Alternative RNA Processing in Cancers

RNA-Seq has brought forth significant discoveries concerning aberrations in RNA processing, implicating RNA variants in a variety of diseases. Here, I will discuss cancer-associated RNA variation accompanying either somatic mutations in a splicing factor or changes in RNA base editor abundance. While many splice variants have been examined at an event-level with short reads, identifying full-length isoform changes may better elucidate the functional consequences of these variants in cancer. Thus, we have employed long-read technology to obtain full-length transcript sequences, developing a computational workflow called FLAIR (Full-length Alternative Isoform analysis of RNA) to identify high-confidence transcripts, differential transcript usage, and haplotype-specific transcripts. We performed nanopore sequencing of chronic lymphocytic leukemia patient samples containing SF3B1 mutation. With FLAIR, we are able to find patterns of aberrant splicing and a decrease in unproductive retained introns associated with SF3B1 mutation. Additionally, we have applied FLAIR to direct RNA sequencing reads to facilitate the identification of longer poly(A) tail lengths associated with intron retentions. Finally, we have sequenced H1975 lung adenocarcinoma cells with knockdown of ADAR, an enzyme that mediates A-to-I editing. We further improved our workflow to identify key inosine-isoform associations with the goal of clarifying the prominence of ADAR in tumorigenesis. Ultimately, our work demonstrates the utility of nanopore sequencing for augmenting cancer and splicing research