ABSTRACT
Multiple noncoding natural antisense transcripts (ncNAT) are known to modulate key biological events such as cell growth or differentiation. However, the actual impact of ncNATs on cancer progression remains largely unknown. In this study, we identified a complete list of differentially expressed ncNATs in hepatocellular carcinoma. Among them, a previously undescribed ncNAT HNF4A-AS1L suppressed cancer cell growth by regulating its sense gene HNF4A, a well-known cancer driver, through a promoter-specific mechanism. HNF4A-AS1L selectively activated the HNF4A P1 promoter via HNF1A, which upregulated expression of tumor suppressor P1-driven isoforms, while having no effect on the oncogenic P2 promoter. RNA-seq data from 23 tissue and cancer types identified approximately 100 ncNATs whose expression correlated specifically with the activity of one promoter of their associated sense gene. Silencing of two of these ncNATs ENSG00000259357 and ENSG00000255031 (antisense to CERS2 and CHKA, respectively) altered the promoter usage of CERS2 and CHKA. Altogether, these results demonstrate that promoter-specific regulation is a mechanism used by ncNATs for context-specific control of alternative isoform expression of their counterpart sense genes. SIGNIFICANCE: This study characterizes a previously unexplored role of ncNATs in regulation of isoform expression of associated sense genes, highlighting a mechanism of alternative promoter usage in cancer.
Subject(s)
Carcinoma, Hepatocellular/pathology , Choline Kinase/metabolism , Hepatocyte Nuclear Factor 4/metabolism , Liver Neoplasms/pathology , Membrane Proteins/metabolism , Promoter Regions, Genetic , RNA, Antisense/genetics , Sphingosine N-Acyltransferase/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Choline Kinase/antagonists & inhibitors , Choline Kinase/genetics , Gene Expression Regulation, Neoplastic , Hepatocyte Nuclear Factor 4/antagonists & inhibitors , Hepatocyte Nuclear Factor 4/genetics , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Male , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Mice , Mice, SCID , Prognosis , Sphingosine N-Acyltransferase/antagonists & inhibitors , Sphingosine N-Acyltransferase/genetics , Tumor Cells, Cultured , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/genetics , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Adenosine Deaminase/metabolism , Neoplasms/genetics , RNA Precursors/genetics , RNA-Binding Proteins/metabolism , Adenosine Deaminase/genetics , Alternative Splicing , Animals , Carrier Proteins/genetics , Cell Line, Tumor , Exons , Gene Expression Regulation, Neoplastic , Humans , Membrane Proteins/genetics , Mice, Inbred NOD , Nonsense Mediated mRNA Decay , RNA Editing , RNA Splice Sites , RNA Splicing , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , Serine-Arginine Splicing Factors/genetics , Splicing Factor U2AF/geneticsABSTRACT
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.
