Abstract 4458: Identification of splicing regulators using retroviral vectors carrying intron-containing expression cassettes

Abstract

Abstract Exon inclusion is controlled by both positive (e.g., SR proteins) and negative (e.g., hnRNPs) regulators; yet high-throughput methods to comprehensively identify all relevant RNA-binding proteins are currently lacking. Thus, our long-term goal is to develop a genome-wide CRISPR sgRNA library-based screen, where cells with desired phenotypes are identified by flow cytometry and analyzed as pools using next generation sequencing. To achieve this goal, we took advantage of previous work in our laboratory demonstrating expression of the Δex2 CD19 isoform in B-cell leukemias and lymphomas resistant to CD19-directed immunotherapy (Sotillo et al., 2015; Yu et al., 2016). Skipping of exon 2 results in the truncated protein which fails to localize to the plasma membrane, yielding cells that are CD19-negative by flow cytometry (Bagashev et al., 2015). To study splicing regulation of exon 2, we designed a series of retroviral vectors containing the entire CD19 open reading frame retaining intronic sequences flanking exon 2 (i.e., intron 1 and intron 2). It is known that the nuclear splicing of provirus containing introns completely removes intron from the packaged viral RNA genome (Li et al., 1998). However, when viruses were stably transducedto B-lymphoid cell lines rendered null for endogenous CD19, the retroviral long terminal repeat (LTR) promoter-driven CD19 transcription resulted in skipping of exon2. Further addition of cognate 5'UTR sequence downstream of the retroviral LTR did not promote inclusion of this alternative exon. Similarly, the retroviral based expression of CD19 gene containing intronic sequences flanking constitutive exon10 did not allow proper splicing of exon10 and instead resulted into transcript retaining both introns (9 and 10). Interestingly, the flanking intronic sequences of exon10 allowed precise splicing of upstream exon2 when the expression cassette contained both exon2 and exon10 flanked by their respective introns. Thus, downstream placement of constitutive exon10 and its surrounding introns affected the splicing of upstream alternative exon2. Subsequent introduction of sgRNA libraries into cells transduced with such intron-containing expression cassettes will allow us to identify both positive and negative regulators of CD19 exon2 inclusion and potentially - novel general regulators of splicing. Bagashev, A., Sotillo, E., Wu, G., and Thomas-Tikhonenko, A. (2015). The importance of CD19 exon 2 for surface localization: Closing the Ig-like loop. Blood 126, 3433. Sotillo, E., Barrett, D.M., Black, K.L., Bagashev, A., Oldridge, D., Lanauze, C., Gazzara, M.R., et al. (2015). Convergence of acquired mutations and alternative splicing of CD19 enables resistance to CART-19 immunotherapy. Cancer Discov 5, 1282-1295. Li, K., Garoff, H. (1998). Packaging of intron-containing genes into retrovirus vectors by alphavirus vectors. Proc Natl Acad Sci USA. Citation Format: Mukta Asnani. Identification of splicing regulators using retroviral vectors carrying intron-containing expression cassettes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4458.