BRCA1 is a breast and ovarian cancer suppressor protein, the in vivo biochemical operations of which are incompletely understood. Our long-term objective is to use molecular, cellular and genomic approaches to explore the molecular mechanism underlying the BRCA1-associated cancer as an essential prerequisite to improve medical treatment and to eventually reduce burdens of this deadly disease. The p220 product of BRCA1 is dedicated, in part, to genome integrity control. It also colocalizes with the inactive X chromosome (Xi) in female somatic cells, interacts directly or indirectly with XIST RNA - a Xi-associated non-coding RNA (ncRNA) species, and promotes proper XIST localization on Xi. BRCA1 also interacts with certain chromodomain-containing proteins, and chromodomains display RNA binding activity. Furthermore, there is emerging circumstantial evidence suggesting that BRCA1 associates in the nucleus with other ncRNA species after DNA damage. In this application, we have proposed (in Specific Aim 1) to search for specific interactions (direct or indirect) of BRCA1 with ncRNAs beyond XIST by double-tag affinity purification and, if detected, to attempt to understand the physiological significance of the relevant interactions by various molecular and cellular methods such as a combined immunostaining and RNA-FISH. In addition, given the knowledge that microRNAs are biologically important ncRNA species and RNA polymerase II products, the new evidence that expression of selected species is deregulated in multiple human tumor species, that they can, when ectopically overexpressed, markedly influence cancer development, and that BRCA1 can influence the pol ll-dependent transcription of certain genes and interacts with multiple transcription control and RNA processing proteins, we will (in Specific Aim 2) implement a genome-wide small RNA cloning and sequencing strategy to search for specific microRNA species whose expression is activated or suppressed by BRCA1. If detected, the physiological importance of these BRCA1-influenced microRNAs will be explored by overexpression or sequence specific inhibition of these microRNAs, again with the objective of shedding new light on how BRCA1 operates in vivo. Description (in lay language): BRCA1 is a tumor suppressor protein that is closely relevant to breast and ovarian cancer. By studying the RNA molecules that interact with or are affected by BRCA1, we would be able to further understand the tumor suppression function of BRCA1 and to establish a knowledge base to develop novel and more effective therapeutics for this deadly disease. ? ? ?
