Breast cancer is a worldwide devastating disease and the most prevalent cancer in women. The State of Qatar is no exception with breast cancer having the highest incidence rate at 31% of all cancers in women (rates acquired from Qatar Cancer registry) and leads mortality rates as well. Significant advances in our understanding of the molecular underpinnings of breast cancer have undoubtedly aided in our ability to diagnose this disease and come up with better treatment, however, there remains a significant need for in-depth profiling of the cancer transcriptome especially in the context of the different subtypes of breast cancer. Given the complexity of cancer, the molecular changes that promote its progression cannot be simplified into a set of biomarkers that fit all cancer types. Instead, each subtype needs detailed characterization in order to discover unique and consequential alterations. Currently, most of the available expression profiling data of breast cancer patient samples are focused on exon data and do not take into account intron or alternative splicing data. The objective of this project is to expand our current knowledge by profiling the understudied yet critical aspect of our transcriptome: minor intron splicing. This approach explores novel biomarkers and potentially innovative targets for therapy. The majority of genes in humans are expressed as pre-mRNAs containing introns (intervening sequences) that interrupt exons (expressed sequences). Introns are removed (spliced out) by a multi-megadalton complex named the spliceosome. Because pre-mRNA splicing is a critical and essential step in gene expression regulation, aberrant splicing can cause a wide range of diseases. A number of recent studies provided a plethora of evidence that in many cancers the splicing program is altered. These cancer-specific splicing profiles play a key role in cancer progression. In fact, all established hallmarks of cancer involve reprogrammed splicing of essential key genes. For example, several of the genes required for apoptosis adopt anti-apoptotic splice isoforms to lower the cancer cell’s susceptibility to cell death, including Bclx, Capsase2, and Fas. Also, the splicing of pre-mRNAs from genes involved in invasion and migration, such as RON, is altered to favor an epithelial-to-mesenchymal transition program. Additionally, cancer cell metabolism relies on altered splicing in genes such as PKM, whose cancer-specific splicing isoform is essential for the Warburg effect. However, despite a growing list of cancer-promoting splicing alterations, our understanding of the underlining mechanisms and how much they contribute to cancer progression is limited, especially for breast cancer. The Younis lab is interested in a subset of introns (~600 introns referred to as minor introns) in the human genome that require a specialized spliceosome. The interest in these introns stems from the fact that they cluster in conserved genes that function in cellular signaling and information relay, proliferation, transformation, and DNA damage repair. Given that these functions are essential cellular process for cell cycle and maintaining genome integrity, their dysregulation has been shown to lead to cancer. The overall aim of this project is to test whether minor intron splicing in breast cancer is dysregulated leading to aberrations in major cellular processes, specifically DNA damage repair, and eventually breast cancer progression. In this project, the team will seek to do the following: (1) Determine the subset of minor introns with altered splicing in breast cancer. For this, data for >1200 breast cancer patient samples from The Cancer Genome Atlas (TCGA, NIH) as well as 56 breast cancer cell lines from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) will be acquired. These large datasets will be mined using standard pipelines for expression of minor intron-containing genes. As minor intron splicing has direct impact on the mRNAs that contain them, this bioinformatics approach will identify minor introns that are dysregulated in cancer cells. The outcome of this part is a database of altered splicing in breast adenocarcinomas. (2) Determine the role of RNA binding proteins (RBPs) in altering minor intron splicing in breast cancer cells. For this, the database of altered minor introns, will be confirmed and mechanistic studies will be performed in a panel of breast cancer cell lines that cover a range of breast carcinoma subtypes (Luminal, Basal and Claudin-low), including MDA-MB-231, MDA-MB-468, MCF7, T47D, and MCF10A (non-transformed breast cells). The outcome of this part is a confirmed shortlist (potential biomarkers) of introns and RBPs whose altered expression is consequential to the breast cancer phenotype. (3) Determine the consequences of a dysregulated minor spliceosome on DNA damage repair machinery in breast cancer. For this, DNA damage will be induced in the panel of breast cancer cells in which the minor spliceosome efficiency is artificially reduced and DNA damage repair will be assessed. The outcome of this part is molecular insights into the role of minor spliceosome in breast cancer progression and the potential use of anti-minor spliceosome reagents in therapy. The data generated in this project will yield a comprehensive molecular understanding of the regulation of a physiologically significant yet poorly understood mechanism of gene expression regulation. The impact of this project is not limited to a better molecular understanding of a devastating disease but will also add to our repertoire of biomarkers, which will have direct impact on diagnosis, and will provide novel therapeutic targets and approaches to deal with breast cancer.