Biological relevance of alternative splicing in hematologic malignancies.

Alternative splicing (AS) is a strictly regulated process that generates multiple mRNA variants from a single gene, thus contributing to proteome diversity. Transcriptome-wide sequencing studies revealed networks of functionally coordinated splicing events, which produce isoforms with distinct or even opposing functions. To date, several mechanisms of AS are deregulated in leukemic cells, mainly due to mutations in splicing and/or epigenetic regulators and altered expression of splicing factors (SFs). In this review, we discuss aberrant splicing events induced by mutations affecting SFs (SF3B1, U2AF1, SRSR2, and ZRSR2), spliceosome components (PRPF8, LUC7L2, DDX41, and HNRNPH1), and epigenetic modulators (IDH1 and IDH2). Finally, we provide an extensive overview of the biological relevance of aberrant isoforms of genes involved in the regulation of apoptosis (e. g. BCL-X, MCL-1, FAS, and c-FLIP), activation of key cellular signaling pathways (CASP8, MAP3K7, and NOTCH2), and cell metabolism (PKM).

KAP1 stabilizes MYCN mRNA and promotes neuroblastoma tumorigenicity by protecting the RNA m6A reader YTHDC1 protein degradation.

BACKGROUND: Neuroblastoma (NB) patients with amplified MYCN often face a grim prognosis and are resistant to existing therapies, yet MYCN protein is considered undruggable. KAP1 (also named TRIM28) plays a crucial role in multiple biological activities. This study aimed to investigate the relationship between KAP1 and MYCN in NB. METHODS: Transcriptome analyses and luciferase reporter assay identified that KAP1 was a downstream target of MYCN. The effects of KAP1 on cancer cell proliferation and colony formation were explored using the loss-of-function assays in vitro and in vivo. RNA stability detection was used to examine the influence of KAP1 on MYCN expression. The mechanisms of KAP1 to maintain MYCN mRNA stabilization were mainly investigated by mass spectrum, immunoprecipitation, RIP-qPCR, and western blotting. In addition, a xenograft mouse model was used to reveal the antitumor effect of STM2457 on NB. RESULTS: Here we identified KAP1 as a critical regulator of MYCN mRNA stability by protecting the RNA N6-methyladenosine (m6A) reader YTHDC1 protein degradation. KAP1 was highly expressed in clinical MYCN-amplified NB and was upregulated by MYCN. Reciprocally, KAP1 knockdown reduced MYCN mRNA stability and inhibited MYCN-amplified NB progression. Mechanistically, KAP1 regulated the stability of MYCN mRNA in an m6A-dependent manner. KAP1 formed a complex with YTHDC1 and RNA m6A writer METTL3 to regulate m6A-modified MYCN mRNA stability. KAP1 depletion decreased YTHDC1 protein stability and promoted MYCN mRNA degradation. Inhibiting MYCN mRNA m6A modification synergized with chemotherapy to restrain tumor progression in MYCN-amplified NB. CONCLUSIONS: Our research demonstrates that KAP1, transcriptionally activated by MYCN, forms a complex with YTHDC1 and METTL3, which in turn maintain the stabilization of MYCN mRNA in an m6A-dependent manner. Targeting m6A modification by STM2457, a small-molecule inhibitor of METTL3, could downregulate MYCN expression and attenuate tumor proliferation. This finding provides a new alternative putative therapeutic strategy for MYCN-amplified NB.

MAPP unravels frequent co-regulation of splicing and polyadenylation by RNA-binding proteins and their dysregulation in cancer.

Maturation of eukaryotic pre-mRNAs via splicing and polyadenylation is modulated across cell types and conditions by a variety of RNA-binding proteins (RBPs). Although there exist over 1,500 RBPs in human cells, their binding motifs and functions still remain to be elucidated, especially in the complex environment of tissues and in the context of diseases. To overcome the lack of methods for the systematic and automated detection of sequence motif-guided pre-mRNA processing regulation from RNA sequencing (RNA-Seq) data we have developed MAPP (Motif Activity on Pre-mRNA Processing). Applying MAPP to RBP knock-down experiments reveals that many RBPs regulate both splicing and polyadenylation of nascent transcripts by acting on similar sequence motifs. MAPP not only infers these sequence motifs, but also unravels the position-dependent impact of the RBPs on pre-mRNA processing. Interestingly, all investigated RBPs that act on both splicing and 3' end processing exhibit a consistently repressive or activating effect on both processes, providing a first glimpse on the underlying mechanism. Applying MAPP to normal and malignant brain tissue samples unveils that the motifs bound by the PTBP1 and RBFOX RBPs coordinately drive the oncogenic splicing program active in glioblastomas demonstrating that MAPP paves the way for characterizing pre-mRNA processing regulators under physiological and pathological conditions.

SF3B3-regulated mTOR alternative splicing promotes colorectal cancer progression and metastasis.

BACKGROUND: Aberrant alternative splicing (AS) is a pervasive event during colorectal cancer (CRC) development. SF3B3 is a splicing factor component of U2 small nuclear ribonucleoproteins which are crucial for early stages of spliceosome assembly. The role of SF3B3 in CRC remains unknown. METHODS: SF3B3 expression in human CRCs was analyzed using publicly available CRC datasets, immunohistochemistry, qRT-PCR, and western blot. RNA-seq, RNA immunoprecipitation, and lipidomics were performed in SF3B3 knockdown or overexpressing CRC cell lines. CRC cell xenografts, patient-derived xenografts, patient-derived organoids, and orthotopic metastasis mouse models were utilized to determine the in vivo role of SF3B3 in CRC progression and metastasis. RESULTS: SF3B3 was upregulated in CRC samples and associated with poor survival. Inhibition of SF3B3 by RNA silencing suppressed the proliferation and metastasis of CRC cells in vitro and in vivo, characterized by mitochondria injury, increased reactive oxygen species (ROS), and apoptosis. Mechanistically, silencing of SF3B3 increased mTOR exon-skipped splicing, leading to the suppression of lipogenesis via mTOR-SREBF1-FASN signaling. The combination of SF3B3 shRNAs and mTOR inhibitors showed synergistic antitumor activity in patient-derived CRC organoids and xenografts. Importantly, we identified SF3B3 as a critical regulator of mTOR splicing and autophagy in multiple cancers. CONCLUSIONS: Our findings revealed that SF3B3 promoted CRC progression and metastasis by regulating mTOR alternative splicing and SREBF1-FASN-mediated lipogenesis, providing strong evidence to support SF3B3 as a druggable target for CRC therapy.

Association of poly(rC)-binding protein-2 with sideroflexin-3 through TOM20 as an iron entry pathway to mitochondria.

Iron is essential for all the lives and mitochondria integrate iron into heme and Fe-S clusters for diverse use as cofactors. Here, we screened mitochondrial proteins in KU812 human chronic myelogenous leukemia cells by glutathione S-transferase pulldown assay with PCBP2 to identify mitochondrial receptors for PCBP2, a major cytosolic Fe(II) chaperone. LC-MS analyses identified TOM20, sideroflexin-3 (SFXN3), SFXN1 and TOM70 in the affinity-score sequence. Stimulated emission depletion microscopy and proteinase-K digestion of mitochondria in HeLa cells revealed that TOM20 is located in the outer membrane of mitochondria whereas SFXN3 is located in the inner membrane. Although direct association was not observed between PCBP2 and SFXN3 with co-immunoprecipitation, proximity ligation assay demonstrated proximal localization of PCBP2 with TOM20 and there was a direct binding between TOM20 and SFXN3. Single knockdown either of PCBP2 and SFXN3 in K562 leukemia cells significantly decreased mitochondrial catalytic Fe(II) and mitochondrial maximal respiration. SFXN3 but not MFRN1 knockout (KO) in mouse embryonic fibroblasts decreased FBXL5 and heme oxygenase-1 (HO-1) but increased transferrin uptake and induced ferritin, indicating that mitochondrial iron entry through SFXN3 is distinct. MFRN1 KO revealed more intense mitochondrial Fe(II) deficiency than SFXN3 KO. Insufficient mitochondrial heme synthesis was evident under iron overload both with SFXN3 and MFRN KO, which was partially reversed by HO-1 inhibitor. Conversely, SFXN3 overexpression caused cytosolic iron deficiency with mitochondrial excess Fe(II), which further sensitized HeLa cells to RSL3-induced ferroptosis. In conclusion, we discovered a novel pathway of iron entry into mitochondria from cytosol through PCBP2-TOM20-SFXN3 axis.

GPATCH8 modulates mutant SF3B1 mis-splicing and pathogenicity in hematologic malignancies.

Mutations in the RNA splicing factor gene SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing, yet there is currently no therapeutic means to correct this mis-splicing. Here, we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain-containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SURP and G-patch domain containing 1 (SUGP1), a G-patch protein recently implicated in SF3B1-mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1-dependent splicing defects and was sufficient to improve dysfunctional hematopoiesis in SF3B1-mutant mice and primary human progenitors. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and highlight the therapeutic impact of correcting aberrant splicing in SF3B1-mutant cancers.

SF3A2 promotes progression and cisplatin resistance in triple-negative breast cancer via alternative splicing of MKRN1.

Triple-negative breast cancer (TNBC) is the deadliest subtype of breast cancer owing to the lack of effective therapeutic targets. Splicing factor 3a subunit 2 (SF3A2), a poorly defined splicing factor, was notably elevated in TNBC tissues and promoted TNBC progression, as confirmed by cell proliferation, colony formation, transwell migration, and invasion assays. Mechanistic investigations revealed that E3 ubiquitin-protein ligase UBR5 promoted the ubiquitination-dependent degradation of SF3A2, which in turn regulated UBR5, thus forming a feedback loop to balance these two oncoproteins. Moreover, SF3A2 accelerated TNBC progression by, at least in part, specifically regulating the alternative splicing of makorin ring finger protein 1 (MKRN1) and promoting the expression of the dominant and oncogenic isoform, MKRN1-T1. Furthermore, SF3A2 participated in the regulation of both extrinsic and intrinsic apoptosis, leading to cisplatin resistance in TNBC cells. Collectively, these findings reveal a previously unknown role of SF3A2 in TNBC progression and cisplatin resistance, highlighting SF3A2 as a potential therapeutic target for patients with TNBC.

Accelerated DNA replication fork speed due to loss of R-loops in myelodysplastic syndromes with SF3B1 mutation.

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.

Myelodysplastic Neoplasms (MDS) with Ring Sideroblasts or SF3B1 Mutations: The Improved Clinical Utility of World Health Organization and International Consensus Classification 2022 Definitions, a Single-Centre Retrospective Chart Review.

Myelodysplastic neoplasms (MDS) with ring sideroblasts (RS) are diagnosed via bone marrow aspiration in the presence of either (i) ≥15% RS or (ii) 5-14% RS and an SF3B1 mutation. In the MEDALIST trial and in an interim analysis of the COMMANDS trial, lower-risk MDS-RS patients had decreased transfusion dependency with luspatercept treatment. A total of 6817 patients with suspected hematologic malignancies underwent molecular testing using a next-generation-sequencing-based genetic assay and 395 MDS patients, seen at our centre from 1 January 2018 to 31 May 2023, were reviewed. Of these, we identified 39 evaluable patients as having lower-risk MDS with SF3B1 mutations: there were 20 (51.3%) males and 19 (48.7%) females, with a median age of 77 years (range of 57 to 92). Nineteen (48.7%) patients had an isolated SF3B1 mutation with a mean variant allele frequency of 35.2% +/- 8.1%, ranging from 7.4% to 46.0%. There were 29 (74.4%) patients with ≥15% RS, 6 (15.4%) with 5 to 14% RS, one (2.6%) with 1% RS, and 3 (7.7%) with no RS. Our study suggests that a quarter of patients would be missed based on the morphologic criterion of only using RS greater than 15% and supports the revised 2022 definitions of the World Health Organization (WHO) and International Consensus Classification (ICC), which shift toward molecularly defined subtypes of MDS and appropriate testing.

The splicing factor WBP11 mediates MCM7 intron retention to promote the malignant progression of ovarian cancer.

Accumulating studies suggest that splicing factors play important roles in many diseases including human cancers. Our study revealed that WBP11, a core splicing factor, is highly expressed in ovarian cancer (OC) tissues and associated with a poor prognosis. WBP11 inhibition significantly impaired the proliferation and mobility of ovarian cancer cells in vitro and in vivo. Furthermore, FOXM1 transcriptionally activated WBP11 expression by directly binding to its promoter in OC cells. Importantly, RNA-seq and alternative splicing event analysis revealed that WBP11 silencing decreased the expression of MCM7 by regulating intron 4 retention. MCM7 inhibition attenuated the increase in malignant behaviors of WBP11-overexpressing OC cells. Overall, WBP11 was identified as an oncogenic splicing factor that contributes to malignant progression by repressing intron 4 retention of MCM7 in OC cells. Thus, WBP11 is an oncogenic splicing factor with potential therapeutic and prognostic implications in OC.

NSUN5/TET2-directed chromatin-associated RNA modification of 5-methylcytosine to 5-hydroxymethylcytosine governs glioma immune evasion.

Malignant glioma exhibits immune evasion characterized by highly expressing the immune checkpoint CD47. RNA 5-methylcytosine(m5C) modification plays a pivotal role in tumor pathogenesis. However, the mechanism underlying m5C-modified RNA metabolism remains unclear, as does the contribution of m5C-modified RNA to the glioma immune microenvironment. In this study, we demonstrate that the canonical 28SrRNA methyltransferase NSUN5 down-regulates ß-catenin by promoting the degradation of its mRNA, leading to enhanced phagocytosis of tumor-associated macrophages (TAMs). Specifically, the NSUN5-induced suppression of ß-catenin relies on its methyltransferase activity mediated by cysteine 359 (C359) and is not influenced by its localization in the nucleolus. Intriguingly, NSUN5 directly interacts with and deposits m5C on CTNNB1 caRNA (chromatin-associated RNA). NSUN5-induced recruitment of TET2 to chromatin is independent of its methyltransferase activity. The m5C modification on caRNA is subsequently oxidized into 5-hydroxymethylcytosine (5hmC) by TET2, which is dependent on its binding affinity for Fe2+ and α-KG. Furthermore, NSUN5 enhances the chromatin recruitment of RBFOX2 which acts as a 5hmC-specific reader to recognize and facilitate the degradation of 5hmC caRNA. Notably, hmeRIP-seq analysis reveals numerous mRNA substrates of NSUN5 that potentially undergo this mode of metabolism. In addition, NSUN5 is epigenetically suppressed by DNA methylation and is negatively correlated with IDH1-R132H mutation in glioma patients. Importantly, pharmacological blockage of DNA methylation or IDH1-R132H mutant and CD47/SIRPα signaling synergistically enhances TAM-based phagocytosis and glioma elimination in vivo. Our findings unveil a general mechanism by which NSUN5/TET2/RBFOX2 signaling regulates RNA metabolism and highlight NSUN5 targeting as a potential strategy for glioma immune therapy.

Identification of splicing factors signature predicting prognosis risk and the mechanistic roles of novel oncogenes in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is the most frequent subtype of head and neck cancer, generally with a poor prognosis and limited therapeutic options due to its highly heterogeneous malignancy. In this study, we screened functional splicing regulatory RNA binding proteins (RBPs) that were closely related with the prognosis of HNSCC patients and showed significant expression differences between HNSCC tumors and normal tissues. Based on this finding, we chose six candidate genes (HNRNPC, ZCRB1, RBM12B, SF3A2, SF3B3, and SRSF11) to generate a prognostic prediction model and validated the accuracy of the prognostic model for predicting patient survival outcomes. We found that the risk score predicted by our model can serve as an independent prognostic predictor. Notably, HNSCC tumors showing higher expression of SF3B3, HNRNPC, or ZCRB1 possessed higher risk scores in the discovered prediction model. The investigation of the underlying mechanism validated that knockdown of SF3B3, HNRNPC, and ZCRB1 separately induced a substantial impairment of HNSCC cell survival. Conversely, overexpression of each of the three genes promoted tumor cellular proliferation. High throughput RNA sequencing analysis revealed that changes in the expression of SF3B3 and HNRNPC remarkably affected alternative splicing of genes related to cell cycle regulation, whereas the depletion of ZCRB1 contributed to aberrant splicing events involving in DNA damage response. In addition, the prognostic prediction model's risk score was demonstrated to be related with the immune infiltration score. Particularly, SF3B3 has a negative correlation with CD8A expression. Therefore, our findings provide promising prognosis predictors and potential therapeutic targets for better treatment efficacy of HNSCC.

SF3B1 mutations provide genetic vulnerability to copper ionophores in human acute myeloid leukemia.

In a phenotypical screen of 56 acute myeloid leukemia (AML) patient samples and using a library of 10,000 compounds, we identified a hit with increased sensitivity toward SF3B1-mutated and adverse risk AMLs. Through structure-activity relationship studies, this hit was optimized into a potent, specific, and nongenotoxic molecule called UM4118. We demonstrated that UM4118 acts as a copper ionophore that initiates a mitochondrial-based noncanonical form of cell death known as cuproptosis. CRISPR-Cas9 loss-of-function screen further revealed that iron-sulfur cluster (ISC) deficiency enhances copper-mediated cell death. Specifically, we found that loss of the mitochondrial ISC transporter ABCB7 is synthetic lethal to UM4118. ABCB7 is misspliced and down-regulated in SF3B1-mutated leukemia, creating a vulnerability to copper ionophores. Accordingly, ABCB7 overexpression partially rescued SF3B1-mutated cells to copper overload. Together, our work provides mechanistic insights that link ISC deficiency to cuproptosis, as exemplified by the high sensitivity of SF3B1-mutated AMLs. We thus propose SF3B1 mutations as a biomarker for future copper ionophore-based therapies.

WTAP-mediated N6-methyladenosine modification promotes the inflammation, mitochondrial damage and ferroptosis of kidney tubular epithelial cells in acute kidney injury by regulating LMNB1 expression and activating NF-κB and JAK2/STAT3 pathways.

Acute kidney injury (AKI) is a serious complication of sepsis patients, but the pathogenic mechanisms underlying AKI are still largely unclear. In this view, the roles of the key component of N6-methyladenosine (m6A)-wilms tumor 1 associated protein (WTAP) in AKI progression were investigated. AKI mice model was established by using cecal ligation and puncture (CLP). AKI cell model was established by treating HK-2 cells with LPS. Cell apoptosis was analyzed by TdT-mediated dUTP Nick-End Labeling (TUNEL) staining and flow cytometry analysis. Cell viability was analyzed by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The concentrations of inflammatory factors were examined with ELISA kits. Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) and Fe2+ levels were detected with related kits. Gene expression was detected by western blot assay or quantitative real-time polymerase chain reaction (qRT-PCR) assay. The relation between WTAP and lamin B1 (LMNB1) was verified by Methylated RNA Immunoprecipitation (meRIP) assay, RIP assay, dual-luciferase reporter assay and Actinomycin D assay. CLP induced significant pathological changes in kidney tissues in mice and promoted inflammation, mitochondrial damage and ferroptosis. LMNB1 level was induced in HK-2 cells by LPS. LMNB1 knockdown promoted LPS-mediated HK-2 cell viability and inhibited LPS-mediated HK-2 cell apoptosis, inflammation, mitochondrial damage and ferroptosis. Then, WTAP was demonstrated to promote LMNB1 expression by m6A Methylation modification. Moreover, WTAP knockdown repressed LPS-treated HK-2 cell apoptosis, inflammation, mitochondrial damage and ferroptosis, while LMNB1 overexpression reversed the effects. Additionally, WTAP affected the pathways of NF-κB and JAK2/STAT3 by LMNB1. WTAP-mediated m6A promoted the inflammation, mitochondrial damage and ferroptosis in LPS-induced HK-2 cells by regulating LMNB1 expression and activating NF-κB and JAK2/STAT3 pathways.

Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

METTL3-mediated the m6A modification of SF3B4 facilitates the development of non-small cell lung cancer by enhancing LSM4 expression.

BACKGROUND: Splicing factor B subunit 4 (SF3B4) has been confirmed to participate in the progression of many cancers and is considered to be a potential target for non-small cell lung cancer (NSCLC). Thus, the role and molecular mechanism of SF3B4 in NSCLC progression deserves further study. METHODS: Quantitative real-time PCR and western blot were employed to detect the mRNA and protein levels of SF3B4, Sm-like protein 4 (LSM4) and methyltransferase-like 3 (METTL3). Cell proliferation, apoptosis, invasion, migration and stemness were tested by cell counting kit-8, colony formation, flow cytometry, transwell, wound healing, and sphere formation assays. The interaction between SF3B4 and METTL3 or LSM4 was confirmed by MeRIP, RIP and Co-IP assays. Mice xenograft models were constructed to assess the effects of METTL3 and SF3B4 on NSCLC tumorigenesis. RESULTS: SF3B4 had high expression in NSCLC tissues and was associated with the shorter overall survival of NSCLC patients. Knockdown of SF3B4 suppressed NSCLC cell proliferation, invasion, migration and stemness, while inducing apoptosis. METTL3 promoted SF3B4 mRNA stability by m6A modification, and its knockdown inhibited NSCLC cell growth, metastasis and stemness by downregulating SF3B4. SF3B4 could interact with LSM4, and sh-SF3B4-mediated the inhibition on NSCLC cell functions could be reversed by LSM4 overexpression. In addition, reduced METTL3 expression restrained NSCLC tumor growth, and this effect was reversed by SF3B4 overexpression. CONCLUSION: METTL3-stablized SF3B4 promoted NSCLC cell growth, metastasis and stemness via positively regulating LSM4.

Exogenous hydrogen sulphide promotes plant flowering through the Arabidopsis splicing factor AtU2AF65a.

Alternative splicing (AS) is an important regulatory mode at the post-transcriptional level, through which many flowering genes regulate floral transition by producing multiple transcripts, and splicing factors have essential roles in this process. Hydrogen sulphide (H2S) is a newly found gasotransmitter that has critical physiological roles in plants, and one of its potential modes of action is via persulfidation of target proteins at specific cysteine sites. Previously, it has been shown that both the splicing factor AtU2AF65a and H2S are involved in the regulation of plant flowering. This study found that, in Arabidopsis, the promoting effect of H2S on flowering was abolished in atu2af65a-4 mutants. Transcriptome analyses showed that when AtU2AF65a contained mutations, the regulatory function of H2S during the AS of many flowering genes (including SPA1, LUH, LUG and MAF3) was inhibited. The persulfidation assay showed that AtU2AF65a can be persulfidated by H2S, and the RNA immunoprecipitation data indicated that H2S could alter the binding affinity of AtU2AF65a to the precursor messenger RNA of the above-mentioned flowering genes. Overall, our results suggest that H2S may regulate the AS of flowering-related genes through persulfidation of splicing factor AtU2AF65a and thus lead to early flowering in plants.

Splicing Factor PQBP1 Curtails BAX Expression to Promote Ovarian Cancer Progression.

Splicing factor polyglutamine binding protein-1 (PQBP1) is abundantly expressed in the central nervous system during development, and mutations in the gene cause intellectual disability. However, the roles of PQBP1 in cancer progression remain largely unknown. Here, it is shown that PQBP1 overexpression promotes tumor progression and indicates worse prognosis in ovarian cancer. Integrative analysis of spyCLIP-seq and RNA-seq data reveals that PQBP1 preferentially binds to exon regions and modulates exon skipping. Mechanistically, it is shown that PQBP1 regulates the splicing of genes related to the apoptotic signaling pathway, including BAX. PQBP1 promotes BAX exon 2 skipping to generate a truncated isoform that undergoes degradation by nonsense-mediated mRNA decay, thus making cancer cells resistant to apoptosis. In contrast, PQBP1 depletion or splice-switching antisense oligonucleotides promote exon 2 inclusion and thus increase BAX expression, leading to inhibition of tumor growth. Together, the results demonstrate an oncogenic role of PQBP1 in ovarian cancer and suggest that targeting the aberrant splicing mediated by PQBP1 has therapeutic potential in cancer treatment.

SETDB1 promotes progression through upregulation of SF3B4 expression and regulates the immunity in ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is the most lethal gynecologic malignant tumour. The mechanism promoting OC initiation and progression remains unclear. SET domain bifurcated histone lysine methyltransferase 1(SETDB1) acts as an oncogene in a variety of tumours. This study aims to explore the role of SETDB1 in OC. METHODS: GEO, TCGA, CSIOVDB and CPTAC databases jointly analysed SETDB1 mRNA and protein expression. Effect of SETDB1 expression on the clinical prognosis of OC patients was analysed through online Kaplan‒Meier plotter and CSIOVDB database. Then, the effect of SETDB1 in OC cells progression and mobility was examined using MTT, EdU, colony formation and transwell assay. Additionally, Cistrome DB database was used to visualize the binding of SETDB1 protein and splicing factor 3b subunit 4 (SF3B4) promoter, and dual-luciferase reporter gene assay was performed to confirm the interaction. Finally, bioinformatics analysis was employed to reveal the relationship between SETDB1 and the microenvironment of OC. RESULTS: In the present study, we found that SETDB1 was obviously upregulated in OC and its overexpression predicted poor prognosis of OC patients. Then, we verified that SETDB1 promoted the progression and motility of OC cells in vitro. Knockdown of SETDB1 had the opposite effect. Further research showed that SETDB1 acted as a transcription factor to activate SF3B4 expression. SF3B4 knockdown impaired the effect of SETDB1 to promote the proliferative capacity and motility of OC cells. Finally, the results of bioinformatics analysis confirmed that SETDB1 regulated the immune microenvironment of ovarian cancer. CONCLUSION: SETDB1 promoted ovarian cancer progression by upregulating the expression of SF3B4 and inhibiting the tumour immunity. SETDB1 may be a promising prognostic and therapeutic marker for OC.

Long noncoding RNA ABHD11-AS1 interacts with SART3 and regulates CD44 RNA alternative splicing to promote lung carcinogenesis.

Hexavalent chromium [Cr(VI)] is a common environmental pollutant and chronic exposure to Cr(VI) causes lung cancer in humans, however, the mechanism of Cr(VI) carcinogenesis has not been well understood. Lung cancer is the leading cause of cancer-related death, although the mechanisms of how lung cancer develops and progresses have been poorly understood. While long non-coding RNAs (lncRNAs) are found abnormally expressed in cancer, how dysregulated lncRNAs contribute to carcinogenesis remains largely unknown. The goal of this study is to investigate the mechanism of Cr(VI)-induced lung carcinogenesis focusing on the role of the lncRNA ABHD11 antisense RNA 1 (tail to tail) (ABHD11-AS1). It was found that the lncRNA ABHD11-AS1 expression levels are up-regulated in chronic Cr(VI) exposure-transformed human bronchial epithelial cells, chronically Cr(VI)-exposed mouse lung tissues, and human lung cancer cells as well. Bioinformatics analysis revealed that ABHD11-AS1 levels are up-regulated in lung adenocarcinomas (LUADs) tissues and associated with worse overall survival of LUAD patients but not in lung squamous cell carcinomas. It was further determined that up-regulation of ABHD11-AS1 expression plays an important role in chronic Cr(VI) exposure-induced cell malignant transformation and tumorigenesis, and the stemness of human lung cancer cells. Mechanistically, it was found that ABHD11-AS1 directly binds SART3 (spliceosome associated factor 3, U4/U6 recycling protein). The interaction of ABHD11-AS1 with SART3 promotes USP15 (ubiquitin specific peptidase 15) nuclear localization. Nuclear localized USP15 interacts with pre-mRNA processing factor 19 (PRPF19) to increase CD44 RNA alternative splicing activating ß-catenin and enhancing cancer stemness. Together, these findings indicate that lncRNA ABHD11-AS1 interacts with SART3 and regulates CD44 RNA alternative splicing to promote cell malignant transformation and lung carcinogenesis.