Poster Presentation Australasian RNA Biology and Biotechnology Association 2026 Conference

The Long and Short of It: Novel Transcript Discovery in Melanoma Using Nanopore Long-read RNA Sequencing (#152)

Jun Yao Teow 1 2 , Ricardo De Paoli-Iseppi 3 , Aparna Rao 1 2 , Naomi Warren 1 2 , Andrew Lonsdale 1 2 , Rotem Aharon 1 2 , James Wilmott 4 5 , Sonia Mailer 1 2 6 , Michael Clark 3 , Alicia Oshlack 1 2 , Grant McArthur 1 2 , Lorey Smith 1 2
  1. Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia
  2. Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia
  3. Department of Anatomy and Physiology, University of Melbourne, Melbourne, Victoria, Australia
  4. Faculty of Medicine and Health, University of Sydney, Sydney, New South Wales, Australia
  5. Melanoma Institute of Australia, University of Sydney, Sydney, New South Wales, Australia
  6. Melanoma Research Victoria, Melbourne, Victoria, Australia

Background: Dysregulated alternative splicing is an established driver of cancer progression and therapeutic resistance. In melanoma, aberrant splicing patterns significantly correlate with patient survival and confer resistance towards targeted therapies, highlighting the clinical significance of alternative splicing for melanoma patients. However, the splicing-driven transcriptome in melanoma remains understudied and incomplete due to the limitations of conventional short-read sequencing that lacks full-length resolution of mRNA isoforms.

Method: We have deeply profiled 54 treatment-naïve melanoma samples collected from 47 patients with primary and/or lymph-node metastatic disease using Nanopore long-read RNA sequencing. A bioinformatic pipeline was applied to discover novel melanoma-specific isoforms that are absent from existing human transcriptome annotations. Novel isoform structure and expression were validated in multiple independent melanoma patient cohorts, and expression compared to normal tissues, using publicly available short-read data.

Results: Long-read RNA sequencing revealed a melanoma transcript repertoire of 86,752 distinct isoforms, including 7,929 high-confidence novel isoforms and 1,927 novel gene loci absent from current annotations. Transcript expression analysis demonstrated that many novel isoforms are broadly expressed in up to 80% of melanoma patients but nearly undetectable in benign tissues, highlighting specificity to melanoma tumours. Motif analysis revealed multiple lineage-restricted transcription factors (TFs) and RNA binding proteins (RBPs), including the well-characterised melanocyte TF MITF and neural crest-restricted TFs and RBPs that are completely undescribed in melanoma. Further analysis of the melanoma-specific transcriptome in an experimental model of melanoma initiation from the neural crest and melanocyte lineage, identified association with the chromatin remodelling SWI/SNF complex. SWI/SNF inhibition completely ablates melanoma-specific isoform expression in established melanoma cell lines thus strongly supporting a role in melanoma-restricted transcription and splicing. How SWI/SNF interacts with lineage specific TFs and RBPs to control melanoma transcript production is under further investigation.

Conclusion: To our knowledge, this work is the first to leverage long-read RNA sequencing to comprehensively profile transcript diversity in a large cohort of melanoma patients. Our findings demonstrate the power of deep transcriptome profiling to identify new actionable targets for clinical management of cancer owing to cancer specific transcripts that are broadly expressed across patients. Our work discovered the previously under-described role of chromatin remodelling and cell lineage-restricted transcription as key mechanisms modulating cancer-specific splicing and novel isoform expression in melanoma. Future work will further dissect the regulatory mechanisms controlling melanoma specific transcription and splicing, and investigate melanoma-specific isoforms for their potential as new biomarkers and targets for mRNA-based therapies.