Uniformed Services University of the Health Sciences
Department of Biochemistry and Molecular Biology
4301 Jones Bridge Road, C1094
Bethesda, Maryland 20814-4799
Fax: (301) 295-3512
Lab: (301) 295-3563
We are working on understanding the mechanism of pre-mRNA splicing. Pre-mRNA splicing takes place in a large particle called the spliceosome, which consists of the pre-mRNA, five small nuclear RNAs (snRNAs), and a large number proteins. Splicing occurs via a two-step pathway. In the first step, the pre-mRNA the lariat intermediate and exon 1 are generated from the pre-mRNA. In the second step, the two intermediates are converted into the mRNA and released lariat intron. We have focused on the second step of splicing during which a transesterification reaction produces the final mRNA from the splicing intermediates. The second-step spliceosome is composed of the U2, U5, and U6 snRNPs together with many proteins; a number of proteins enter the spliceosome at specific stages following the first step, causing structural changes in the spliceosome that culminate in the second splicing reaction.
Our work has been centered on the Prp18 protein, which joins the spliceosome just prior to the transesterification reaction that generates the final spliced products. We showed that Prp18 acts in concert with the U5 snRNP during the second step of splicing and that an evolutionarily conserved region of Prp18 stabilizes the interaction of the splicing intermediates with the U5 snRNA. Analysis of the splicing of pre-mRNA substrates with mutations near the ends of the exons led us to propose a revised model of the interactions of the splicing intermediates with the U5 snRNA at the time of the second transesterification reaction (see Figure). In this model the exons are aligned to form a continuous helix that facilitates the second transesterification reaction. The model suggests that the interaction of exon1 with loop 1 of U5 changes from the first step to the second.
We are also studying the cyclophilin USA-CyP (PPi H). 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. USA-CyP was initially identified as a component of the U4/U6 snRNP through its strong association with the hPrp3 and hPrp4 proteins. We showed 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.
L. B. Crotti, D. Bacíková, and D. S. Horowitz, "Prp18 Stabilizes the Interaction of Both Exons with the U5 snRNA during the Second Step of pre-mRNA Splicing," Genes & Dev., 21, 1204-1216 (2007)
D. Bacíková and D. S. Horowitz, "Genetic and Functional Interaction of Evolutionarily Conserved Regions of the Prp18 Protein and the U5 snRNA," Mol. Cell. Biol., 25, 2107-2116 (2005)
D. Bacíková and D. S. Horowitz, "Mutational Analysis Identifies Two Separable Roles of the Saccharomyces cerevisiae Splicing Factor Prp18", RNA, 8, 1280-1293 (2002)
D. S. Horowitz, E. J. Lee, S. Mabon, and T. Misteli, "The Cyclophilin USA-CyP Functions in pre-mRNA Splicing," EMBO J, 21, 470-480 (2002) [highlighted in Science, 295, 931 (2002)]