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1.
Int J Oncol ; 65(1)2024 Jul.
Article in English | MEDLINE | ID: mdl-38847231

ABSTRACT

Signal recognition particles (SRPs) are essential for regulating intracellular protein transport and secretion. Patients with tumors with high SRP9 expression tend to have a poorer overall survival. However, to the best of our knowledge, no reports have described the relationship between SRP9 localization and prognosis in pancreatic cancer. Thus, the present study aimed to investigate this relationship. Immunohistochemical staining for SRP9 using excised specimens from pancreatic cancer surgery cases without preoperative chemotherapy or radiotherapy showed that SRP9 was preferentially expressed in the nucleus of the cancerous regions in some cases, which was hardly detected in other cases, indicating that SRP9 was transported to the nucleus in the former cases. To compare the prognosis of patients with SRP9 nuclear translocation, patients were divided into two groups: Those with a nuclear translocation rate of >50% and those with a nuclear translocation rate of ≤50%. The nuclear translocation rate of >50% group had a significantly better recurrence­free survival than the nuclear translocation rate of ≤50% group (P=0.037). Subsequent in vitro experiments were conducted; notably, the nuclear translocation rate of SRP9 was reduced under amino acid­deficient conditions, suggesting that multiple factors are involved in this phenomenon. To further study the function of SRP9 nuclear translocation, in vitro experiments were performed by introducing SRP9 splicing variants (v1 and v2) and their deletion mutants lacking C­terminal regions into MiaPaCa pancreatic cancer cells. The results demonstrated that both splicing variants showed nuclear translocation regardless of the C­terminal deletions, suggesting the role of the N­terminal regions. Given that SRP9 is an RNA­binding protein, the study of RNA immunoprecipitation revealed that signaling pathways involved in cancer progression and protein translation were downregulated in nuclear­translocated v1 and v2. Undoubtedly, further studies of the nuclear translocation of SRP9 will open an avenue to optimize the precise evaluation and therapeutic control of pancreatic cancer.


Subject(s)
Cell Nucleus , Pancreatic Neoplasms , Humans , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/mortality , Prognosis , Male , Female , Cell Nucleus/metabolism , Middle Aged , Aged , Cell Line, Tumor , Signal Recognition Particle/metabolism , Signal Recognition Particle/genetics , Active Transport, Cell Nucleus , Serine-Arginine Splicing Factors/metabolism , Serine-Arginine Splicing Factors/genetics , Adult , Gene Expression Regulation, Neoplastic
2.
J Cancer Res Clin Oncol ; 150(6): 292, 2024 Jun 06.
Article in English | MEDLINE | ID: mdl-38842611

ABSTRACT

BACKGROUND: Glioblastoma (GBM) is a highly aggressive and prevalent brain tumor that poses significant challenges in treatment. SRSF9, an RNA-binding protein, is essential for cellular processes and implicated in cancer progression. Yet, its function and mechanism in GBM need clarification. METHODS: Bioinformatics analysis was performed to explore differential expression of SRSF9 in GBM and its prognostic relevance to glioma patients. SRSF9 and CDK1 expression in GBM cell lines and patients' tissues were quantified by RT-qPCR, Western blot or immunofluorescence assay. The role of SRSF9 in GBM cell proliferation and migration was assessed by MTT, Transwell and colony formation assays. Additionally, transcriptional regulation of CDK1 by SRSF9 was investigated using ChIP-PCR and dual-luciferase assays. RESULTS: The elevated SRSF9 expression correlates to GBM stages and poor survival of glioma patients. Through gain-of-function and loss-of-function strategies, SRSF9 was demonstrated to promote proliferation and migration of GBM cells. Bioinformatics analysis showed that SRSF9 has an impact on cell growth pathways including cell cycle checkpoints and E2F targets. Mechanistically, SRSF9 appears to bind to the promoter of CDK1 gene and increase its transcription level, thus promoting GBM cell proliferation. CONCLUSIONS: These findings uncover the cellular function of SRSF9 in GBM and highlight its therapeutic potential for GBM.


Subject(s)
Brain Neoplasms , CDC2 Protein Kinase , Cell Movement , Cell Proliferation , Glioblastoma , Serine-Arginine Splicing Factors , Humans , Glioblastoma/pathology , Glioblastoma/genetics , Glioblastoma/metabolism , CDC2 Protein Kinase/metabolism , CDC2 Protein Kinase/genetics , Serine-Arginine Splicing Factors/metabolism , Serine-Arginine Splicing Factors/genetics , Brain Neoplasms/pathology , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Cell Line, Tumor , Prognosis , Female , Male , Middle Aged
3.
Beijing Da Xue Xue Bao Yi Xue Ban ; 56(3): 495-504, 2024 Jun 18.
Article in Chinese | MEDLINE | ID: mdl-38864136

ABSTRACT

OBJECTIVE: To investigate the function and underlying mechanism of cysteine and glycine-rich protein 2 (CSRP2) in neuroblastoma (NB). METHODS: The correlation between the expression level of CSRP2 mRNA and the prognosis of NB children in NB clinical samples was analyzed in R2 Genomics Analysis and Visualization Platform. The small interfering RNA (siRNA) targeting CSRP2 or CSRP2 plasmid were transfected to NB cell lines SK-N-BE(2) and SH-SY5Y. Cell proliferation was observed by crystal violet staining and real-time cellular analysis. The ability of colony formation of NB cells was observed by colony-forming unit assay. Immunofluorescence assay was used to detect the expression of the proliferation marker Ki-67. Flow cytometry analysis for cell cycle proportion was used with cells stained by propidium iodide (PI). Annexin V/7AAD was used to stain cells and analyze the percentage of cell apoptosis. The ability of cell migration was determined by cell wound-healing assay. The level of protein and mRNA expression of CSRP2 in NB primary tumor and NB cell lines were detected by Western blot and quantitative real-time PCR (RT-qPCR). RESULTS: By analyzing the NB clinical sample databases, it was found that the expression levels of CSRP2 in high-risk NB with 3/4 stages in international neuroblastoma staging system (INSS) were significantly higher than that in low-risk NB with 1/2 INSS stages. The NB patients with high expression levels of CSRP2 were shown lower overall survival rate than those with low expression levels of CSRP2. We detected the protein levels of CSRP2 in the NB samples by Western blot, and found that the protein level of CSRP2 in 3/4 INSS stages was significantly higher than that in 1/2 INSS stages. Knockdown of CSRP2 inhibited cell viability and proliferation of NB cells. Overexpression of CSRP2 increased the proliferation of NB cells. Flow cytometry showed that the proportion of sub-G1, G0/G1 and S phase cells and Annexin V positive cells were increased after CSRP2 deficiency. In the cell wound-healing assay, the healing rate of NB cells was significantly attenuated after knockdown of CSRP2. Further mechanism studies showed that the proportion of the proliferation marker Ki-67 and the phosphorylation levels of extracellular signal-regulated kinases 1/2 (ERK1/2) were significantly decreased after CSRP2 knockdown. CONCLUSION: CSRP2 is highly expressed in high-risk NB with 3/4 INSS stages, and the expression levels of CSRP2 are negatively correlated with the overall survival of NB patients. CSRP2 significantly increased the proliferation and cell migration of NB cells and inhibited cell apoptosis via the activation of ERK1/2. All these results indicate that CSRP2 promotes the progression of NB by activating ERK1/2, and this study will provide a potential target for high-risk NB therapy.


Subject(s)
Apoptosis , Cell Movement , Cell Proliferation , Neuroblastoma , Humans , Neuroblastoma/metabolism , Neuroblastoma/pathology , Neuroblastoma/genetics , Cell Line, Tumor , RNA, Small Interfering/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Prognosis , Cell Cycle , Disease Progression , Ki-67 Antigen/metabolism , Serine-Arginine Splicing Factors/metabolism , Serine-Arginine Splicing Factors/genetics
4.
BMC Cancer ; 24(1): 580, 2024 May 12.
Article in English | MEDLINE | ID: mdl-38735973

ABSTRACT

BACKGROUND: SRSF1, a member of Serine/Arginine-Rich Splicing Factors (SRSFs), has been observed to significantly influence cancer progression. However, the precise role of SRSF1 in osteosarcoma (OS) remains unclear. This study aims to investigate the functions of SRSF1 and its underlying mechanism in OS. METHODS: SRSF1 expression level in OS was evaluated on the TCGA dataset, TAGET-OS database. qRT-PCR and Western blotting were employed to assess SRSF1 expression in human OS cell lines as well as the interfered ectopic expression states. The effect of SRSF1 on cell migration, invasion, proliferation, and apoptosis of OS cells were measured by transwell assay and flow cytometry. RNA sequence and bioinformatic analyses were conducted to elucidate the targeted genes, relevant biological pathways, and alternative splicing (AS) events regulated by SRSF1. RESULTS: SRSF1 expression was consistently upregulated in both OS samples and OS cell lines. Diminishing SRSF1 resulted in reduced proliferation, migration, and invasion and increased apoptosis in OS cells while overexpressing SRSF1 led to enhanced growth, migration, invasion, and decreased apoptosis. Mechanistically, Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and Gene Set Enrichment Analysis (GSEA) revealed that the biological functions of SRSF1 were closely associated with the dysregulation of the protein targeting processes, location of the cytosolic ribosome, extracellular matrix (ECM), and proteinaceous extracellular matrix, along with the PI3K-AKT pathway, Wnt pathway, and HIPPO pathway. Transcriptome analysis identified AS events modulated by SRSF1, especially (Skipped Exon) SE events and (Mutually exclusive Exons) MXE events, revealing potential roles of targeted molecules in mRNA surveillance, RNA degradation, and RNA transport during OS development. qRT-PCR confirmed that SRSF1 knockdown resulted in the occurrence of alternative splicing of SRRM2, DMKN, and SCAT1 in OS. CONCLUSIONS: Our results highlight the oncogenic role of high SRSF1 expression in promoting OS progression, and further explore the potential mechanisms of action. The significant involvement of SRSF1 in OS development suggests its potential utility as a therapeutic target in OS.


Subject(s)
Apoptosis , Bone Neoplasms , Cell Movement , Cell Proliferation , Disease Progression , Gene Expression Regulation, Neoplastic , Osteosarcoma , Serine-Arginine Splicing Factors , Humans , Serine-Arginine Splicing Factors/genetics , Serine-Arginine Splicing Factors/metabolism , Osteosarcoma/genetics , Osteosarcoma/pathology , Osteosarcoma/metabolism , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Bone Neoplasms/metabolism , Cell Proliferation/genetics , Cell Line, Tumor , Apoptosis/genetics , Cell Movement/genetics , Up-Regulation , Alternative Splicing
5.
Proc Natl Acad Sci U S A ; 121(21): e2322974121, 2024 May 21.
Article in English | MEDLINE | ID: mdl-38743621

ABSTRACT

SRSF1 is the founding member of the SR protein family. It is required-interchangeably with other SR proteins-for pre-mRNA splicing in vitro, and it regulates various alternative splicing events. Dysregulation of SRSF1 expression contributes to cancer and other pathologies. Here, we characterized SRSF1's interactome using proximity labeling and mass spectrometry. This approach yielded 190 proteins enriched in the SRSF1 samples, independently of the N- or C-terminal location of the biotin-labeling domain. The detected proteins reflect established functions of SRSF1 in pre-mRNA splicing and reveal additional connections to spliceosome proteins, in addition to other recently identified functions. We validated a robust interaction with the spliceosomal RNA helicase DDX23/PRP28 using bimolecular fluorescence complementation and in vitro binding assays. The interaction is mediated by the N-terminal RS-like domain of DDX23 and both RRM1 and the RS domain of SRSF1. During pre-mRNA splicing, DDX23's ATPase activity is essential for the pre-B to B spliceosome complex transition and for release of U1 snRNP from the 5' splice site. We show that the RS-like region of DDX23's N-terminal domain is important for spliceosome incorporation, while larger deletions in this domain alter subnuclear localization. We discuss how the identified interaction of DDX23 with SRSF1 and other SR proteins may be involved in the regulation of these processes.


Subject(s)
DEAD-box RNA Helicases , RNA Splicing , Serine-Arginine Splicing Factors , Spliceosomes , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , Humans , Spliceosomes/metabolism , Serine-Arginine Splicing Factors/metabolism , Serine-Arginine Splicing Factors/genetics , RNA Precursors/metabolism , RNA Precursors/genetics , Protein Binding , HeLa Cells
6.
PLoS One ; 19(5): e0296565, 2024.
Article in English | MEDLINE | ID: mdl-38781195

ABSTRACT

Epigenetic silencing through methylation is one of the major mechanisms for downregulation of tumor suppressor miRNAs in various malignancies. The aim of this study was to identify novel tumor suppressor miRNAs which are silenced by DNA hypermethylation and investigate the role of at least one of these in oral squamous cell carcinoma (OSCC) pathogenesis. We treated cells from an OSCC cell line SCC131 with 5-Azacytidine, a DNA methyltransferase inhibitor, to reactivate tumor suppressor miRNA genes silenced/downregulated due to DNA methylation. At 5-day post-treatment, total RNA was isolated from the 5-Azacytidine and vehicle control-treated cells. The expression of 2,459 mature miRNAs was analysed between 5-Azacytidine and control-treated OSCC cells by the microRNA microarray analysis. Of the 50 miRNAs which were found to be upregulated following 5-Azacytidine treatment, we decided to work with miR-6741-3p in details for further analysis, as it showed a mean fold expression of >4.0. The results of qRT-PCR, Western blotting, and dual-luciferase reporter assay indicated that miR-6741-3p directly targets the oncogene SRSF3 at the translational level only. The tumor-suppressive role of miR-6741-3p was established by various in vitro assays and in vivo study in NU/J athymic nude mice. Our results revealed that miR-6741-3p plays a tumor-suppressive role in OSCC pathogenesis, in part, by directly regulating SRSF3. Based on our observations, we propose that miR-6741-3p may serve as a potential biological target in tumor diagnostics, prognostic evaluation, and treatment of OSCC and perhaps other malignancies.


Subject(s)
Carcinoma, Squamous Cell , Gene Expression Regulation, Neoplastic , MicroRNAs , Mouth Neoplasms , Serine-Arginine Splicing Factors , MicroRNAs/genetics , MicroRNAs/metabolism , Humans , Mouth Neoplasms/genetics , Mouth Neoplasms/pathology , Animals , Cell Line, Tumor , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Mice , Gene Expression Regulation, Neoplastic/drug effects , Serine-Arginine Splicing Factors/genetics , Serine-Arginine Splicing Factors/metabolism , DNA Methylation , Introns/genetics , Mice, Nude , Azacitidine/pharmacology , Oncogenes/genetics
7.
Cell Mol Biol Lett ; 29(1): 78, 2024 May 22.
Article in English | MEDLINE | ID: mdl-38778254

ABSTRACT

Alternative splicing of pre-mRNAs is a fundamental step in RNA processing required for gene expression in most metazoans. Serine and arginine-rich proteins (SR proteins) comprise a family of multifunctional proteins that contain an RNA recognition motif (RRM) and the ultra-conserved arginine/serine-rich (RS) domain, and play an important role in precise alternative splicing. Increasing research supports SR proteins as also functioning in other RNA-processing-related mechanisms, such as polyadenylation, degradation, and translation. In addition, SR proteins interact with N6-methyladenosine (m6A) regulators to modulate the methylation of ncRNA and mRNA. Dysregulation of SR proteins causes the disruption of cell differentiation and contributes to cancer progression. Here, we review the distinct biological characteristics of SR proteins and their known functional mechanisms during carcinogenesis. We also summarize the current inhibitors that directly target SR proteins and could ultimately turn SR proteins into actionable therapeutic targets in cancer therapy.


Subject(s)
Neoplasms , Humans , Neoplasms/metabolism , Neoplasms/drug therapy , Neoplasms/genetics , Animals , Serine-Arginine Splicing Factors/metabolism , Serine-Arginine Splicing Factors/genetics , Alternative Splicing/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics
8.
Leukemia ; 38(6): 1334-1341, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38714876

ABSTRACT

We investigated data from 180 consecutive patients with myelodysplastic/myeloproliferative neoplasms with SF3B1 mutation and thrombocytosis (MDS/MPN-SF3B1-T) who were diagnosed according to the 2022 World Health Organization (WHO) classification of myeloid neoplasms to identify covariates associated with survival. At a median follow-up of 48 months (95% confidence interval [CI] 35-61 months), the median survival was 69 months (95% CI 59-79 months). Patients with bone marrow ring sideroblasts (RS) < 15% had shorter median overall survival (OS) than did those with bone marrow RS ≥ 15% (41 months [95% CI 32-50 months] versus 76 months [95% CI 59-93 months]; P < 0.001). According to the univariable analyses of OS, age ≥ 65 years (P < 0.001), hemoglobin concentration (Hb) < 80 g/L (P = 0.090), platelet count (PLT) ≥ 800 × 10E + 9/L (P = 0.087), bone marrow RS < 15% (P < 0.001), the Revised International Prognostic Scoring System (IPSS-R) cytogenetic category intermediate/poor/very poor (P = 0.005), SETBP1 mutation (P = 0.061) and SRSF2 mutation (P < 0.001) were associated with poor survival. Based on variables selected from univariable analyses, two separate survival prediction models, a clinical survival model, and a clinical-molecular survival model, were developed using multivariable analyses with the minimum value of the Akaike information criterion (AIC) to specifically predict outcomes in patients with MDS/MPN-SF3B1-T according to the 2022 WHO classification.


Subject(s)
Mutation , Myelodysplastic-Myeloproliferative Diseases , Phosphoproteins , RNA Splicing Factors , Thrombocytosis , Humans , RNA Splicing Factors/genetics , Male , Female , Thrombocytosis/genetics , Aged , Phosphoproteins/genetics , Middle Aged , Myelodysplastic-Myeloproliferative Diseases/genetics , Myelodysplastic-Myeloproliferative Diseases/mortality , Myelodysplastic-Myeloproliferative Diseases/pathology , Prognosis , Aged, 80 and over , Adult , Survival Rate , Follow-Up Studies , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/mortality , Myelodysplastic Syndromes/pathology , Serine-Arginine Splicing Factors/genetics
9.
Anal Chem ; 96(23): 9713-9720, 2024 Jun 11.
Article in English | MEDLINE | ID: mdl-38795036

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that results in the degeneration of neurons in the brain and spinal cord. Although a substantial number of studies have been conducted, much remains to be learned about the cellular mechanisms underlying ALS. In this study, we employed an engineered ascorbate peroxidase (APEX)-based proximity biotinylation, together with affinity pull-down of the ensuing biotinylated peptides, to investigate the proximity proteomes of human SOD1 and its two ALS-linked mutants, G85R and G93A. We were able to identify 25 common biotinylated peptides with preferential enrichment in the proximity proteomes of SOD1G85R and SOD1G93A over wild-type SOD1. Our coimmunoprecipitation followed by Western blot analyses revealed that one of these proteins, SRSF2, binds more strongly with the two SOD1 mutants than its wild-type counterpart. We also observed aberrant splicing of mRNAs in cells with ectopic expression of the two SOD1 mutants relative to cells expressing the wild-type protein. In addition, the aberrations in splicing elicited by the SOD1 variants were markedly attenuated upon knockdown of SRSF2. Collectively, we uncovered that ALS-liked SOD1G85R and SOD1G93A mutants interact more strongly with SRSF2, where the aberrant interactions perturbed mRNA splicing. Thus, our work offered novel mechanistic insights into the contributions of the ALS-linked SOD1 mutants to disease etiology.


Subject(s)
Amyotrophic Lateral Sclerosis , Mutation , RNA Splicing , Serine-Arginine Splicing Factors , Superoxide Dismutase-1 , Serine-Arginine Splicing Factors/metabolism , Serine-Arginine Splicing Factors/genetics , Humans , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Superoxide Dismutase-1/metabolism , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , HEK293 Cells , Biotinylation
10.
Nat Commun ; 15(1): 2819, 2024 Apr 01.
Article in English | MEDLINE | ID: mdl-38561338

ABSTRACT

Previous genetic studies of venous thromboembolism (VTE) have been largely limited to common variants, leaving the genetic determinants relatively incomplete. We performed an exome-wide association study of VTE among 14,723 cases and 334,315 controls. Fourteen known and four novel genes (SRSF6, PHPT1, CGN, and MAP3K2) were identified through protein-coding variants, with broad replication in the FinnGen cohort. Most genes we discovered exhibited the potential to predict future VTE events in longitudinal analysis. Notably, we provide evidence for the additive contribution of rare coding variants to known genome-wide polygenic risk in shaping VTE risk. The identified genes were enriched in pathways affecting coagulation and platelet activation, along with liver-specific expression. The pleiotropic effects of these genes indicated the potential involvement of coagulation factors, blood cell traits, liver function, and immunometabolic processes in VTE pathogenesis. In conclusion, our study unveils the valuable contribution of protein-coding variants in VTE etiology and sheds new light on its risk stratification.


Subject(s)
Venous Thromboembolism , Humans , Venous Thromboembolism/genetics , Risk Factors , Blood Coagulation Factors/genetics , Exome , Genome-Wide Association Study , Serine-Arginine Splicing Factors/genetics , Phosphoproteins/genetics
11.
Zhonghua Bing Li Xue Za Zhi ; 53(5): 430-438, 2024 May 08.
Article in Chinese | MEDLINE | ID: mdl-38678322

ABSTRACT

Objective: To investigate the effect of serine/arginine-rich splicing factor 2 (SRSF2) on ferroptosis and its possible mechanism in glioblastoma cells. Methods: The online database of gene expression profiling interactive analysis 2 (GEPIA 2) and Chinese Glioma Genome Atlas were used to analyze the expression of SRSF2 in glioblastoma tissue and its association with patients prognosis. To validate the findings of the online databases, the pathological sections of glioblastoma and non-tumor brain tissues from Tianjin Medical University General Hospital, Tianjin, China were collected and analyzed by using immunohistochemistry. Silencing SRSF2 gene expression in glioblastoma cells by siRNA was analyzed with Western blot. The proliferation index was detected by using CCK8 assay. The rescued experiment was conducted by using expression plasmid of pcDNA3.1(+)-SRSF2. The activity of ferroptosis was assessed by using the levels of iron ions and malondialdehyde in glioblastoma cells and the changes in the ratio of glutathione to oxidized glutathione. The changes of gene expression and differential pre-mRNA alternative splicing (PMAS) induced by SRSF2 were monitored by using the third-generation sequencing technology analysis, namely Oxford nanopore technologies (ONT) sequencing analysis. Results: SRSF2 expression was higher in glioblastoma tissues than non-tumor brain tissues. Immunohistochemistry also showed a positive rate of 88.48%±4.60% in glioblastoma tissue which was much higher than the 9.97%±4.57% in non-tumor brain tissue. The expression of SRSF2 was inversely correlated with overall and disease-free disease survivals (P<0.01). The proliferation index of glioblastoma cells was significantly reduced by silencing with SRSF2 siRNA (P<0.01) and could be reversed with transfection of exogenous SRSF2. The levels of intracellulariron ions and malondialdehyde increased (P<0.05), but the glutathione/oxidized glutathione ratio and the expression of key proteins in the glutathione pathway remained unchanged (P>0.05). ONT sequencing results showed that silencing SRSF2 in glioblastoma cells could induce a significant alternative 3' splice site change on ferroptosis suppressor protein 1 (FSP1). Conclusion: SRSF2 inhibits the ferroptosis in glioblastoma cells and promotes their proliferation, which may be achieved by regulating FSP1 PMAS.


Subject(s)
Alternative Splicing , Brain Neoplasms , Cell Proliferation , Ferritins , Ferroptosis , Glioblastoma , Oxidoreductases , Serine-Arginine Splicing Factors , Humans , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , Ferroptosis/genetics , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Glioblastoma/pathology , Glioblastoma/metabolism , Prognosis , RNA, Small Interfering/genetics , Serine-Arginine Splicing Factors/genetics , Serine-Arginine Splicing Factors/metabolism
12.
Genome Res ; 34(3): 376-393, 2024 Apr 25.
Article in English | MEDLINE | ID: mdl-38609186

ABSTRACT

Exon-intron circRNAs (EIciRNAs) are a circRNA subclass with retained introns. Global features of EIciRNAs remain largely unexplored, mainly owing to the lack of bioinformatic tools. The regulation of intron retention (IR) in EIciRNAs and the associated functionality also require further investigation. We developed a framework, FEICP, which efficiently detected EIciRNAs from high-throughput sequencing (HTS) data. EIciRNAs are distinct from exonic circRNAs (EcircRNAs) in aspects such as with larger length, localization in the nucleus, high tissue specificity, and enrichment mostly in the brain. Deep learning analyses revealed that compared with regular introns, the retained introns of circRNAs (CIRs) are shorter in length, have weaker splice site strength, and have higher GC content. Compared with retained introns in linear RNAs (LIRs), CIRs are more likely to form secondary structures and show greater sequence conservation. CIRs are closer to the 5'-end, whereas LIRs are closer to the 3'-end of transcripts. EIciRNA-generating genes are more actively transcribed and associated with epigenetic marks of gene activation. Computational analyses and genome-wide CRISPR screening revealed that SRSF1 binds to CIRs and inhibits the biogenesis of most EIciRNAs. SRSF1 regulates the biogenesis of EIciLIMK1, which enhances the expression of LIMK1 in cis to boost neuronal differentiation, exemplifying EIciRNA physiological function. Overall, our study has developed the FEICP pipeline to identify EIciRNAs from HTS data, and reveals multiple features of CIRs and EIciRNAs. SRSF1 has been identified to regulate EIciRNA biogenesis. EIciRNAs and the regulation of EIciRNA biogenesis play critical roles in neuronal differentiation.


Subject(s)
Exons , Introns , RNA, Circular , RNA, Circular/genetics , RNA, Circular/metabolism , Humans , Serine-Arginine Splicing Factors/genetics , Serine-Arginine Splicing Factors/metabolism , High-Throughput Nucleotide Sequencing , Computational Biology/methods
13.
EMBO Rep ; 25(5): 2220-2238, 2024 May.
Article in English | MEDLINE | ID: mdl-38600345

ABSTRACT

Perturbation of protein phosphorylation represents an attractive approach to cancer treatment. Besides kinase inhibitors, protein phosphatase inhibitors have been shown to have anti-cancer activity. A prime example is the small molecule LB-100, an inhibitor of protein phosphatases 2A/5 (PP2A/PP5), enzymes that affect cellular physiology. LB-100 has proven effective in pre-clinical models in combination with immunotherapy, but the molecular underpinnings of this synergy remain understood poorly. We report here a sensitivity of the mRNA splicing machinery to phosphorylation changes in response to LB-100 in colorectal adenocarcinoma. We observe enrichment for differentially phosphorylated sites within cancer-critical splicing nodes of U2 snRNP, SRSF and hnRNP proteins. Altered phosphorylation endows LB-100-treated colorectal adenocarcinoma cells with differential splicing patterns. In PP2A-inhibited cells, over 1000 events of exon skipping and intron retention affect regulators of genomic integrity. Finally, we show that LB-100-evoked alternative splicing leads to neoantigens that are presented by MHC class 1 at the cell surface. Our findings provide a potential explanation for the pre-clinical and clinical observations that LB-100 sensitizes cancer cells to immune checkpoint blockade.


Subject(s)
Colonic Neoplasms , RNA Splicing , Humans , Colonic Neoplasms/genetics , Colonic Neoplasms/immunology , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , RNA Splicing/drug effects , Phosphorylation , Cell Line, Tumor , RNA, Messenger/genetics , RNA, Messenger/metabolism , Alternative Splicing , Antigens, Neoplasm/metabolism , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Serine-Arginine Splicing Factors/metabolism , Serine-Arginine Splicing Factors/genetics , Protein Phosphatase 2/metabolism , Enzyme Inhibitors/pharmacology
14.
Exp Cell Res ; 438(1): 114026, 2024 May 01.
Article in English | MEDLINE | ID: mdl-38604522

ABSTRACT

The emergence of AR-V7, a truncated isoform of AR upon androgen deprivation therapy treatment, leads to the development of castration resistant prostate cancer (CRPC). Understanding mechanisms that regulate AR-V7 expression is critical for developing newer therapeutic strategies. In this study, we have investigated the regulation of AR-V7 during cell cycle and identified a distinct pattern of periodic fluctuation, peaking during G2/M phase. This fluctuation correlates with the expression of Cdc-2 like kinase 1 (CLK1) and phosphorylated serine/arginine-rich splicing factor 1 (p-SRSF1) during these phases, pointing towards their role in AR-V7 generation. Functional assays reveal that CLK1 knockdown prolongs the S phase, leading to altered cell cycle distribution and increased accumulation of AR-V7 and pSRSF1 in G1/S phase. Conversely, CLK1 overexpression rescues AR-V7 and p-SRSF1 levels in the G2/M phase, consistent with observed cell cycle alterations upon AR-V7 knockdown and overexpression in CRPC cells. Furthermore, overexpression of kinase-deficient CLK1 mutant leads to diminished AR-V7 levels during G2/M, underlining the essential contribution of CLK1's kinase activity in modulating AR-V7 expression. Collectively, our findings, for the first time, show periodic regulation of AR-V7 expression, its effect on cell cycle progression and the critical role of CLK1-pSRSF1 axis in modulating AR-V7 expression throughout the cell cycle.


Subject(s)
G2 Phase Cell Cycle Checkpoints , Prostatic Neoplasms, Castration-Resistant , Receptors, Androgen , Humans , Male , Cell Line, Tumor , Cell Proliferation/genetics , G2 Phase/genetics , G2 Phase Cell Cycle Checkpoints/genetics , Gene Expression Regulation, Neoplastic , Phosphorylation , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/pathology , Prostatic Neoplasms, Castration-Resistant/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Receptors, Androgen/metabolism , Receptors, Androgen/genetics , Serine-Arginine Splicing Factors/metabolism , Serine-Arginine Splicing Factors/genetics
15.
J Bioenerg Biomembr ; 56(3): 235-245, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38613636

ABSTRACT

Acute myocardial infarction (AMI) is one of the most prevalent cardiovascular diseases, accounting for a high incidence rate and high mortality worldwide. Hypoxia/reoxygenation (H/R)-induced myocardial cell injury is the main cause of AMI. Several studies have shown that circular RNA contributes significantly to the pathogenesis of AMI. Here, we established an AMI mouse model to investigate the effect of circDiaph3 in cardiac function and explore the functional role of circDiaph3 in H/R-induced cardiomyocyte injury and its molecular mechanism. Bioinformatics tool and RT-qPCR techniques were applied to detect circDiaph3 expression in human patient samples, heart tissues of AMI mice, and H/R-induced H9C2 cells. CCK-8 was used to examine cell viability, while annexin-V/PI staining was used to assess cell apoptosis. Myocardial reactive oxygen species (ROS) levels were detected by immunofluorescence. Western blot was used to detect the protein expression of anti-apoptotic Bcl-2 while pro-apoptotic Bax and cleaved-Caspase-3. Furthermore, ELISA was used to detect inflammatory cytokines production. While bioinformatics tool and RNA pull-down assay were used to verify the interaction between circDiaph3 and miR-338-3p. We found that circDiaph3 expression was high in AMI patients and mice, as well as in H/R-treated H9C2 cells. CircDiaph3 silencing ameliorated apoptosis and inflammatory response of cardiomyocytes in vivo. Moreover, the knockdown of cirDiaph3 mitigated H/R-induced apoptosis and the release of inflammatory mediators like IL-1ß, IL-6, and TNF-α in H9C2 cells. Mechanistically, circDiaph3 induced cell apoptosis and inflammatory responses in H/R-treated H9C2 cells by sponging miR-338-3p. Overexpressing miR-338-3p in H/R-treated cells prominently reversed circDiaph3-induced effects. Notably, miR-338-3p inhibited SRSF1 expression in H/R-treated H9C2 cells. While overexpressing SRSF1 abrogated miR-338-3p-mediated alleviation of apoptosis and inflammation after H/R treatment. To summarize, circDiaph3 aggravates H/R-induced cardiomyocyte apoptosis and inflammation through the miR-338-3p/SRSF1 axis. These findings suggest that the circDiaph3/miR-338-3pp/SRSF1 axis could be a potential therapeutic target for treating H/R-induced myocardial injury.


Subject(s)
Apoptosis , Inflammation , MicroRNAs , Myocytes, Cardiac , RNA, Circular , Animals , Humans , Male , Mice , Inflammation/metabolism , Inflammation/pathology , Mice, Inbred C57BL , MicroRNAs/metabolism , MicroRNAs/genetics , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , RNA, Circular/metabolism , RNA, Circular/genetics , Serine-Arginine Splicing Factors/metabolism , Serine-Arginine Splicing Factors/genetics
16.
Leuk Res ; 141: 107500, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38636413

ABSTRACT

Mutations in spliceosome genes (SRSF2, SF3B1, U2AF1, ZRSR2) correlate with inferior outcomes in patients treated with intensive chemotherapy for Acute Myeloid Leukemia. However, their prognostic impact in patients treated with less intensive protocols is not well known. This study aimed to evaluate the impact of Spliceosome mutations in patients treated with Venetoclax and Azacitidine for newly diagnosed AML. 117 patients treated in 3 different hospitals were included in the analysis. 34 harbored a mutation in at least one of the spliceosome genes (splice-mut cohort). K/NRAS mutations were more frequent in the splice-mut cohort (47% vs 19%, p=0.0022). Response rates did not differ between splice-mut and splice-wt cohorts. With a median follow-up of 15 months, splice mutations were associated with a lower 18-month LFS (p=0.0045). When analyzing splice mutations separately, we found SRSF2 mutations to be associated with poorer outcomes (p=0.034 and p=0.037 for OS and LFS respectively). This negative prognostic impact remained true in our multivariate analysis. We believe this finding should warrant further studies aimed at overcoming this negative impact.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Leukemia, Myeloid, Acute , Mutation , Serine-Arginine Splicing Factors , Humans , Serine-Arginine Splicing Factors/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/mortality , Male , Female , Middle Aged , Prognosis , Aged , Adult , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Aged, 80 and over , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Azacitidine/therapeutic use , Azacitidine/administration & dosage , Young Adult , Spliceosomes/genetics , Sulfonamides
17.
Nucleic Acids Res ; 52(8): 4676-4690, 2024 May 08.
Article in English | MEDLINE | ID: mdl-38567732

ABSTRACT

SRSF1 governs splicing of over 1500 mRNA transcripts. SRSF1 contains two RNA-recognition motifs (RRMs) and a C-terminal Arg/Ser-rich region (RS). It has been thought that SRSF1 RRMs exclusively recognize single-stranded exonic splicing enhancers, while RS lacks RNA-binding specificity. With our success in solving the insolubility problem of SRSF1, we can explore the unknown RNA-binding landscape of SRSF1. We find that SRSF1 RS prefers purine over pyrimidine. Moreover, SRSF1 binds to the G-quadruplex (GQ) from the ARPC2 mRNA, with both RRMs and RS being crucial. Our binding assays show that the traditional RNA-binding sites on the RRM tandem and the Arg in RS are responsible for GQ binding. Interestingly, our FRET and circular dichroism data reveal that SRSF1 unfolds the ARPC2 GQ, with RS leading unfolding and RRMs aiding. Our saturation transfer difference NMR results discover that Arg residues in SRSF1 RS interact with the guanine base but not other nucleobases, underscoring the uniqueness of the Arg/guanine interaction. Our luciferase assays confirm that SRSF1 can alleviate the inhibitory effect of GQ on gene expression in the cell. Given the prevalence of RNA GQ and SR proteins, our findings unveil unexplored SR protein functions with broad implications in RNA splicing and translation.


Subject(s)
G-Quadruplexes , Protein Binding , Serine-Arginine Splicing Factors , Serine-Arginine Splicing Factors/metabolism , Serine-Arginine Splicing Factors/genetics , Serine-Arginine Splicing Factors/chemistry , Humans , Binding Sites , RNA Splicing , RNA Recognition Motif/genetics , RNA, Messenger/metabolism , RNA, Messenger/genetics , RNA, Messenger/chemistry , RNA/metabolism , RNA/genetics , RNA/chemistry
18.
Adv Sci (Weinh) ; 11(21): e2306871, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38569495

ABSTRACT

RNA splicing dysregulation and the involvement of specific splicing factors are emerging as common factors in both obesity and metabolic disorders. The study provides compelling evidence that the absence of the splicing factor SRSF1 in mature adipocytes results in whitening of brown adipocyte tissue (BAT) and impaired thermogenesis, along with the inhibition of white adipose tissue browning in mice. Combining single-nucleus RNA sequencing with transmission electron microscopy, it is observed that the transformation of BAT cell types is associated with dysfunctional mitochondria, and SRSF1 deficiency leads to degenerated and fragmented mitochondria within BAT. The results demonstrate that SRSF1 effectively binds to constitutive exon 6 of Ndufs3 pre-mRNA and promotes its inclusion. Conversely, the deficiency of SRSF1 results in impaired splicing of Ndufs3, leading to reduced levels of functional proteins that are essential for mitochondrial complex I assembly and activity. Consequently, this deficiency disrupts mitochondrial integrity, ultimately compromising the thermogenic capacity of BAT. These findings illuminate a novel role for SRSF1 in influencing mitochondrial function and BAT thermogenesis through its regulation of Ndufs3 splicing within BAT.


Subject(s)
Adipocytes, Brown , Homeostasis , Mitochondria , Serine-Arginine Splicing Factors , Thermogenesis , Animals , Thermogenesis/genetics , Thermogenesis/physiology , Serine-Arginine Splicing Factors/genetics , Serine-Arginine Splicing Factors/metabolism , Mice , Adipocytes, Brown/metabolism , Mitochondria/metabolism , Mitochondria/genetics , Homeostasis/genetics , Homeostasis/physiology , RNA Splicing/genetics , Electron Transport Complex I/genetics , Electron Transport Complex I/metabolism , NADH Dehydrogenase/genetics , NADH Dehydrogenase/metabolism , Male
19.
Nat Commun ; 15(1): 3016, 2024 Apr 08.
Article in English | MEDLINE | ID: mdl-38589367

ABSTRACT

Myelodysplastic syndromes (MDS) with mutated SF3B1 gene present features including a favourable outcome distinct from MDS with mutations in other splicing factor genes SRSF2 or U2AF1. Molecular bases of these divergences are poorly understood. Here we find that SF3B1-mutated MDS show reduced R-loop formation predominating in gene bodies associated with intron retention reduction, not found in U2AF1- or SRSF2-mutated MDS. Compared to erythroblasts from SRSF2- or U2AF1-mutated patients, SF3B1-mutated erythroblasts exhibit augmented DNA synthesis, accelerated replication forks, and single-stranded DNA exposure upon differentiation. Importantly, histone deacetylase inhibition using vorinostat restores R-loop formation, slows down DNA replication forks and improves SF3B1-mutated erythroblast differentiation. In conclusion, loss of R-loops with associated DNA replication stress represents a hallmark of SF3B1-mutated MDS ineffective erythropoiesis, which could be used as a therapeutic target.


Subject(s)
Myelodysplastic Syndromes , R-Loop Structures , Humans , Splicing Factor U2AF/genetics , Serine-Arginine Splicing Factors/genetics , RNA Splicing Factors/genetics , Myelodysplastic Syndromes/drug therapy , Myelodysplastic Syndromes/genetics , Mutation , Transcription Factors/genetics , Phosphoproteins/genetics
20.
Adv Sci (Weinh) ; 11(20): e2307442, 2024 May.
Article in English | MEDLINE | ID: mdl-38520084

ABSTRACT

Chondrodysplasia is closely associated with low birth weight and increased susceptibility to osteoarthritis in adulthood. Prenatal prednisone exposure (PPE) can cause low birth weight; however, its effect on offspring cartilage development remains unexplored. Herein, rats are administered clinical doses of prednisone intragastrically on gestational days (GDs) 0-20 and underwent long-distance running during postnatal weeks (PWs) 24-28. Knee cartilage is assayed for quality and related index changes on GD20, PW12, and PW28. In vitro experiments are performed to elucidate the mechanism. PPE decreased cartilage proliferation and matrix synthesis, causing offspring chondrodysplasia. Following long-distance running, the PPE group exhibited more typical osteoarthritis-like changes. Molecular analysis revealed that PPE caused cartilage circRNomics imbalance in which circGtdc1 decreased most significantly and persisted postnatally. Mechanistically, prednisolone reduced circGtdc1 expression and binding with Srsf1 to promote degradation of Srsf1 via K48-linked polyubiquitination. This further inhibited the formation of EDA/B+Fn1 and activation of PI3K/AKT and TGFß pathways, reducing chondrocyte proliferation and matrix synthesis. Finally, intra-articular injection of offspring with AAV-circGtdc1 ameliorated PPE-induced chondrodysplasia, but this effect is reversed by Srsf1 knockout. Altogether, this study confirms that PPE causes chondrodysplasia and susceptibility to osteoarthritis by altering the circGtdc1-Srsf1-Fn1 axis; in vivo, overexpression of circGtdc1 can represent an effective intervention target for ameliorating PPE-induced chondrodysplasia.


Subject(s)
Disease Models, Animal , Osteoarthritis , Prednisone , Prenatal Exposure Delayed Effects , RNA, Circular , Signal Transduction , Animals , Female , Rats , Pregnancy , RNA, Circular/genetics , RNA, Circular/metabolism , Prenatal Exposure Delayed Effects/metabolism , Prenatal Exposure Delayed Effects/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Prednisone/pharmacology , Osteoarthritis/genetics , Osteoarthritis/metabolism , Serine-Arginine Splicing Factors/metabolism , Serine-Arginine Splicing Factors/genetics , Rats, Sprague-Dawley
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