Validation and pathway dissection of disease genes using endoribonucelase prepared siRNAs

Coordinator:    Prof. Dr. Frank Buchholz
Institution: Medizinische Fakultät der TU Dresden, Universitäts Krebszentrum (UCC), Universitätsklinikum Carl Gustav Carus

The scientific goal of this project was to obtain functional data on protein-protein interactions via loss of function studies. Using the highly efficient and specific endoribonuclease prepared (e)siRNA resources developed in our laboratory we were able to determine protein complexes and to study comprehensively protein landscapes via esiRNA knockdown of specific protein candidates. Based on a genome-wide RNAi screen several novel disease-related genes could be classified. In one study, a novel protein complex related to the paraplegia hereditary disease (HSP) was identified and functional interactions between HSP and DNA repair could be determined. Moreover, esiRNA knockdowns in TP53 positive and TP53 negative human cell lines have identified factors selectively required for the growth of TP53 negative cells. Knockdown of the gene UNRIP specifically affected TP53 negative cells and was studied in detail, uncovering a possible vulnerability of TP53-negative tumors. Because TP53 is one of the most frequently mutated genes in cancer this discovery might reveal an “Achilles heel” of many cancer cell types. Furthermore three main protein complexes were studied in detail via RNAi. Transgenic BAC cell lines (bacterial artificial chromosomal cell lines) were generated for all complex parts and the protein levels as well as the location of binding proteins were studied after knockdown of all complex components. It could be demonstrated that the knockdown of single complex components lead to a complete instability of the whole protein complex as the measured cell protein level was reduced after knockdown. These results will most likely enable us to measure global protein-level dependencies in the future but also to specifically regulate proteins of a complex. In addition, we identified HOT1 as direct telomere-binding protein in vertebrates. Depletion and overexpression experiments classified HOT1 as a positive regulator of telomere lengths and proved that HOT1 associates with the active telomerase complex. As the length of telomeres is directly related to the cellular aging process and to cancer, the positive functional role of HOT1 shown in our laboratory represents a significant therapeutic target.

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Fig: Immuno-fluorescence staining of HOT1 (green) at chromosome ends
of mouse pachytene chromosome spreads (red) is shown.

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