ABSTRACT
Aberrant gene expression in cardiomyocyte has been revealed to be the fundamental essence of pathological cardiac hypertrophy. However, the detailed mechanisms are not fully understood. The underlying regulators of gene expression involved in cardiac hypertrophy remain to be further identified. Here, we report that the RNA-binding protein RNA-binding motif protein 4 (RBM4) functions as an endogenic protector that is able to fight against cardiomyocyte hypertrophy in vitro. Under pro-hypertrophic stimulation of angiotensin II (Ang II), the protein level of RBM4 in cardiomyocyte and myocardium is elevated. Knockdown of RBM4 can further aggravate cardiomyocyte hypertrophy, while over-expression of RBM4 represses cardiomyocyte hypertrophy. Mechanistically, RBM4 is localized in the nucleus and down-regulates the expression of polypyrimidine tract-binding protein 1 (PTBP1), which has been shown to aggravate cardiomyocyte hypertrophy. In addition, we suggest that the up-regulation of RBM4 in cardiomyocyte hypertrophy is caused by N6-methyladenosine (m6A). Ang II induces m6A methylation of RBM4 mRNA, which further enhances the YTH domain-containing family protein 1 (YTHDF1)-mediated translation of RBM4. Thus, our results reveal a novel pathway consisting of m6A, RBM4 and PTBP1, which is involved in cardiomyocyte hypertrophy.
ABSTRACT
BACKGROUND: Aberrant splicing has been closely associated with human cancer, though the precise underlying mechanisms linking the two remain not fully understood. Investigating the role of splicing factors in cancer progression may aid in the development of targeted therapies for dysregulated splicing, thereby opening up new avenues for cancer treatment. RNA-binding motif 4 (RBM4) has been identified as a critical participant in the condensin II complex, which is involved in chromosome condensation and stabilization during mitosis. Its significance in tumors is currently gaining attention. The genetic characteristics of RBM4 suggest its potential to elucidate the malignant progression of tumors in a broader context, encompassing various types of cancer, known as pan-cancer. METHODS: This study aims to comprehensively explore the potential function of RBM4 in pan-cancer by leveraging existing databases such as The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx). RESULTS: RBM4 is found to be overexpressed in almost all tumors and exhibits significant prognostic and diagnostic efficacy. The correlation between RBM4 and immune signatures, including immune cell infiltration and immune checkpoint genes, indicates that RBM4 could serve as a guiding factor for immunotherapy. CONCLUSIONS: As a member of the pan-oncogene, RBM4 has the potential to become a biomarker and therapeutic target for various malignant tumors, offering novel possibilities for precision medicine.
Subject(s)
Alternative Splicing , Neoplasms , Humans , Prognosis , Neoplasms/genetics , RNA Splicing , RNA-Binding Motifs , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Ebola virus (EBOV) belongs to Filoviridae family possessing single-stranded negative-sense RNA genome, which is a serious threat to human health. Nowadays, no therapeutics have been proven to be successful in efficiently decreasing the mortality rate. RNA binding proteins (RBPs) are reported to participate in maintaining cell integrity and regulation of viral replication. However, little is known about whether and how RBPs participate in regulating the life cycle of EBOV. In our study, we found that RNA binding motif protein 4 (RBM4) inhibited the replication of EBOV in HEK293T and Huh-7 cells by suppressing viral mRNA production. Such inhibition resulted from the direct interaction between the RRM1 domain of RBM4 and the "CU" enrichment elements located in the PE1 and TSS of the 3'-leader region within the viral genome. Simultaneously, RBM4 could upregulate the expression of some cytokines involved in the host innate immune responses to synergistically exert its antiviral function. The findings therefore suggest that RBM4 might serve as a novel target of anti-EBOV strategy.
Subject(s)
Ebolavirus , Hemorrhagic Fever, Ebola , Humans , Ebolavirus/genetics , RNA , HEK293 Cells , Virus Replication , RNA-Binding Motifs , Genomics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
AMG232 effectively inhibits cancers with wild-type p53 (also known as TP53) by reactivating p53, but whether it inhibits glioma angiogenesis remains unclear. This study confirms that AMG232 inhibits the proliferation of glioma endothelial cells (GECs) in a dose-dependent manner and inhibits the angiogenesis of GECs. p53 and RNA-binding motif protein 4 (RBM4) were expressed at low levels in GECs, while MDM2 and vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR) were highly expressed. In vitro and in vivo experiments confirmed that AMG232 upregulated p53 and RBM4, and downregulated MDM2 and VEGFR2 by blocking the MDM2-p53 interaction. Both p53 silencing and RBM4 silencing significantly upregulated the expression of VEGFR2, promoted the proliferation, migration and tube formation of GECs, and reversed the effects of AMG232 on downregulating VEGFR2 and inhibiting the angiogenesis of GECs. AMG232 increased RBM4 expression by upregulating p53, and p53 bound to RBM4 and promoted its transcription. RBM4 bound to and shortened the half-life of VEGFR2, promoting its degradation. Finally, AMG232 produced a significant decrease in new vessels and hemoglobin content in vivo. This study proves that AMG232 inhibits glioma angiogenesis by blocking the MDM2-p53 interaction, in which the p53-RBM4-VEGFR2 pathway plays an important role.
Subject(s)
Endothelial Cells , Glioma , Humans , Cell Movement , Cell Proliferation/physiology , Endothelial Cells/metabolism , Glioma/drug therapy , Glioma/genetics , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , RNA-Binding Proteins/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
RBM4 has been reported as a tumor suppressor gene in cancers, including lung cancer, colon cancer and gastric cancer. However, the role of RBM4 in clear cell renal cell carcinoma (ccRCC) remains unclear. Therefore, the present study investigated the expression and biological function of RBM4 in ccRCC. Analysis of the differential expression of RBM4 and its relationship with clinicopathological features using ccRCC samples data from TCGA database deminstrated that RBM4 expression in tumor samples of ccRCC was lower than that in normal samples, and RBM4 expression was closely related to the survival time of patients. RBM4 overexpression (RBM4-oe) cell lines were constructed to investigate the effect of RBM4 on biological function using CCK-8, EdU, flow cytometry and wound-healing assays. In addition, the regulatory effect of RBM4 on signaling pathways was investigated by GSEA and WB assays. RBM4-oe significantly reduced the proliferation of ccRCC cells by controlling the p53 signaling pathway, inhibited cell cycle progression and promoted apoptosis. In addition, RBM4-oe suppressed the migration and invasion of cells by EMT. Mechanistically, RBM4-oe facilitated the activity of the p53 signaling pathway by enhancing the stability of p53 mRNA. Finally, RBM4-oe markedly inhibited the growth of tumors formed with 786-O cells in vivo. In summary, there findings suggeated that RBM4 inhibits the progression of ccRCC by promoting p53 signaling pathway activity by enhancing the stability of p53 mRNA, suggesting that RBM4 may be a potential target for the treatment of patients.
Subject(s)
Carcinoma, Renal Cell , Carcinoma , Kidney Neoplasms , Humans , Carcinoma, Renal Cell/pathology , Tumor Suppressor Protein p53/genetics , RNA, Messenger/genetics , Cell Proliferation/genetics , Kidney Neoplasms/pathology , Cell Line, Tumor , RNA-Binding Proteins/geneticsABSTRACT
Increased angiogenesis in the liver plays a critical role in the progression of hepatocellular carcinoma (HCC). However, the molecular mechanism underlying increased angiogenesis in HCC is not well understood. Current study was designed to identify the potential angiogenic effect of RNA-binding motif 4 (RBM4)through a small-scale overexpression screening, followed by comparison of the expression level of RBM4 in cancer and adjacent tissues in multiple malignancies to explore the relationship between RBM4 and CD31 protein expression level and related clinical indicators, and understand the role of RBM4 in the hepatocellular carcinoma. To understand the specific mechanism of RBM4 in detail, transcriptome sequencing, mass spectrometry and multiple molecular cytological studies were performed. These cellular level results were verified by experiments in animal models of nude mice. The increased expression of RBM4 in cancer tissues, suggested its use as a prognostic biomarker. The RBM4 expression was found to be strongly correlated with tumor microvessel density. Mechanistically, RBM4 mediated its effects via interaction with HNRNP-M through the latter's WDR15 domain, which then stabilized RelA/p65 mRNA. Consequently, RBM4 induced the activation of the NF-kB signaling pathway, upregulating the expression of proangiogenic factor VEGF-A. The results confirmed the mechanism by which RBM4 promotes angiogenesis in hepatocellular carcinoma suggesting RBM4 as a crucial promoter of angiogenesis in HCC, helping understand regulation of NF-kB signaling in HCC.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Animals , Humans , Mice , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Liver Neoplasms/metabolism , Mice, Nude , Neovascularization, Pathologic/metabolism , NF-kappa B/metabolism , RNA-Binding Motifs , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
The human rhomboid-5 homolog-1 (RHBDF1) is a multi-transmembrane protein present mainly on the endoplasmic reticulum. RHBDF1 has been implicated in the activation of epidermal growth factor receptor (EGFR)-derived cell growth signals and other activities critical to cellular responses to stressful conditions, but details of this activation mechanism are unclear. Here, we report a RHBDF1 mRNA transcript alternative splicing variant X6 (RHBDF1 X6 or RHX6) that antagonizes RHBDF1 activities. We found that while the RHBDF1 gene is marginally expressed in breast tumor-adjacent normal tissues, it is markedly elevated in the tumor tissues. In sharp contrast, the RHX6 mRNA represents the primary RHBDF1 variant in normal breast epithelial cells and tumor-adjacent normal tissues but is diminished in breast cancer cells and tumors. We demonstrate that, functionally, RHX6 acts as an inhibitor of RHBDF1 activities. We show that artificially overexpressing RHX6 in breast cancer cells leads to retarded proliferation, migration, and decreased production of epithelial-mesenchymal transition-related adhesion molecules. Mechanically, RHX6 is able to inhibit the maturation of TACE, a protease that processes pro-TGFα, a pro-ligand of EGFR, and to prevent intracellular transportation of pro-TGFα to the cell surface. Additionally, we show that the production of RHX6 is under the control of the alternative splicing regulator RNA binding motif protein-4 (RBM4). Our findings suggest that differential splicing of the RHBDF1 gene transcript may have a regulatory role in the development of epithelial cell cancers.
Subject(s)
Alternative Splicing , Breast Neoplasms , ErbB Receptors , Membrane Proteins , Breast Neoplasms/genetics , Cell Line, Tumor , ErbB Receptors/metabolism , Female , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Transforming Growth Factor alpha/metabolismABSTRACT
Circ-RBM4 (mm9_circ_013935) has been revealed to have low expression in the renal tissues of diabetic nephropathy (DN) mice, and its underlying regulatory mechanism remains unexplored. The high glucose (HG) - treated mouse podocytes were used to establish DN cell models. A cell counting kit-8 assay was used to examine the viability of mouse podocytes. The expression of proteins related to fibrosis (collagen I, collagen III, fibronectin) was detected using Western blot. The concentration of inflammation cytokines (tumor necrosis factor α, interleukin 1ß (IL-1ß), IL-8) in mouse podocytes was assessed by ELISA. The interaction between genes was explored by luciferase reporter assays. HG treatment decreased the viability and elevated the expression of fibrosis and inflammation factors in mouse podocytes. Circ-RBM4 expression was downregulated in HG-treated mouse podocytes. Circ-RBM4 overexpression reversed HG-induced increase in levels of proteins related to fibrosis and the concentration of inflammation factors. The miR-153-3p was revealed to bind with circ-RBM4 and directly targeted nuclear factor I/C (NFIC) in mouse podocytes. Rescue assays indicated that circ-RBM4 attenuated HG-induced fibrosis and inflammation response in mouse podocytes by inhibiting miR-153-3p expression or upregulating NFIC expression. Circ-RBM4 alleviated the renal inflammation and renal fibrosis in DN by targeting the miR-153-3p/NFIC axis.
Subject(s)
Diabetic Nephropathies/genetics , Diabetic Nephropathies/pathology , Gene Expression/genetics , Gene Expression/physiology , Kidney/pathology , MicroRNAs/genetics , MicroRNAs/metabolism , NFI Transcription Factors/genetics , NFI Transcription Factors/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Animals , Cells, Cultured , Disease Models, Animal , Fibrosis , Inflammation , Kidney/metabolism , Mice , Podocytes/metabolism , Podocytes/pathology , RNA-Binding Proteins/physiologyABSTRACT
BACKGROUND: RNA splicing is a key post-transcriptional mechanism that generates protein diversity and contributes to the fine-tuning of gene expression, which may facilitate adaptation to environmental challenges. Here, we employ a systems approach to study alternative splicing changes upon enteric infection in females from classical Drosophila melanogaster strains as well as 38 inbred lines. RESULTS: We find that infection leads to extensive differences in isoform ratios, which results in a more diverse transcriptome with longer 5' untranslated regions (5'UTRs). We establish a role for genetic variation in mediating inter-individual splicing differences, with local splicing quantitative trait loci (local-sQTLs) being preferentially located at the 5' end of transcripts and directly upstream of splice donor sites. Moreover, local-sQTLs are more numerous in the infected state, indicating that acute stress unmasks a substantial number of silent genetic variants. We observe a general increase in intron retention concentrated at the 5' end of transcripts across multiple strains, whose prevalence scales with the degree of pathogen virulence. The length, GC content, and RNA polymerase II occupancy of these introns with increased retention suggest that they have exon-like characteristics. We further uncover that retained intron sequences are enriched for the Lark/RBM4 RNA binding motif. Interestingly, we find that lark is induced by infection in wild-type flies, its overexpression and knockdown alter survival, and tissue-specific overexpression mimics infection-induced intron retention. CONCLUSION: Our collective findings point to pervasive and consistent RNA splicing changes, partly mediated by Lark/RBM4, as being an important aspect of the gut response to infection.
Subject(s)
Bacterial Infections/genetics , Drosophila Proteins/metabolism , RNA Splicing , RNA-Binding Proteins/metabolism , 5' Untranslated Regions , Animals , Bacterial Infections/microbiology , Base Composition , Drosophila Proteins/genetics , Drosophila melanogaster , Female , Intestines/microbiology , Introns , Pseudomonas/pathogenicity , Quantitative Trait Loci , RNA-Binding Proteins/geneticsABSTRACT
Regulatory factors function by modulating a variety of cascade mechanisms in cells. RBM4 is a multifunctional RNA-binding protein in post-transcriptional gene regulation. Cytoplasmic RBM4 interacts with Ago2 to regulate inflammatory responses by affecting mRNA decay and cap-dependent translation. However, it is unclear whether RBM4 functions in inflammation regulation by its splicing factor role. Here, the cell biology, gene expression profile and alternative splicing pattern of HeLa cells with RBM4 overexpression (RBM-OE) were compared with the control. The results showed that RBM4-OE inhibited proliferation. RBM4-OE extensively affects the transcriptional level of genes involved in cell surface receptor signalling pathway, inflammatory responses and the response to lipopolysaccharide. RBM4 broadly regulated the alternative splicing of hundreds of genes with functions of protein binding, helicase activity, DNA binding and transcription co-activator. RBM4-regulated splicing of these genes plays an important role in apoptotic process and gene transcription regulation. As an example, exon inclusion of TNIP1 mediated by RBM4 affects the expression of its targets in inflammatory pathways. These results indicated that RBM4 can mediate the inflammatory response via splicing regulation, which adds to the understanding of the critical role of RBM4 in cancer complicated by inflammation. In conclusion, this study indicated a mechanism in which the dysregulation of alternative splicing can influence cellular biology and lead to various immune-related diseases.
Subject(s)
Alternative Splicing/genetics , Cell Proliferation/genetics , Inflammation/genetics , RNA-Binding Proteins/genetics , Transcription Factors/genetics , Apoptosis/genetics , Cell Line, Tumor , DNA-Binding Proteins/genetics , Exons/genetics , HeLa Cells , Humans , RNA Splicing/genetics , RNA, Messenger/genetics , Signal Transduction/genetics , Transcription, Genetic/genetics , Transcriptional Activation/genetics , Transcriptome/geneticsABSTRACT
Browning of white adipocytes (WAs) (also referred as beige cells) was demonstrated to execute thermogenesis by consuming stored lipids as do brown adipocytes (BAs), and this is highly related to metabolic homeostasis. Alternative splicing (AS) constitutes a pivotal mechanism for defining cellular fates and functional specifications. Nevertheless, the impacts of AS regulation on the browning of WAs have not been comprehensively investigated. In this study, we first identified the discriminative expression and splicing profiles of the muscleblind-like 1 (MBNL1) gene in postnatal brown adipose tissues (BATs) compared to those of embryonic BATs. A shift in the MBNL1+ex 5 isoform 7 (MBNL17) to MBNL1-ex 5 isoform 1 (MBNL11) was characterized throughout BAT development or during the in vitro browning of pre-WAs, 3T3-L1 cells. The interplay between MBNL1 and the exonic CCUG motif constitutes an autoregulatory mechanism for excluding MBNL1 exon 5. The simultaneous association of RNA-binding motif protein 4a (RBM4a) with exonic and intronic CU elements collaboratively mediates the skipping of MBNL1 exon 5. Overexpressing the MBNL11 isoform exhibited a more-prominent effect than that of the MBNL17 isoform on programming its own transcripts and beige cell-related splicing events in a CCUG motif-mediated manner. In addition to splicing regulation, overexpression of the MBNL11 and MBNL17 isoforms differentially enhanced beige adipogenic signatures of 3T3-L1 cells. Our findings demonstrated that MBNL1 constitutes an emerging and autoregulatory mechanism involved in development of beige cells.
Subject(s)
Adipogenesis/genetics , Adipose Tissue, Brown/embryology , Adipose Tissue, Brown/growth & development , Alternative Splicing , DNA-Binding Proteins/genetics , RNA-Binding Proteins/genetics , 3T3-L1 Cells , Adipose Tissue, Brown/metabolism , Animals , DNA-Binding Proteins/metabolism , Exons , Mice , Mice, Inbred C57BL , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA-Binding Proteins/metabolismABSTRACT
Alternative splicing (AS) constitutes a pivotal mechanism for expanding the transcriptome and proteome diversity in higher eukaryotes. In contrast, misregulated AS events are relevant to carcinogenic signatures, including migration, angiogenesis, immortality, and drug resistance of cancer cells. Using a transcriptome analysis, discriminative splicing profiles of hypoxia-inducible factor (HIF)-1α transcripts were identified in tumorous tissues compared to adjacent normal tissues of lung cancer (LC) patients. In cancerous tissues or LC-derived cells, relatively high levels of HIF-1α-ex14 transcripts encoding the HIF-1αS isoform were noted compared to adjacent normal tissues and non-cancerous cells. The HIF-1αS isoform exhibited a more-prominent effect than that of the HIF-1αL isoform translated from HIF-1α+ex14 transcripts on enhancing promoter activities of the vascular endothelial growth factor receptor 2 (VEGFR2), serine/arginine splicing factor 1 (SRSF1), and c13orf25 genes. An increase in the SRSF1 protein facilitated the generation of HIF-1α-ex14 transcripts, whereas overexpression of RNA-binding motif protein 4 (RBM4) enhanced the expression of HIF-1α+ex14 transcripts in the A549 cells. Results of splicing reporter assays demonstrated the differential impacts of RBM4 and SRSF1 on the utilization of HIF-1α exon 14 in a CU element-dependent manner. In addition to transcriptional regulation, overexpression of the HIF-1αS and HIF-1αL isoforms differentially enhanced the metastatic signatures of A549 cells. Taken together, SRSF1 and RBM4 constitute an antagonistic mechanism on regulating the splicing profiles of HIF-1α gene, which is relevant to the oncogenic signatures of LC cells.
Subject(s)
Alternative Splicing/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Lung Neoplasms/genetics , RNA-Binding Proteins/metabolism , Serine-Arginine Splicing Factors/metabolism , A549 Cells , Carcinogenesis/genetics , Cells, Cultured , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathologyABSTRACT
Induced brown adipocytes (also referred to as beige cells) execute thermogenesis, as do the classical adipocytes by consuming stored lipids, being related to metabolic homeostasis. Treatment of phytochemicals, including berberine (BBR), was reported to induce conversion from white adipocytes to beige cells. In this study, results of microRNA (miRNA)-seq analyses revealed a decrease in miR-92a, of which the transcription is driven by the c13orf25 promoter in BBR-treated 3T3-L1 cells. BBR treatment manipulated the expressions of SP1 and MYC, in turn, reducing the activity of the c13orf25 promoter. A decrease in miR-92a led to an increase in RNA-binding motif protein 4a (RBM4a) expression, which facilitated the beige adipogenesis. Overexpression of miR-92a or depletion of RBM4a reversely interfered with the impact of BBR treatment on the beige adipogenic signatures, gene expressions, and splicing events in 3T3-L1 cells. Our findings demonstrated that BBR treatment enhanced beige adipogenesis of 3T3-L1 cells through transcription-coupled post-transcriptional regulation.
Subject(s)
Adipocytes, Beige/metabolism , Adipogenesis/genetics , Berberine/pharmacology , Transcription, Genetic , 3T3-L1 Cells , Adipocytes, Beige/drug effects , Adipocytes, Brown/drug effects , Adipocytes, Brown/metabolism , Adipogenesis/drug effects , Animals , Base Sequence , Gene Expression Regulation/drug effects , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Open Reading Frames/genetics , Promoter Regions, Genetic/genetics , RNA Splicing/drug effects , RNA Splicing/genetics , RNA-Binding Proteins/metabolism , Transcription, Genetic/drug effectsABSTRACT
Brown adipocytes (BAs) exhibit an energy-expending signature that is important in balancing metabolic homeostasis. In this study, results of transcriptome analyses revealed the reprogrammed splicing profile of the PR domain containing 16 (PRDM16) gene, a key transcription factor involved in brown adipogenesis, throughout development of wild-type brown adipose tissues (BATs). Moreover, discriminative splicing patterns of PRDM16 transcripts were noted in embryonic and postnatal RBM4a-/- BATs. Overexpression of RBM4a enhanced the relative levels of PRDM16-ex 16 transcripts by simultaneously interacting with exonic and intronic CU elements, which encoded the PRDM16S isoform containing a distinct C-terminus. The presence of the overexpressed PRDM16S isoform showed a stronger effect than the overexpressed PRDM16L isoform on enhancing transcriptional activity of the RBM4a and the PGC-1α promoter. Overexpression of the PRDM16S isoform exerted more-prominent effects on enhancing the BAT-related gene program and energy expenditure compared to those of PRDM16L-overexpressing cells. Our studies demonstrated that RBM4a-regulated alternative splicing constituted another regulatory mechanism for strengthening the influence of PRDM16 on the development of brown adipocytes.
Subject(s)
Adipocytes, Brown/metabolism , Adipose Tissue, Brown/metabolism , Alternative Splicing , DNA-Binding Proteins/genetics , Gene Expression Regulation , RNA-Binding Proteins/genetics , Transcription Factors/genetics , Adipogenesis/genetics , Animals , Cellular Reprogramming/genetics , DNA-Binding Proteins/metabolism , Exons , Fibroblasts/metabolism , Gene Expression Profiling , Introns , Mice , Models, Biological , Protein Binding , Protein Isoforms , RNA-Binding Proteins/metabolism , Transcription Factors/metabolism , TranscriptomeABSTRACT
An increase in mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) reportedly attenuates insulin-mediated signaling which participates in the development of brown adipose tissues (BATs). Nevertheless, the effect of MAP4K4 on brown adipogenesis remains largely uncharacterized. In this study, results of a transcriptome analysis (also referred as RNA-sequencing) showed differential expressions of MAP4K4 or SRSF3 transcripts isolated from distinct stages of embryonic BATs. The discriminative splicing profiles of MAP4K4 or SRSF3 were noted as well in brown adipocytes (BAs) with RNA-binding motif protein 4-knockout (RBM4-/-) compared to the wild-type counterparts. Moreover, the relatively high expressions of authentic SRSF3 transcripts encoding the splicing factor functioned as a novel regulator toward MAP4K4 splicing during brown adipogenesis. The presence of alternatively spliced MAP4K4 variants exerted differential effects on the phosphorylation of c-Jun N-terminal protein kinase (JNK) which was correlated with the differentiation or metabolic signature of BAs. Collectively, the RBM4-SRSF3-MAP4K4 splicing cascade constitutes a novel molecular mechanism in manipulating the development of BAs through related signaling pathways.
Subject(s)
Adipocytes, Brown/cytology , Adipogenesis , Alternative Splicing , Protein Serine-Threonine Kinases/genetics , RNA-Binding Proteins/genetics , Serine-Arginine Splicing Factors/genetics , Adipocytes, Brown/metabolism , Animals , Cell Differentiation , Cell Line , Gene Expression Profiling , Gene Knockout Techniques , Genetic Variation , MAP Kinase Kinase 4/metabolism , Mice , Phosphorylation , Sequence Analysis, RNA , Signal Transduction , NF-kappaB-Inducing KinaseABSTRACT
Alternative splicing has been widely demonstrated to function as pivotal regulation in specifying cellular fates and biological functions. The relative expression or cellular localization of a splicing factor constitutes an important mechanism in spatiotemporal programming of cell- and stage-specific splicing profiles. In this study, results of deep RNA-sequencing (RNA-Seq) analyses first revealed the reprogrammed splicing profile and reduced expression of serine/arginine-rich splicing factor protein kinase 1 (SRPK1) throughout the development of brown adipose tissue (BAT). A gradual increase in the exon 10-skipped SRPK1 transcript, a potential target of a nonsense-mediated decay (NMD) mechanism, was noted during brown adipogenesis. Elevated RBM4a constituted the regulatory mechanism that led to skipping of SRPK1 exon 10. Moreover, brown adipogenesis-induced upregulation of microRNA (miR)-485 interfered with SRPK1 expression by targeting its 3'-untranslated region (UTR). Depletion of endogenous SRPK1 enhanced the development of C3H10T1/2 cells toward brown adipocytes. Taking our results together, multiple post-transcriptional regulations reduced SRPK1 expression, which subsequently affected brown adipogenesis.
Subject(s)
Adipocytes, Brown/metabolism , Adipogenesis/genetics , Gene Expression Regulation , Protein Serine-Threonine Kinases/metabolism , Transcription, Genetic , Adipose Tissue, Brown/metabolism , Alternative Splicing/genetics , Animals , Base Sequence , Cell Line , Exons/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Mice , MicroRNAs/genetics , MicroRNAs/metabolism , Models, Biological , Polypyrimidine Tract-Binding Protein/metabolism , Protein Biosynthesis/drug effects , Protein Transport/genetics , RNA Stability/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Transcriptome/geneticsABSTRACT
Alternative splicing (AS) of pre-messenger (m)RNA is a pivotal mechanism in expanding proteomic diversity, which determines the functions of mammalian cells. By conducting transcriptome analyses to profile splicing events in human colorectal cancer (CRC) tissues compared to adjacent normal counterparts, we noted differential splicing profiles of serine/arginine-rich splicing factor 3 (SRSF3) and mitogen-activated protein 4 kinase 4 (MAP4K4) in cancerous tissues of CRC compared to adjacent normal tissues. In addition to SRSF3-mediated autoregulation, RNA-binding motif protein 4 (RBM4) constituted another mechanism in reprogramming the splicing profile of SRSF3. Upregulated expressions of SRSF3 in CRC cells modulated utilization of MAP4K4 exon 16 in a sequence-dependent manner. Alternatively spliced MAP4K4 variants exhibited differential effects on the phosphorylation of c-Jun N-terminal protein kinase 1 (JNK1) which subsequently modulated expression profiles of E-cadherin, N-cadherin, and vimentin, all of which are involved in the migration and invasion of CRC cells. Collectively, RBM4-SRSF3-MAP4K4 constitutes a novel mechanism for manipulating the metastasis of CRC cells through the JNK1 signaling pathway.
Subject(s)
Colorectal Neoplasms/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , MAP Kinase Signaling System , Neoplasm Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA Splicing , RNA-Binding Proteins/metabolism , Serine-Arginine Splicing Factors/metabolism , Colorectal Neoplasms/genetics , Female , Humans , Intracellular Signaling Peptides and Proteins/genetics , Male , Neoplasm Metastasis , Neoplasm Proteins/genetics , Protein Serine-Threonine Kinases/genetics , RNA-Binding Proteins/genetics , Serine-Arginine Splicing Factors/geneticsABSTRACT
LncRNAs play a vital role in alternative splicing of target genes. However, the mechanisms underlying lncRNAs involvement in splicing are poorly understood. In the present study, we identified a previously uncharacterized lncRNA, which is denoted as TPM1-AS, is reverse-transcribed from the fourth intronic region of the tropomyosin I (TPM1). In situ hybridization and RNA immunoprecipitation assays demonstrated that TPM1-AS was located in the nucleus and interacted with RNA-binding motif protein 4 (RBM4) in human esophageal cancer cells. TPM1-AS overexpression or RBM4 knockdown decreased endogenous exon 2a expression of TPM1, resulting in specifically down-regulation of TPM1variant V2 and V7 in human esophageal cancer cells. Mechanismly, the interaction of TPM1-AS with RBM4 hindered binding of RBM4 to TPM1 pre-mRNA and inhibited RBM4 to promote endogenous exon 2a inclusion of TPM1. Importantly, overexpression of TPM1-AS inhibited migration and filopodium formation, whereas TPM1variant V2 and V7 promoted these behaviors of human esophageal cancer cells. Taken together, the results suggest that a natural antisense TPM1-AS regulates the alternative splicing of TPM1 through an interaction with RBM4 and involves in TPM1-mediated filopodium formation and migration of cancer cells.
Subject(s)
Alternative Splicing , RNA, Antisense/genetics , RNA-Binding Proteins/metabolism , Tropomyosin/genetics , Cell Line, Tumor , Cell Nucleus/metabolism , Esophageal Neoplasms/pathology , Exons/genetics , Humans , Protein Binding , Pseudopodia/metabolismABSTRACT
RNA-binding motif 4 (RBM4) has been reported to play an important role in many human tumors such as lung cancer, breast cancer, ovarian cancer and gastric cancer by regulating alternative splicing and messenger RNA (mRNA) translation. However, little is known about the role of RBM4 in the development of hepatocellular carcinoma (HCC). This study aimed to investigate the expression of RBM4 in HCC tissues. Expression of RBM4 was detected by immunohistochemistry in 95 cases of HCC. Correlations of RBM4 expression with the overall survival and disease-free survival of HCC were also studied. Patients with high RBM4 expression had better overall survival rate and disease-free survival rate than those with low RBM4 expression (P<0.001, P=0.007, respectively). RBM4 expression, together with tumor numbers, capsular formation, vascular invasion and Barcelona clinic liver cancer (BCLC) stage, was an independent prognostic factor for overall survival rate and disease-free survival rate of HCC. Our data implicate RBM4 as a novel prognostic marker and a potential therapeutic target for HCC.
ABSTRACT
Alternative splicing (AS) is a pivotal mechanism for the expansion of gene diversity, which determines the cellular fate or specification. However, the effect of AS networks on brown adipogenesis has not been comprehensively investigated. In this study, we identified the discriminative splicing profiles of RNA-binding motif protein 4a-knockout (RBM4a-/-) brown adipocytes (BAs) and compared them with those of their wild-type counterparts through deep RNA-sequencing. Among these candidates, RBM4a ablation enhanced the relative level of exon 4-excluded neuro-oncological ventral antigen 1 (Nova1-4) transcripts, which were predominantly generated in embryonic BAs. By contrast, most of the Nova1 transcripts were exon 4-included (Nova1+4) in mature BAs. The Nova1 isoforms exhibited differential effects on repressing the development of BAs. Moreover, overexpression of Nova1 proteins reduced the serine/arginine splicing factor 6 (SRSF6) level by enhancing the generation of intron 2-included (SRSF6+intron 2) transcripts, which are a putative candidate of the AS-coupled nonsense-mediated decay mechanism. Furthermore, we observed the positive effect of SRSF6 on BA development. These results highlight the hierarchical role of RBM4a in an AS cascade that manipulates brown adipogenesis.