Interfering with protein-protein interactions in the spliceosme
Final Report Abstract
Pre-mRNA splicing is carried out by the spliceosome, which is assembled anew on each pre-mRNA intron by the stepwise interaction of snRNPs and non-snRNP splicing factors. During assembly, maturation and catalysis, the spliceosome‟s RNA-RNA, RNA-protein and protein-protein interaction networks are repeatedly remodeled. To stall the spliceosome at particular, perhaps novel, assembly stages, we had proposed to characterize spliceosomal protein-protein complexes and to develop small molecule modulators of these interactions. We determined a structure of the complex between the yeast (y) Aar2 protein and the RNaseH-like and Jab1/MPN-like domains of the yPrp8 protein and screened for modulators of this interaction using an in silico approach. We identified a number of spliceosomal proteins interacting at the N-terminal domain of the human (h) Prp38 protein, determined the structure of this domain, mapped interaction sites using a surface-scanning mutagenesis approach and initiated a fragment-based search for small molecule modulators of these interactions. Using a time-resolved FRET-based high-throughput assay, we identified small molecules that interfere with the interaction of the yBrr2 helicase and a C-terminal fragment of yPrp8, validated the modulators using ALPHA Screen technology and characterized the activities of the substances by surface plasmon resonance, RNA unwinding and in vitro splicing assays. Finally, we developed a stage-specific highthroughput in vitro splicing assay that allowed us to identify a group of psoromic acid derivatives and of zinc chelating small molecules as novel families of pre-mRNA splicing inhibitors. The results provide a number of novel leads which now can be optimized to develop efficient spliceosome inhibitors.
Publications
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(2011). Mechanism for Aar2p function as a U5 snRNP assembly factor. Genes Dev., 25, 1601-1612
Weber, G., Cristao, V.F., Alves, F. L., Santos, K.F., Holton, N., Rappsilber, J., Beggs, J.D., Wahl, M.C.
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(2012). Molecular architecture of zinc chelating small molecules that inhibit spliceosome assembly at an early stage. RNA, 18, 1605-1611
Patil, V., Canzoneri, J.C., Samatov, T.R., Lührmann, R., Oyelere, A.K.
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(2012). Psoromic acid derivatives: a new family of small-molecule pre-mRNA splicing inhibitors discovered by a stage-specific high-throughput in vitro splicing assay. Chembiochem., 13, 640-644
Samatov, T.R., Wolf, A., Odenwälder, P., Bessonov, S., Deraeve, C., Bon, R.S., Waldmann, H., Lührmann, R.
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(2013). Structural basis for dual roles of Aar2p in U5 snRNP assembly. Genes Dev., 27, 525-540
Weber, G., Cristao, V.F., Santos, K.F., Mozaffari Jovin, S., Heroven, A.C., Holton, N., Lührmann, R., Beggs, J.D., Wahl M.C.
