Abstract
The human TRA2B gene consists of 10 exons and 9 introns and produces 5 splice isoforms (TRA2β1 to TRA2β5). TRA2B exon 2 encodes multiple premature termination codons. TRA2β1 lacks exon 2 and is translated into a functional transformer 2β (Tra2β) protein, whereas TRA2β4 contains 10 exons and works as a functional RNA. Overexpressed Tra2β and ectopic expression of TRA2β4 may be oncogenic. We found that heterogeneous nuclear ribonucleoprotein (hnRNP)A1 and hnRNPU interacted with TRA2β4 exon 2. Minigene assays revealed that hnRNPA1 facilitated inclusion of exon 2, whereas hnRNPU promoted its skipping. However, knockdown of hnRNPA1 or hnRNPU reduced both TRA2β1 and TRA2β4 levels, and overexpression of these hnRNPs increased levels of both isoforms, suggesting that hnRNPA1 and hnRNPU mainly regulate the transcription of TRA2B. In fact, hnRNPA1 and hnRNPU positively regulated the promoter activity of TRA2B. Circular dichroism analyses, electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated the presence of G-quadruplex (G4) formation in the promoter of TRA2B. Formation of G4 suppressed TRA2B transcription, whereas hnRNPA1, but not hnRNPU, interacted with the G4 to facilitate transcription. Our results suggest that hnRNPA1 may modulate TRA2B transcription through its regulation of G4 formation in its promoter in colon cancer cells.
Highlights
The human TRA2B gene consists of 10 exons and 9 introns and generates 5 mRNA isoforms (TRA2β1 to TRA2β5) through alternative splicing and usage of alternative promoters or polyadenylation sites[11]
We were interested in hnRNPA1 and hnRNPU as exon 2-binding proteins, since these 2 RNA-binding proteins are known to be associated with accelerated cell growth[29,30,31]
Additional biotinylated RNA pull-down assays (Supplementary Fig. S1b) and RNA immunoprecipitation assays (Supplementary Fig. S1c,d) demonstrated that both hnRNPA1 and hnRNPU were preferentially associated with TRA2β4, compared with TRA2β1 or Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Summary
The human TRA2B gene consists of 10 exons and 9 introns and generates 5 mRNA isoforms (TRA2β1 to TRA2β5) through alternative splicing and usage of alternative promoters or polyadenylation sites[11]. Tra2β protein interacts with the BCL2 3′-UTR and promotes abnormal growth of colon cancer cells[16]. Based on these findings, both Tra2β and TRA2β4 are considered to be www.nature.com/scientificreports/. The hnRNP family functions as major splicing factors as well as transcriptional regulators for various genes[17]. Both hnRNPA1 and hnRNPU could upregulate the promoter activity of TRA2B. We suggest that hnRNPA1 may regulate TRA2B transcription through interaction with G4 in its promoter region
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