RNA editing and splicing are the two major processes that dynamically regulate human transcriptome diversity. Despite growing evidence of crosstalk between RNA editing enzymes (mainly ADAR1) and splicing machineries, detailed mechanistic explanations and their biological importance in diseases, such as cancer are still lacking. Herein, we identify approximately a hundred high-confidence splicing events altered by ADAR1 and/or ADAR2, and ADAR1 or ADAR2 protein can regulate cassette exons in both directions. We unravel a binding tendency of ADARs to dsRNAs that involves GA-rich sequences for editing and splicing regulation. ADAR1 edits an intronic splicing silencer, leading to recruitment of SRSF7 and repression of exon inclusion. We also present a mechanism through which ADAR2 binds to dsRNA formed between GA-rich sequences and polypyrimidine (Py)-tract and precludes access of U2AF65 to 3′ splice site. Furthermore, we find these ADARs-regulated splicing changes per se influence tumorigenesis, not merely byproducts of ADARs editing and binding.

Adaptation to hypoxia, a hallmark feature of many tumors, is an important driver of cancer cell survival, proliferation and the development of resistance to chemotherapy. Hypoxia-induced stabilization of hypoxia-inducible factors (HIFs) leads to transcriptional activation of a network of hypoxia target genes involved in angiogenesis, cell growth, glycolysis, DNA damage repair and apoptosis. Although the transcriptional targets of hypoxia have been characterized, the alternative splicing of transcripts that occurs during hypoxia and the roles they play in oncogenesis are much less understood. To identify and quantify hypoxia-induced alternative splicing events in human cancer cells, we performed whole transcriptome RNA-Seq in breast cancer cells that are known to provide robust transcriptional response to hypoxia. We found 2005 and 1684 alternative splicing events including intron retention, exon skipping and alternative first exon usage that were regulated by acute and chronic hypoxia where intron retention was the most dominant type of hypoxia-induced alternative splicing. Many of these genes are involved in cellular metabolism, transcriptional regulation, actin cytoskeleton organisation, cancer cell proliferation, migration and invasion, suggesting they may modulate or be involved in additional features of tumorigenic development that extend beyond the known functions of canonical full-length transcripts.

Adenosine-to-inosine (A-to-I) RNA editing entails the enzymatic deamination of adenosines to inosines by adenosine deaminases acting on RNA (ADARs). Dysregulated A-to-I editing has been implicated in various diseases, including cancers. However, the precise factors governing the A-to-I editing and their physiopathological implications remain as a long-standing question. Herein, we unravel that DEAH box helicase 9 (DHX9), at least partially dependent of its helicase activity, functions as a bidirectional regulator of A-to-I editing in cancer cells. Intriguingly, the ADAR substrate specificity determines the opposing effects of DHX9 on editing as DHX9 silencing preferentially represses editing of ADAR1-specific substrates, whereas augments ADAR2-specific substrate editing. Analysis of 11 cancer types from The Cancer Genome Atlas (TCGA) reveals a striking overexpression of DHX9 in tumors. Further, tumorigenicity studies demonstrate a helicase-dependent oncogenic role of DHX9 in cancer development. In sum, DHX9 constitutes a bidirectional regulatory mode in A-to-I editing, which is in part responsible for the dysregulated editome profile in cancer.