Long-read sequencing uncovers alternative transcript isoforms essential for hypoxic adaptation in hepatocellular carcinoma

Abstract

Alternative promoter or transcription start site has been revealed as a key transcriptomic signature for various types of cancer. However, the transcript isoforms arisen from such drastic change in promoter usage are barely mentioned. Recent success in long-read sequencing has proven its ability in discovering novel and accurate full-length isoforms. In order to explore how alternative transcription start site contributes to tumorigenesis through isoform switch, nanopore long-read cDNA sequencing was applied to study hepatocellular carcinoma (HCC) under hypoxic stress, which is commonly seen in solid tumors and governed by the master transcription factor, hypoxia-inducible factor 1 alpha (HIF-1ɑ).

A large number of unannotated transcripts were identified in both normoxia and hypoxia conditions, echoing previous studies on the necessity of utilizing long-read sequencing to study isoforms. Among various splicing events, alternative first exon (AFE) was the most prominently upregulated splicing event under hypoxia, which align with the hypothesis, as a switch in first exon must occur as a consequence of alternative transcription start site. Given the fact that HIF-1, as a master transcription factor, is stabilized under hypoxia for the induction of stress response genes, HIF-1ɑ ChIP-sequencing was performed and its contribution to AFEs was highly noticeable.

UGP2, encoding a glycogen precursor synthesizing enzyme, was found as one of the top upregulated isoforms among all tested cell lines. The canonical and hypoxia-induced isoforms share high similarity in their protein coding sequences but are totally different in 5’ untranslated regions (5’UTRs). The first exon switch was confirmed to allow bypassing the translationally inhibitory upstream open reading frames (uORFs) in canonical first exon and increasing translation efficiency. Functionally, knockout of UGP2 suppressed glycogen synthesis and cell proliferation in hypoxia, proving its importance in hypoxic adaptation.

Another hypoxia-induced isoform with HIF-1ɑ-mediated AFE, DDX41, was found encoding a protein isoform with 127 less amino acids at the N-terminus, resulting in the loss of the nuclear localization sequence (NLS) compared to the canonical isoform. The change in localization was confirmed by both subcellular fractionation followed by western blot and immunofluorescent experiments. Surprisingly, RNA-sequencing of DDX41 isoform-specific knockdown cells revealed a widespread decrease in the expression of hypoxia-induced genes, including a substantial number of HIF-1ɑ direct targets. The protein level of HIF-1ɑ was later found downregulated upon knockdown of DDX41 hypoxia-induced isoform, while mRNA expression remained unaffected, suggesting DDX41 is involved in regulating protein level of HIF-1ɑ. This finding highlights the importance of DDX41 isoform in hypoxic adaptation although further exploration is required to comprehend the underlying mechanism.

In summary, this project revealed the transcriptomic isoform landscape of HCC under normoxia and hypoxia conditions. It also demonstrated how HIF-1ɑ-mediated isoforms participate in the adaptation in low oxygen environment, fulfilling the aim to enhance our understanding in hypoxia-induced tumor progression and contribute to novel isoforms identification for potential biomarkers and effective targeted treatment for HCC patients.