The molecular basis of 7SK RNA methylation in non-small cell lung cancer

PROJECT SUMMARY Lung cancer is a leading cause of cancer death in the United States, with non-small cell lung cancer (NSCLC) being the major subtype accounting for approximately 85% of cases. Effective treatments for NSCLC are currently lacking due to the complexity of its molecular subtypes and the incomplete understanding of its underlying mechanisms. RNA N6-methyladenosine (m6A) modification is increasingly recognized as being deregulated in human cancers, however, specific m6A events in NSCLC are yet to be identified. We found elevated m6A modification on 7SK small nuclear RNA in NSCLC cells and a NSCLC mouse model. Targeted reduction of m6A-7SK inhibits NSCLC cell growth in soft agar colony formation assays. 7SK regulates RNA Polymerase II (Pol II) transcription and is a central player in controlling Pol II transcriptional pausing and elongation. Therefore, m6A modification of 7SK may underlie NSCLC formation and phenotype. The goal of this study is to dissect the molecular pathways that regulate m6A-7SK and decipher the mechanisms by which m6A-7SK affects gene regulatory networks in NSCLC. The first aim is to identify the m6A sites using real-time PCR-based m6A detection assay, and to identify writer and eraser enzymes of 7SK by short hairpin RNA knockdown. The second aim is to determine how m6A-7SK influences gene expression and NSCLC formation. CRISPR dCasRx-ALKBH5 fusion protein will be used to modulate m6A-7SK in NSCLC cells, and cell proliferation and migration assays will be employed. To determine the molecular details of m6A-7SK-mediated gene regulation, SHAPE-MaP sequencing will probe the structure of m6A-7SK; nascent RNA-seq, ChIP-seq of Pol II, and ChIRP-seq of 7SK will be performed to survey transcriptional program in NSCLC cells with different levels of m6A-7SK. The results of this study will reveal novel molecular details of m6A modifications on 7SK and how they regulate cancer gene expression. This information will provide the basis for future research to develop novel NSCLC therapeutics by altering RNA m6A levels.