RNA-binding Motif Protein Research Articles

Deletion of RBM20 exon 9 impairs skeletal muscle growth and satellite cell function in pigs

Maintaining healthy skeletal tissue is essential for overall well-being and quality of life. Skeletal muscle plays a key role in this process, yet models for studying its detailed function are limited. While RNA-binding motif protein 20 (RBM20) is primarily associated with dilated cardiomyopathy (DCM), its role in skeletal muscle remains largely unexplored. This study investigates RBM20 function in skeletal muscle using an RBM20 exon 9 deletion pig model (RBM20E9D). The deletion of exon 9 resulted in loosely arranged muscle fibers, large inter-fiber gaps, and irregular organization, leading to impaired muscle growth and development. Analysis of skeletal muscle satellite cells revealed significantly reduced proliferation, diminished myotube formation in vitro, and disrupted sarcomere structure due to exon 9 deletion. Given the critical role of satellite cell proliferation and differentiation in muscle repair, RBM20E9D pigs offer a novel model for studying the mechanisms underlying skeletal muscle injury, repair, and growth.

Identification of Goat Supernumerary Teat Phenotype Using Wide-Genomic Copy Number Variants.

Supernumerary teats (SNTs) or nipples often emerge around the mammary line. This study performed a genome-wide selective sweep analysis (GWS) at the copy number variant (CNV) level using two selected signal calculation methods (VST and FST) to identify candidate genes associated with SNTs in goats. A total of 12,310 CNVs were identified from 37 animals and 123 CNVs, with the top 1% VST values including 84 candidate genes (CDGs). Of these CDGs, minichromosome maintenance complex component 3, ectodysplasin A receptor associated via death domain, and cullin 5 demonstrated functions closely related to mammary gland development. In addition, 123 CNVs with the top 1% FST values were annotated to 97 CDGs. 5-Hydroxytryptamine receptor 2A, CCAAT/enhancer-binding protein alpha, and the polymeric immunoglobulin receptor affect colostrum secretion through multiple signaling pathways. Two genes, namely, RNA-binding motif protein 46 and β-1,3-galactosyltransferase 5, showed a close relation to mammary gland development. Six CNVs were identified and annotated to five genes by intersecting the top 1% of candidate CNVs with both parameters. These genes include LOC102185621, LOC102190481, and UDP-glucose pyrophosphorylase 2, which potentially affect the occurrence of BC through multiple biological processes, such as cell detoxification, glycogen synthesis, and phospholipid metabolism. In conclusion, we discovered numerous genes related to mammary development and breast cancer (BC) through a GWS, which suggests the mechanism of SNTs in goats and a certain association between mammary cancer and SNTs.

RBM15-mediated the m6A modification of MAT2A promotes osteosarcoma cell proliferation, metastasis and suppresses ferroptosis.

Methionine adenosyltransferase 2A (MAT2A) has been found to mediate osteosarcoma (OS) progression. Therefore, more roles and mechanisms of MAT2A in the development of OS deserve further exploration. The mRNA and protein levels of MAT2A and RNA binding motif protein 15 (RBM15) were tested by quantitative real-time PCR and western blot (WB). Cell proliferation and metastasis were examined using EdU assay and transwell assay. The protein levels of metastasis-related markers and ferroptosis-related marker were measured by WB. Cell ferroptosis was assessed via testing GSH, ROS, and Fe2+levels. Mice xenograft model was constructed to explore the roles of MAT2A and RBM15 in vivo. RBM15 and MAT2A interaction was assessed by MeRIP assay and dual-luciferase reporter assay. High expression of MAT2A was observed in OS tumor tissues and cells. MAT2A knockdown reduced OS cell proliferation, migration, invasion and enhanced ferroptosis. Silencing of MAT2A inhibited OS tumor growth in vivo. RBM15 was upregulated in OS tumor tissues and cells, which could promote MAT2A expression by N6-methyladenosine (m6A) modification. Downregulation of RBM15 repressed OS cell behaviors and tumorigenesis by decreasing MAT2A expression. In conclusion, MAT2A, regulated by RBM15-mediated m6A modification, accelerated OS malignant progression by increasing cell proliferation, metastasis and decreasing ferroptosis.

RBM15 facilitates osimertinib resistance of lung adenocarcinoma through m6A-dependent epigenetic silencing of SPOCK1.

Lung cancer is the leading cause of cancer-related mortality globally. N6-methyladenosine (m6A) is the most abundant modification in mammalian mRNA and is involved in the biological regulation of tumors, including lung cancer. However, the role of m6A-related proteins, such as RNA-binding motif protein 15 (RBM15), in lung cancer progression remains largely unknown. Our study indicated that RBM15 is significantly overexpressed in lung adenocarcinoma, serving as an independent prognostic factor for poor outcomes and facilitating tumor cell proliferation and migration. RBM15 was markedly elevated in patients with EGFR mutations, correlating with a poorer prognosis, while it had negligible prognostic value in EGFR wild-type patients. As EGFR-tyrosine kinase inhibitors (TKIs) are the standard treatment for patients with EGFR mutations, we subsequently determined that RBM15 drives osimertinib resistance via a novel mechanism: enhancing m6A modification of cwcv- and kazal-like domains proteoglycan 1 (SPOCK1) mRNA, promoting epithelial-mesenchymal transition-mediated osimertinib resistance through a bypass activation pathway. These findings were validated in osimertinib-resistant H1975 cells and organoids from patients with osimertinib-resistant lung adenocarcinoma. Furthermore, the RBM15-SPOCK1 axis was activated in drug-tolerant persister cells, indicating that early targeting of RBM15 during EGFR-TKI treatment could dramatically extend the patient response and benefit from TKI therapy. Our results emphasize the critical role of RBM15 in reversing EGFR-TKI resistance and propose it as a promising therapeutic target for prolonging TKI treatment benefits in patients with lung adenocarcinoma.

RBM20 promotes doxorubicin-induced cardiotoxicity by Atp6v0a1 alternative splicing

Abstract Background & Objective: Doxorubicin (dox), an effective chemotherapy drug, is extremely limited in clinical practice by its lethal cardiotoxicity. Whereas dysregulation of splice-regulatory networks has been reported in cardiomyopathies, the role of RNA binding motif protein 20 (RBM20), an adaptive splice regulator, in doxorubicin-induced cardiotoxicity (DIC) remained to be elucidated. Purpose Here we aimed to show the role of RBM20 in DIC and elucidate the relationship between alternative splicing and lysosome acidification impairment. Method Mice were treated with intraperitoneal injection of cumulative 20mg/kg doxorubicin with four equal injections over a period of 2 weeks to induce DIC. The contribution of RBM20 in cardiotoxicity was evaluated in conditional cardiomyocyte-specific RBM20 overexpression (RBM20tg/+/Myh6-CreERT) mice. The regulatory factor of RBM20 was determined through luciferase assay and chromatin immunoprecipitation in vitro. Autophagic flux was evaluated by transmission electron microscope in RBM20tg/+/Myh6-CreERT mice and by lysotracker and a tandem fluorescence RFP-GFP-LC3 reporter system in RBM20 overexpressed or knockdown cardiomyocyte. RBM20-dependent splicing events were identified by rMATS 3.2.5 on the RNA-sequencing data. Results In the DIC human myocardium and murine model, we observed significant elevation of RBM20 in cardiomyocytes. Cardiomyocyte-specific RBM20 overexpression resulted in severe cardiac dysfunction reflected by dilated ventricles, declined left ventricular ejection fraction, increased chamber stiffness and lower survival rate. After screening for potential upstream transcription factors, c-Jun was identified to bind and upregulate RBM20 directly. The inhibition of c-Jun by SR 11302 could partially mitigate DIC in vitro. Mechanistically, RBM20 was positively related with autophagic flux blockade and impaired lysosome acidification by shifting Atp6v0a1, the a1-subunit of the V0 domain of vacuolar H+-ATPases, toward an exon 6-7 exclusion isoform without altering total transcript levels. By restoring exon 6-7-containing ATP6V0a1 level, we were able to reduce DIC by restoring normal autophagic flux. Conclusion Overall, our study reveals that elevation of RBM20 disturbed lysosome acidification in doxorubicin induced cardiotoxicity via the alternative splicing of Atp6v0a1, leading to heart failure.JUN-Rbm20 upregulattion promoted DICRBM20 alternative spliced atp6v0a1

Reconstructing the sequence specificities of RNA-binding proteins across eukaryotes.

RNA-binding proteins (RBPs) are key regulators of gene expression. Here, we introduce EuPRI (Eukaryotic Protein-RNA Interactions) - a freely available resource of RNA motifs for 34,736 RBPs from 690 eukaryotes. EuPRI includes in vitro binding data for 504 RBPs, including newly collected RNAcompete data for 174 RBPs, along with thousands of reconstructed motifs. We reconstruct these motifs with a new computational platform - Joint Protein-Ligand Embedding (JPLE) - which can detect distant homology relationships and map specificity-determining peptides. EuPRI quadruples the number of known RBP motifs, expanding the motif repertoire across all major eukaryotic clades, and assigning motifs to the majority of human RBPs. EuPRI drastically improves knowledge of RBP motifs in flowering plants. For example, it increases the number of Arabidopsis thaliana RBP motifs 7-fold, from 14 to 105. EuPRI also has broad utility for inferring post-transcriptional function and evolutionary relationships. We demonstrate this by predicting a role for 12 Arabidopsis thaliana RBPs in RNA stability and identifying rapid and recent evolution of post-transcriptional regulatory networks in worms and plants. In contrast, the vertebrate RNA motif set has remained relatively stable after its drastic expansion between the metazoan and vertebrate ancestors. EuPRI represents a powerful resource for the study of gene regulation across eukaryotes.

RBM15 drives the progression of lung adenocarcinoma by regulating N6-methyladenosine-mediated LDHA mRNA stability

Abnormal N6-methyladenosine (m6A) methylation in RNA plays a pivotal role in the pathogenesis of many types of tumors by influencing mRNA metabolism, alternative splicing, translocation, stability and translation. However, the specific regulators and underlying mechanisms of m6A modification in the progression of lung adenocarcinoma are not well understood. In this study, we analyzed the RNA-seq transcriptome data downloaded from The Cancer Genome Atlas (TCGA) database, and identified “m6A writer” RNA binding motif protein 15 (RBM15) expression was significantly elevated in lung adenocarcinoma (LUAD) biopsies, and the higher RBM15 levels were correlated with the poorer overall survival (OS) of LUAD patients. Further study confirmed RBM15 was prominently expressed in LUAD tissues and cell lines. Moreover, silencing RBM15 in PC9 and H1299 cells reduced cell proliferation both in vitro and in vivo, while overexpression of RBM15 in A549 cells promoted cell growth. Mechanistically, lactate dehydrogenase A (LDHA) acted as a downstream target of RBM15. RBM15-mediated m6A modification of LDHA mRNA enhanced its stability to exert an oncogenic role in LUAD. Taken together, our findings suggest that the RBM15/LDHA axis might be a novel and promising therapeutic target for LUAD.

Rbm3 deficiency leads to transcriptome-wide splicing alterations

ABSTRACT Rbm3 (RNA-binding motif protein 3) is a stress responsive gene, which maintains cellular homeostasis and promotes survival upon various harmful cellular stimuli. Rbm3 protein shows conserved structural and molecular similarities to heterogeneous nuclear ribonucleoproteins (hnRNPs), which regulate all steps of the mRNA metabolism. Growing evidence is pointing towards a broader role of Rbm3 in various steps of gene expression. Here, we demonstrate that Rbm3 deficiency is linked to transcriptome-wide pre-mRNA splicing alterations, which can be reversed through Rbm3 co-expression from a cDNA. Using an MS2 tethering assay, we show that Rbm3 regulates splice site selection similar to other hnRNP proteins when recruited between two competing 5 ′ splice sites. Furthermore, we show that the N-terminal part of Rbm3 encompassing the RNA recognition motif (RRM), is sufficient to elicit changes in splice site selection. On the basis of these findings, we propose a novel, undescribed function of Rbm3 in RNA splicing that contributes to the preservation of transcriptome integrity.

RBM15B Promotes Prostate Cancer Cell Proliferation via PCNA m6A Modification.

Prostate cancer (PC) is the most frequently occurring cancer in men, characterized by the abnormal proliferation of cells within the prostate gland. This study explores the role of RNA binding motif protein 15B (RBM15B) in PC. RBM15B expression levels in PC patients were predicted using the Starbase database. The expression of RBM15B and proliferating cell nuclear antigen (PCNA) expression in PC cells was detected. Following RBM15B knockdown, cell proliferation assays were conducted. N6-methyladenosine (m6A) levels in PC cells were quantified, and RNA immunoprecipitation was performed to analyze the binding of m6A and YTH N-methyladenosine RNA binding protein 1 (YTHDF1) on PCNA mRNA. The stability of PCNA mRNA was assessed after treatment with actinomycin D. An in vivo nude mouse xenograft model was created to validate the role of RBM15B. The findings revealed the upregulation of RBM15B in PC. RBM15B knockdown resulted in decreased proliferation, colony formation, and EdU-positive cells. Mechanical analysis showed that RBM15B facilitated m6A modification of PCNA mRNA, leading to increasing m6A methylation. YTHDF1 bound to these m6A sites on PCNA mRNA, thus stabilizing it. Furthermore, PCNA overexpression mitigated the effects of RBM15B knockdown on PC cell proliferation. In conclusion, RBM15B promotes PC cell proliferation by enhancing the stability of PCNA mRNA through YTHDF1-mediated m6A modification.

Circular RNA hsa_circ_0081343 modulates trophoblast autophagy through Rbm8a nuclear translocation

IntroductionFetal growth restriction (FGR) is a kind of obstetric complication that seriously endangers fetal life. Recent studies reported significant reduction of hsa_circ_0081343 in human placenta developed in FGR and is involved in cell migration, invasion, and apoptosis of trophoblast by acting as microRNA sponges. Autophagy is required for invasion of trophoblast cells and for vascular remodeling during placentation. In this study, we aimed to explore the mechanistic link between hsa_circ_0081343 and autophagy. MethodsWe investigated the interactions between hsa_circ_0081343 and RNA-binding proteins were studied by RNA pull-down assay, mass spectrometry and RNA immunoprecipitation assay. The mechanism of nuclear translocation of Rbm8a were assessed by reverse transcription-quantitative PCR, Western blot, immunofluorescence and Co-Immunoprecipitation. Western blot, immunofluorescence and transmission electron microscopy were performed to elucidate the mechanism underlying hsa_circ_0081343 and/or Rbm8a mediated regulation of autophagy. Resultshsa_circ_0081343 served as an RNA-binding protein (RBP) sponge. RNA binding motif protein 8A (Rbm8a) was directly bound to hsa_circ_0081343 in the cytoplasm, while knockdown of hsa_circ_0081343 facilitated Rbm8a localization in the nucleus. We also identified Rbm8a as a potential import cargo for Importin13 (Ipo13), which transported Rbm8a across the nuclear membrane into the nucleus.Ipo13 recognized Rbm8a via a functional nuclear localization signal (NLS). Furthermore, the mechanistic study revealed that hsa_circ_0081343-mediated nuclear translocation of Rbm8a activated trophoblast autophagy. DiscussionOur results suggest that hsa_circ_0081343 could bind to RBP and the interaction between hsa_circ_0081343 and Rbm8a participate in regulating autophagy. These findings offer novel molecular targets and insights for a potential therapeutic strategy against FGR.

Development of a MaizeGerm50K array and application to maize genetic studies and breeding

Genotyping arrays based on single nucleotide polymorphisms (SNPs) provide a low-cost, high-throughput platform. The development of a SNP array that fully reflects the genetic diversity of maize (Zea mays L.) germplasm and is applicable to molecular breeding programs is desirable. In this study, we developed a MaizeGerm50K array comprising 50,852 SNPs selected from the resequencing data of 1604 maize inbred lines and other markers. A genome-wide association study using a landrace panel genotyped with the array permitted mapping of several known genes. We also verified a candidate gene, RNA-binding motif protein 24-like 1 (ZmRBM24L1), delaying flowering through overexpression lines. Genomic selection for yield and agronomic traits showed high prediction accuracy. The MaizeGerm50K array is thus a valuable genomic tool for maize genetic studies and breeding.

Global RNA Interaction and Transcriptome Profiles Demonstrate the Potential Anti-Oncogenic Targets and Pathways of RBM6 in HeLa Cells

Background: The fate and functions of RNAs are coordinately regulated by RNA-binding proteins (RBPs), which are often dysregulated in various cancers. Known as a splicing regulator, RNA-binding motif protein 6 (RBM6) harbors tumor-suppressor activity in many cancers; however, there is a lack of research on the molecular targets and regulatory mechanisms of RBM6. Methods: In this study, we constructed an RBM6 knock-down (shRBM6) model in the HeLa cell line to investigate its functions and molecular targets. Then we applied improved RNA immunoprecipitation coupled with sequencing (iRIP-seq) and whole transcriptome sequencing approaches to investigate the potential role and RNA targets of RBM6. Results: Using The Cancer Genome Atlas dataset, we found that higher expression of RBM6 is associated with a better prognosis in many cancer types. In addition, we found that RBM6 knockdown promoted cell proliferation and inhibited apoptosis, demonstrating that RBM6 may act as an anti-oncogenic protein in cancer cells. RBM6 can regulate the alternative splicing (AS) of genes involved in DNA damage response, proliferation, and apoptosis-associated pathways. Meanwhile, RBM6 knockdown activated type I interferon signaling pathways and inhibited the expression of genes involved in the cell cycle, cellular responses to DNA damage, and DNA repair pathways. The differentially expressed genes (DEGs) by shRBM6 and their involved pathways were likely regulated by the transcription factors undergoing aberrant AS by RBM6 knockdown. For iRIP-seq analysis, we found that RBM6 could interact with a large number of mRNAs, with a tendency for binding motifs GGCGAUG and CUCU. RBM6 bound to the mRNA of cell proliferation- and apoptosis-associated genes with dysregulated AS after RBM6 knockdown. Conclusions: In summary, our study highlights the important role of RBM6, as well as the downstream targets and regulated pathways, suggesting the potential regulatory mechanisms of RBM6 in the development of cancer.

RBM39 Enhances Cholangiocarcinoma Growth Through EZH2-mediated WNT7B/β-catenin Pathway

Background and aimsThe RNA-binding motif protein 39 (RBM39) functions as both an RNA-binding protein and a splicing factor in a variety of cancer types. However, the function of RBM39 in cholangiocarcinoma (CCA) remains undefined. In this study, we aimed to investigate the role of RBM39 in CCA and explore its potential as a therapeutic target. MethodsThe expression of RBM39 in CCA was investigated by analyzing human CCA tumor specimens. CRISPR/Cas9 or shRNA-mediated depletion of RBM39 was performed in vitro and in vivo to document the oncogenic role of RBM39 in CCA. The anti-tumor effect of the RBM39 inhibitor Indisulam in combination with the EZH2 degrader MS177 was assessed in vitro and in vivo. ResultsRBM39 is significantly increased in human CCA tissues and associated with a poor prognosis in CCA patients. Depletion of RBM39 by CRISPR/Cas9 or shRNA inhibited CCA cell proliferation in vitro and prevented CCA development and tumor growth in mice. Mechanistically, our results showed that depletion of RBM39 suppressed EZH2 expression via disrupting its mRNA splicing. RBM39-regulated EZH2 controls WNT7B/β-catenin activity. Pharmacological co-targeting of RBM39 (with Indisulam) and EZH2 (with MS177) resulted in a synergistic antitumor effect, both in vitro and in vivo. ConclusionThis study discloses a novel RBM39-EZH2-β-catenin signaling axis that is crucial for CCA growth. Our findings suggest that simultaneous inhibition of RBM39 and EZH2 presents a promising therapeutic strategy for CCA treatment.

Deubiquitinating enzyme USP39 promotes the growth and metastasis of gastric cancer cells by modulating the degradation of RNA-binding protein RBM39

It has been revealed recently that the RNA-binding motif protein RBM39 is highly expressed in several cancers, which results in poor patient survival. However, how RBM39 is regulated in gastric cancer cells is unknown. Here, affinity purification-mass spectrometry and a biochemical screening are employed to identify the RBM39-interacting proteins and the deubiquitinating enzymes (DUBs) that regulate the RBM39 protein level. Integration of the data obtained from these two approaches uncovers USP39 as the potential DUB that regulates RBM39 stability. Bioinformatic analysis discloses that USP39 is increased in gastric cancer tissues and its elevation shortens the duration of overall survival for gastric cancer patients. Biochemical experiments verify that USP39 and RBM39 interact with each other and highly colocalize in the nucleus. Expression of USP39 elevates while USP39 knockdown attenuates the RBM39 protein level and their interaction regulates this modulation and their colocalization. Mechanistic studies reveal that USP39 reduces the K48-linked polyubiquitin chains on RBM39, thus enhancing its stability and increasing the protein level by preventing its proteasomal degradation. USP39 overexpression promotes while its knockdown attenuates the growth, colony formation, migration, and invasion of gastric cancer cells. Interestingly, overexpression of RBM39 partially restores the impact of USP39 depletion while RBM39 knockdown partially abolishes the effect of USP39 overexpression on the growth, colony formation, migration, and invasion of gastric cancer cells. Collectively, this work identifies the first DUB for RBM39 and elucidates the regulatory functions and the underlying mechanism, providing a possible alternative approach to suppressing RBM39 by inhibiting USP39 in cancer therapy.

Decoding Cold Therapy Mechanisms of Enhanced Bone Repair through Sensory Receptors and Molecular Pathways.

Applying cold to a bone injury can aid healing, though its mechanisms are complex. This study investigates how cold therapy impacts bone repair to optimize healing. Cold was applied to a rodent bone model, with the physiological responses analyzed. Vasoconstriction was mediated by an increase in the transient receptor protein channels (TRPs), transient receptor potential ankyrin 1 (TRPA1; p = 0.012), and transient receptor potential melastatin 8 (TRPM8; p < 0.001), within cortical defects, enhancing the sensory response and blood flow regulation. Cold exposure also elevated hypoxia (p < 0.01) and vascular endothelial growth factor expression (VEGF; p < 0.001), promoting angiogenesis, vital for bone regeneration. The increased expression of osteogenic proteins peroxisome proliferator-activated receptor gamma coactivator (PGC-1α; p = 0.039) and RNA-binding motif protein 3 (RBM3; p < 0.008) suggests that the reparative processes have been stimulated. Enhanced osteoblast differentiation and the presence of alkaline phosphatase (ALP) at day 5 (three-fold, p = 0.021) and 10 (two-fold, p < 0.001) were observed, along with increased osteocalcin (OCN) at day 10 (two-fold, p = 0.019), indicating the presence of mature osteoblasts capable of mineralization. These findings highlight cold therapy's multifaceted effects on bone repair, offering insights for therapeutic strategies.

RBM25 is required to restrain inflammation via ACLY RNA splicing-dependent metabolism rewiring.

Spliceosome dysfunction and aberrant RNA splicing underline unresolved inflammation and immunopathogenesis. Here, we revealed the misregulation of mRNA splicing via the spliceosome in the pathogenesis of rheumatoid arthritis (RA). Among them, decreased expression of RNA binding motif protein 25 (RBM25) was identified as a major pathogenic factor in RA patients and experimental arthritis mice through increased proinflammatory mediator production and increased hyperinflammation in macrophages. Multiomics analyses of macrophages from RBM25-deficient mice revealed that the transcriptional enhancement of proinflammatory genes (including Il1b, Il6, and Cxcl10) was coupled with histone 3 lysine 9 acetylation (H3K9ac) and H3K27ac modifications as well as hypoxia inducible factor-1α (HIF-1α) activity. Furthermore, RBM25 directly bound to and mediated the 14th exon skipping of ATP citrate lyase (Acly) pre-mRNA, resulting in two distinct Acly isoforms, Acly Long (Acly L) and Acly Short (Acly S). In proinflammatory macrophages, Acly L was subjected to protein lactylation on lysine 918/995, whereas Acly S did not, which influenced its affinity for metabolic substrates and subsequent metabolic activity. RBM25 deficiency overwhelmingly increased the expression of the Acly S isoform, enhancing glycolysis and acetyl-CoA production for epigenetic remodeling, macrophage overactivation and tissue inflammatory injury. Finally, macrophage-specific deletion of RBM25 led to inflammaging, including spontaneous arthritis in various joints of mice and inflammation in multiple organs, which could be relieved by pharmacological inhibition of Acly. Overall, targeting the RBM25-Acly splicing axis represents a potential strategy for modulating macrophage responses in autoimmune arthritis and aging-associated inflammation.

RBM8A, a new target of TEAD4, promotes breast cancer progression by regulating IGF1R and IRS-2

BackgroundBreast cancer (BC) is the most common malignant tumor in women worldwide, and further elucidation of the molecular mechanisms involved in BC pathogenesis is essential to improve the prognosis of BC patients. RNA Binding Motif Protein 8 A (RBM8A), with high affinity to a myriad of RNA transcripts, has been shown to play a crucial role in genesis and progression of multiple cancers. We attempted to explore its functional significance and molecular mechanisms in BC.MethodsBioinformatics analysis was performed on publicly available BC datasets. qRT-PCR was used to determine the expression of RBM8A in BC tissues. MTT assay, clone formation assay and flow cytometry were employed to examine BC cell proliferation and apoptosis in vitro. RNA immunoprecipitation (RIP) and RIP-seq were used to investigate the binding of RBM8A/EIF4A3 to the mRNA of IGF1R/IRS-2. RBM8A and EIF4A3 interactions were determined by co-immunoprecipitation (Co-IP) and immunofluorescence. Chromatin immunoprecipitation (Ch-IP) and dual-luciferase reporter assay were carried out to investigate the transcriptional regulation of RBM8A by TEAD4. Xenograft model was used to explore the effects of RBM8A and TEAD4 on BC cell growth in vivo.ResultsIn this study, we showed that RBM8A is abnormally highly expressed in BC and knockdown of RBM8A inhibits BC cell proliferation and induces apoptosis in vitro. EIF4A3, which phenocopy RBM8A in BC, forms a complex with RBM8A in BC. Moreover, EIF4A3 and RBM8A complex regulate the expression of IGF1R and IRS-2 to activate the PI3K/AKT signaling pathway, thereby promoting BC progression. In addition, we identified TEAD4 as a transcriptional activator of RBM8A by Ch-IP, dual luciferase reporter gene and a series of functional rescue assays. Furthermore, we demonstrated the in vivo pro-carcinogenic effects of TEAD4 and RBM8A by xenograft tumor experiments in nude mice.ConclusionCollectively, these findings suggest that TEAD4 novel transcriptional target RBM8A interacts with EIF4A3 to increase IGF1R and IRS-2 expression and activate PI3K/AKT signaling pathway, thereby further promoting the malignant phenotype of BC cells.

RBM3 Inhibits the Cell Cycle of Cutaneous Squamous Cell Carcinoma through the PI3K/AKT Signaling Pathway.

RBM3 is a key RNA-binding protein that has been implicated in various cellular processes, including cell proliferation and cell cycle regulation. However, its role in cutaneous squamous cell carcinoma (cSCC) remains poorly understood. We aimed to investigate the expression levels of RNA-binding motif protein 3 (RBM3) in patients with cSCC and evaluate its effect on cell ability in cSCC and its underlying regulatory mechanisms. The expression of RBM3 in cSCC tissues and A431 cells was determined via immunohistochemistry and western blotting. Plenti-CMV-RBM3- Puro was used to overexpress RBM3. The effect of RBM3 on the proliferation ability of cSCC cells was evaluated using MTT and colony formation assay. Cell apoptosis and cell cycle were determined using flow cytometry, while the protein expressions of BAX, NF-κB, BCL2, CASPASE 3, CYCLIN B, CYCLIN E, CDK1, phosphorylated (P)-CDK1, CDK2, P-CDK2, ERK, P-ERK, P-AMPK, AKT, P-AKT, MDM2, and P53 were assessed using western blotting. RBM3 expression was significantly downregulated in cSCC tissues and A431 cells. RBM3 overexpression significantly inhibited the cell proliferation and colony formation ability of A431. Notably, RNA-seq results showed that the differentially expressed genes associated with RBM3 were primarily involved in the regulation of the cell cycle, oocyte meiosis, and P53 signaling pathway, as well as the modulation of the MAPK, AMPK, Hippo, mTOR, PI3K/AKT, Wnt, FoxO, and NF-κB signaling pathways. Additionally, our findings demonstrated that overexpression of RBM3 inhibited cell proliferation and induced cell cycle arrest of cSCC through modulation of the PI3K/AKT signaling pathway. This study provides novel insights into the suppressive roles of RBM3 in cell proliferation and the cell cycle in cSCC and highlights its therapeutic potential for cSCC.

Abstract P224: RbFox2 is a Rho-related BTB Domain Containing 1 (RhoBTB1)-Cullin 3 (CUL3) Pathway Target that Regulates the Actin Cytoskeleton

Blood pressure (BP) is controlled by an array of cellular pathways. Our prior research revealed that endothelial nitric oxide (NO)-dependent vessel tone and BP are regulated through the RhoBTB1-CUL3 mediated proteasomal degradation of Phosphodiesterase 5 (PDE5). RhoBTB1 is an adaptor protein which delivers protein substrates to the CUL3 E3 ubiquitin ligase for proteasomal degradation. However, the range of protein targets of RhoBTB1 remains unknown. We employed the ascorbate peroxidase (APEX2) proximity labelling system coupled with mass spectroscopy to identify RhoBTB1 targets in A7r5 smooth muscle cells. This analysis identified nearly 75 proteins which putatively bind to the C terminal half of RhoBTB1 (known as B1B2C), the region we previously identified was essential for adaptor function. One of these proteins was RNA binding motif protein 9, known as RBM9 and RbFox2, a key regulator of splicing events. Subsequent co-immunoprecipitation (Co-IP) assays validated the interaction of RbFox2 with RhoBTB1 and its B1B2C domains. Expression of Rbfox2 was increased in response to treatment of A7r5 cells with either MG132, an inhibitor of the proteasome, and MLN4924, an inhibitor of the CUL family. Similarly, RbFox2 expression was increased in CRISPR-Cas9 generated CUL3-deficient HEK293 cells, in aorta isolated from SMC-specific CUL3-deficient mice, and in A7r5 cells expressing an siRNA targeting RhoBTB1. Ang-II treatment of mice, which raised blood pressure by 40 mmHg, also increased RbFox2 expression, presumably through down regulation of RhoBTB1. Targeting RbFox2 by siRNA in A7r5 cells resulted in a decrease in actin fiber formation detected by phalloidin staining. In summary, our findings suggest that RbFox2 regulation may occur through the RhoBTB1-CUL3 proteasomal pathway and may potentially play a mechanistic role in actin cytoskeleton organization and thus arterial stiffness associated with hypertension.

Loss-of-function variants in RNA binding motif protein X-linked induce neuronal defects contributing to amyotrophic lateral sclerosis pathogenesis.

Despite being one of the most prevalent RNA modifications, the role of N6-methyladenosine (m6A) in amyotrophic lateral sclerosis (ALS) remains ambiguous. In this investigation, we explore the contribution of genetic defects of m6A-related genes to ALS pathogenesis. We scrutinized the mutation landscape of m6A genes through a comprehensive analysis of whole-exome sequencing cohorts, encompassing 508 ALS patients and 1660 population-matched controls. Our findings reveal a noteworthy enrichment of RNA binding motif protein X-linked (RBMX) variants among ALS patients, with a significant correlation between pathogenic m6A variants and adverse clinical outcomes. Furthermore, Rbmx knockdown in NSC-34 cells overexpressing mutant TDP43Q331K results in cell death mediated by an augmented p53 response. Similarly, RBMX knockdown in ALS motor neurons derived from induced pluripotent stem cells (iPSCs) manifests morphological defects and activation of the p53 pathway. Transcriptional analysis using publicly available single-cell sequencing data from the primary motor cortex indicates that RBMX-regulated genes selectively influence excitatory neurons and exhibit enrichment in ALS-implicated pathways. Through integrated analyses, our study underscores the emerging roles played by RBMX in ALS, suggesting a potential nexus between the disease and dysregulated m6A-mediated mRNA metabolism.