Minor Intron Genes as Potential Targets for Affecting Survival of Triple Negative Breast Cancer Cells Zoya Farooqui, Dr Ihab Younis, Dr Mazen Sidani Department of Biological Sciences, Carnegie Mellon University Qatar • Triple Negative Breast cancer (TNBC) is an aggressive cancer associated with lower survival, prognosis, and less treatment options. • Minor Intron genes (MIGs) have information processing functions. • Every hallmark of cancer is associated with a MIG. • Previous work on this research explored splicing behaviour of MIGs in breast cancer cells by manipulating levels of U6atac snRNA. Spliced mRNA Unspliced mRNA retaining minor intron After U6atac inhibition Class I: Accumulation of Unspliced mRNA Class II: No Accumulation of Unspliced mRNA Overall Aim: Build on previous research to broaden understanding of MIGs, their behaviour in TNBC and manipulate their splicing for potential thereaptuic advantage. Specific Aim 1: Understand why unspliced mRNA only accumulates for Class I MIGs. -Hypothesis : Unspliced mRNA for Class I genes holds translational potential. Specific Aim 2: Find a lowU6atac AMO dose that is critical for TNBC cells. • El Marabti, E., Malek, J., & Younis, I. (2021). Minor Intron Splicing from Basic Science to Disease. International journal of molecular sciences, 22(11), 6062.https://doi.org/10.3390/ijms2211606 • Hall, S. L., & Padgett, R. A. (1996). Requirement of U12 snRNA for in vivo splicing of a minor class of eukaryotic nuclear pre-mRNA introns. Science (New York, N.Y.), 271(5256),1716- 17181https://doi.org/10.1126/science.271.5256.171 • Younis, I., Dittmar, K., Wang, W., Foley, S. W., Berg, M. G., Hu, K. Y., ... & Dreyfuss, G. (2013). Minor introns are embedded molecular switches regulated by highly unstable U6atacsnRNA. Elife, 2, e00780. Previous Research Exon Intron Categorization of MIGs in two classes: Research Aims Background Methodology Aim 1 Aim 2 Conclusions Future Directions Figure 2: 0.5μM U6atac AMO is a critical dose for MDA-MB-231 cells. (A) RealTime Glo viability assay on transfected MDA-MB-231 cells using MT reagent over 5 day period. * indicates significance of p-value <0. 05. (B) Compilation of percentage decrease in expression of spliced mRNA following 0.5μM U6atac AMO treatment using RT-qPCR data of tested MIGs. MIGs with 50% or more percentage decrease were considered significant. (C) Most significant Gene ontology results on sensitive MIGS. Figure 1:Treatment with U6atac AMO prevents translation of both Class I and Class II MIGs. RT-qPCR (A, C) splicing analysis and Western Blot image (B, D) of Class I (EIF3I) and Class II (MAPK14) MIGs. MDA-MB-231 cells were transfected with Control and U6atac AMO. RNA was extracted, converted to cDNA and used for RT-qPCR using QuantStudio 6 Flex thermocycler.Similarly, protein lysate were extracted from transfected cells, separated by SDS-PAGE, stained with specific antibodies. Results B C References • Protein expression of MIGs directly correlates with the splicing of their minor intron. • Unspliced mRNA for both Class I and Class II genes is not translated. • 0.5μM U6atac AMO dose affects replication and growth of MDA-MB- 231 (TNBC) cells. • Splicing of multiple MIGs is significantly affected by 0.5μM U6atac AMO treatment. • These sensitive MIGs form potential targets whose expression can be prevented to affect survival of TNBC cells. • Exploration of miRNA binding sites within minor introns of Class I and Class II genes to explain splicing trends observed in MIGs. • Testing 0.5μM U6atac AMO in non- breast cancer and other cancer cell lines to observe if its effect is specific to TNBC cells. • Manipulating expression of sensitive MIGs within TNBC cells. Transfection of MDA- MB-231 cells (TNBC) with U6atac AMO Protein Extraction Plating for Viability Assay RNA Extraction and cDNA synthesis Western Blot RealTime Glo Viability Assay Quantitative PCR (qPCR)