The broad long-term objectives to this proposal are to identify new factors involved in histone mRNA 3' end formation both biochemically and genetically and to characterize those proteins in vivo and in vitro.
Specific Aim 1 is designed to use a combination of classical and innovative biochemical techniques to identify and purify proteins binding in a functionally relevant manner to histone pre-mRNA in HeLa nuclear extract.
Specific Aim 2 will involve the use of a novel GFP based genetic screen to be performed in Drosophila melanogaster aimed at identifying loss of function mutants in the process. This screen will be performed to near saturating levels with the aid of an embryo sorter.
Specific Aim 3 is to develop an in vivo system to study the function relevance of identified factors as well as to characterize them. This in vivo assay may also be applied in a novel siRNA-based genetic screen in mammalian cells. An in vitro system will also be developed to study histone mRNA 3' end formation in the context of active transcription. Histone mRNA 3' end formation is intimately related to the cell cycle and DNA replication, thus understanding this process may lead to the production of more effective chemotherapeutic agents.
