found. The yeast and human Prp18 proteins are functionally interchangeable in human splicing. As part of a collaboration, we solved the x-ray crystal structure of yeast Prp18, which has a compact helical structure, as shown in the Figure.

Prp18 has a nearly invariant loop between helices 4 and 5, which was only partly resolved in the structure. Based on the structure and the sequence homology of the Prp18's, we carried out an extensive mutational analysis of yeast Prp18, which has allowed us to identify two roles of Prp18 in splicing. First, helices 1 and 2 of Prp18 interact directly with the second-step splicing factor Slu7, and Prp18 is needed for stable binding of Slu7 to the spliceosome. The second function requires the conserved loop and is independent of Slu7. Prp18 proteins bearing mutations in the conserved loop are dominant negatives, and we are characterizing some of these mutants in vitro and in vivo to understand what this other function of Prp18 is.

Selected Publications

Resources