This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.MicroRNAs are small non-coding regulatory RNAs implicated in both development and cancer. MicroRNA precursors are processed by Microprocessor, a complex consisting of the endonuclease drosha and its partner, the DGCR8 protein. Drosha is also part of another larger nuclear complex, hereafter called Macroprocessor. Macroprocessor contains several RNA binding and unwinding factors as well as EWS, an RNA-binding protein involved in human tumors, but its function and the identities of its substrates are unknown. I intend to characterize the Macroprocessor complex and identify its substrates. In order to study drosha?s function, I have previously employed retroviral siRNA-producing vectors to reduce drosha protein levels in HeLa cells, and found that knockdown cells show reduced viability by a colony formation assay. Rnt1, the sole RNAse III in budding yeast, has several mRNA substrates. To determine if drosha, too, has a role in mRNA processing, I conducted an expression analysis of the knockdowns using microarrays. There were a total of 44 probe sets identified, 17 of which increased in the drosha knockdowns, and 27 of which decreased. One of the 27 targets that decreased (c-jun) was identified with two different probe sets. Another decreasing target in all four comparisons was, as expected, the drosha mRNA itself. None of the targets (except drosha) display homology to any of the siRNA constructs, making it unlikely that they are off-target effects. I am building on my substantial body of preliminary data, capitalizing on my unique and powerful reagents (the affinity-purified anti-drosha antibody and the anti-drosha siRNA knockdown constructs), as well as the extensive biochemical and mass spectrometric resources of the Chait lab to characterize the role of drosha in the Macroprocessor complex.
My specific aims i nclude:1. Isolation and identification of drosha substrates2. Determination of drosha?s dynamics within the cell cycle
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