Abstract 2724: A combination of short-read and long-read RNA sequencing reveals NOVA1’s role in telomere biology

Abstract

Abstract Dysregulated alternative splicing underlies many aspects of cancer cell biology including tumor cell telomere maintenance. We recently described a role for the neuronal splicing factor, NOVA1, in non-small cell lung cancer in the regulation of full-length hTERT splicing, telomerase activity, maintenance of telomere length, and general tumor growth characteristics. We wanted to further define the role of NOVA1 in telomere biology. Cells that maintain telomeres with different mechanisms were studied to be able to decipher telomerase specific versus alternative lengthening of telomeres (ALT) specific perturbations by NOVA1. Both short-read (Illumina) and long-read (Pacific Biosciences) sequencing of control and NOVA1 siRNA knockdown cells in H920 non-small cell lung cancer cells (telomerase positive) and U2OS cells (ALT positive cells) were performed to detail the transcriptomes of these cells. A combination of rMATS analysis of the short-read sequencing, SQANTI analysis of the long-read sequencing, and pathway analysis was performed to identify genes related to telomere biology and alternative splicing related to telomerase. Skipped exon events were the primary focus of this study as this is NOVA1’s main role in alternative splicing. rMATS analysis identified 4,530 significant events mapping to 2,950 genes in H920 cells and 2,534 significant events in 1,863 genes in U2OS cells. SQANTI analyzed 15,443 full-length transcripts (7,530 genes) in control and 14,463 transcripts (7,240 genes) in NOVA1 siRNA treated samples of the H920 cells and 12,133 transcripts (6,618 genes) in control and 10,126 transcripts (5,959 genes) in NOVA1 siRNA treated U2OS cells. There were 2,762 and 2,470 novel transcripts in control and NOVA1 siRNA treated H920 cells, respectively. In U2OS cells there were 2,178 novel transcripts in control and 1,688 novel transcripts in NOVA1 siRNA treated cells. The pathway analysis of rMATS cross-referenced with SQANTI revealed five top candidates that were specifically altered in NOVA1 knockdown cells and related telomere length maintenance by telomerase (POLD1, SUN2, ERCC1, SRSF2, and PCBP2). Interestingly, SRSF2 and PCBP2 were identified in our recent hTERT minigene loss of function screen for splicing regulatory genes of hTERT and have binding sites in hTERT near NOVA1s motif in intron 8. Long-read sequencing determined the full-length nature of these splicing events pointed out by the rMATS analysis. These data indicate that telomerase positive cancer cells with dysregulated expression of NOVA1 utilize different isoforms compared to ALT cells with NOVA1 to maintain telomeres. These new observations reveal that alternative splicing regulated in part by NOVA1 plays a larger and unexpected role in telomere biology. Manipulation of alternative splicing to shorten/deprotect telomeres should be pursued as a means to inhibit survival and growth of cancer cells. Citation Format: Andrew T. Ludlow, Mohammed E. Sayed, Aaron L. Slusher, Mark Ribick, Anisha Pancholi, Brian Sereni, Yu Qui, Elizabeth Tseng, Meredith Ashby, David C. Corney. A combination of short-read and long-read RNA sequencing reveals NOVA1’s role in telomere biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2724.