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HnRNPA1 interacts with G-quadruplex in the TRA2B promoter and stimulates its transcription in human colon cancer cells.

ABSTRACT: The human TRA2B gene consists of 10 exons and 9 introns and produces 5 splice isoforms (TRA2?1 to TRA2?5). TRA2B exon 2 encodes multiple premature termination codons. TRA2?1 lacks exon 2 and is translated into a functional transformer 2? (Tra2?) protein, whereas TRA2?4 contains 10 exons and works as a functional RNA. Overexpressed Tra2? and ectopic expression of TRA2?4 may be oncogenic. We found that heterogeneous nuclear ribonucleoprotein (hnRNP)A1 and hnRNPU interacted with TRA2?4 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 TRA2?1 and TRA2?4 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.

SUBMITTER: Nishikawa T

PROVIDER: S-EPMC6635519 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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HnRNPA1 interacts with G-quadruplex in the TRA2B promoter and stimulates its transcription in human colon cancer cells.

Nishikawa Tatsuya T Kuwano Yuki Y Takahara Yumiko Y Nishida Kensei K Rokutan Kazuhito K

Scientific reports 20190716 1

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. Mini ...[more]

PMID: 31311954

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Project description:BackgroundTransgelin, an actin-binding protein, is associated with cytoskeleton remodeling. Findings from our previous studies demonstrated that transgelin was up-regulated in node-positive colorectal cancer (CRC) versus node-negative disease. Over-expression of TAGLN affected the expression of 256 downstream transcripts and increased the metastatic potential of colon cancer cells in vitro and in vivo. This study aims to explore the mechanisms through which transgelin participates in the metastasis of colon cancer cells.MethodsImmunofluorescence and immunoblotting analysis were used to determine the cellular localization of endogenous and exogenous transgelin in colon cancer cells. Co-immunoprecipitation and subsequently high-performance liquid chromatography/tandem mass spectrometry were performed to identify the proteins that were potentially interacting with transgelin. The 256 downstream transcripts regulated by transgelin were analyzed with bioinformatics methods to discriminate the specific key genes and signaling pathways. The Gene-Cloud of Biotechnology Information (GCBI) tools were used to predict the potential transcription factors (TFs) for the key genes. The predicted TFs corresponded to the proteins identified to interact with transgelin. The interaction between transgelin and the TFs was verified by co-immunoprecipitation and immunofluorescence.ResultsTransgelin was found to localize in both the cytoplasm and nucleus of the colon cancer cells. Approximately 297 proteins were identified to interact with transgelin. The overexpression of TAGLN led to the differential expression of 184 downstream genes. Network topology analysis discriminated seven key genes, including CALM1, MYO1F, NCKIPSD, PLK4, RAC1, WAS and WIPF1, which are mostly involved in the Rho signaling pathway. Poly (ADP-ribose) polymerase-1 (PARP1) was predicted as the unique TF for the key genes and concurrently corresponded to the DNA-binding proteins potentially interacting with transgelin. The interaction between PARP1 and transgelin in human RKO colon cancer cells was further validated by immunoprecipitation and immunofluorescence assays.ConclusionsOur results suggest that transgelin binds to PARP1 and regulates the expression of downstream key genes, which are mainly involved in the Rho signaling pathway, and thus participates in the metastasis of colon cancer.

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HnRNPA1 interacts with G-quadruplex in the TRA2B promoter and stimulates its transcription in human colon cancer cells.

Publications

HnRNPA1 interacts with G-quadruplex in the TRA2B promoter and stimulates its transcription in human colon cancer cells.

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