Ping Yi

SRSF9 Forms Phase-Separated Condensates to Promote Ovarian Cancer Progression by Inducing RNA Alternative Splicing That Is Inhibited by m6A Modification

Abstract Deregulation of RNA alternative splicing and modification can play an important role in tumor initiation and progression. Elucidation of the interplay between alternative splicing and modifications of RNA could provide important insights into cancer biology. In this study, we showed that serine/arginine-rich splicing factor 9 (SRSF9) recognized non-N6-methyladenosine (m6A)–modified NUMB mRNA and induced an oncogenic isoform switch in ovarian cancer. NUMB mRNA m6A modification antagonized SRSF9-mediated alternative splicing. Notably, SRSF9 formed phase-separated condensates within the nucleus, which was indispensable for its splicing function as well as its tumor-promoting effect in ovarian cancer. Furthermore, SRSF9 was aberrantly upregulated in ovarian cancer, correlating with poor patient prognosis. Loss of SRSF9 or antisense oligonucleotide–mediated isoform switch of NUMB mRNA inhibited ovarian cancer growth in vitro and in vivo. In conclusion, this study reveals that SRSF9 condensation promotes ovarian cancer progression through modulation of alternative splicing, in competition with m6A modification. Significance: Phase separation increases activity of the splicing factor SRSF9 to support progression of ovarian cancer by generating an oncogenic isoform of NUMB mRNA competitively with m6A modification, which provides promising therapeutic targets.

hnRNPL phase separation activates PIK3CB transcription and promotes glycolysis in ovarian cancer

Ovarian cancer has the highest mortality rate among gynecologic tumors worldwide, with unclear underlying mechanisms of pathogenesis. RNA-binding proteins (RBPs) primarily direct post-transcriptional regulation through modulating RNA metabolism. Recent evidence demonstrates that RBPs are also implicated in transcriptional control. However, the role and mechanism of RBP-mediated transcriptional regulation in tumorigenesis remain largely unexplored. Here, we show that the RBP heterogeneous ribonucleoprotein L (hnRNPL) interacts with chromatin and regulates gene transcription by forming phase-separated condensates in ovarian cancer. hnRNPL phase separation activates PIK3CB transcription and glycolysis, thus promoting ovarian cancer progression. Notably, we observe that the PIK3CB promoter is transcribed to produce a non-coding RNA which interacts with hnRNPL and promotes hnRNPL condensation. Furthermore, hnRNPL is significantly amplified in ovarian cancer, and its high expression predicts poor prognosis for ovarian cancer patients. By using cell-derived xenograft and patient-derived organoid models, we show that hnRNPL knockdown suppresses ovarian tumorigenesis. Together, our study reveals that phase separation of the chromatin-associated RBP hnRNPL promotes PIK3CB transcription and glycolysis to facilitate tumorigenesis in ovarian cancer. The formed hnRNPL-PIK3CB-AKT axis depending on phase separation can serve as a potential therapeutic target for ovarian cancer.

ALYREF condensation stabilizes m5C-modified PARP10 mRNA and promotes PI3K-AKT signaling in ovarian cancer

Abstract The role of epigenetic regulation of RNAs in the tumorigenesis remains incompletely understood. This study uncovers a critical function of the 5-methylcytosine (m 5 C) RNA modification reader protein ALYREF (also termed, ALY; BEF) in ovarian cancer. ALYREF is elevated in ovarian cancer patient samples, and its depletion reduces ovarian tumorigenesis and metastasis in mice in a m 5 C-dependent manner. Mechanistically, ALYREF binds to the m 5 C-modified mRNA of ADP-ribosyltransferase PARP10, competing with exosome complex component MTR4, and enhancing the stability and nuclear export of PARP10 mRNA. Further, ALYREF forms condensates in the nucleus of ovarian cancer cells, and depletion or mutation of ALYREF’s intrinsically disordered regions rescues its control on PARP10 mRNA nucleoplasmic distribution and stability, reduces tumor growth and is required for promotion of ovarian cancer aggressiveness and proliferation. Finally, ALYREF and PARP10 expression correlate with poor prognosis in ovarian cancer patients. Together, these findings suggest that ALYREF phase separation facilitates the malignant progression of ovarian cancer by promoting PARP10 expression and thereby enhancing PARP10-dependent proliferative pathways in a m 5 C-dependent manner.

IGF2BP3 recruits NUDT21 to regulate SPTBN1 alternative polyadenylation and drive ovarian cancer progression

Ovarian cancer (OC) is one of the deadliest gynecological malignancies. As the prevalent post-transcriptional regulation, alternative polyadenylation (APA) plays a crucial role in various tumors. Here we identify that the APA regulator NUDT21 is upregulated in OC and promotes malignant progression. We further demonstrate that IGF2BP3 interacts with NUDT21, which suggests m6A modification could regulate APA processing. Mechanistically, IGF2BP3, recognizing the m6A-modified site in intron 32 of SPTBN1, recruits NUDT21 to promote the usage of the SPTBN1 proximal polyadenylation site (PAS), thus increasing the generation of short transcripts in OC cells. Intriguingly, the SPTBN1 long variant demonstrates tumor-suppressive properties, whereas the short variant enhances oncogenic activity in OC. Subsequently, we illustrate that the long isoform inhibits tumor growth and metastasis by binding to CDK1 and blocking the G2/M phase of the cell cycle. In conclusion, this study uncovers a previously unrecognized regulatory mechanism in OC, which could provide potential therapeutic strategies for OC.

Genome‐wide profiling of N6‐methyladenosine‐modified pseudogene‐derived long noncoding RNAs reveals the tumour‐promoting and innate immune‐restraining function of RPS15AP12 in ovarian cancer

AbstractBackgroundPseudogene‐derived lncRNAs are widely dysregulated in cancer. Technological advancements have facilitated the functional characterization of increasing pseudogenes in cancer progression. However, the association between pseudogenes and RNA N6‐methyladenosine (m6A) modification in cancer, as well as the underlying mechanisms, remains largely unexplored.MethodsWe analyzed the expression of 12 146 pseudogenes and comprehensively examined the m6A modification of RNAs derived from them and their paralogs. Through integrative analysis of multi‐omics data, we explored the associations between pseudogene dysregulation and m6A, identifying critical pseudogenes involved in HGSOC progression. Tumour promotion role of RPS15AP12 and its cognate parent gene was characterized by cell proliferation, transwell assays, and scratch assays in ovarian cells and xenograft nude mice. RNA decay assays were used to reveal the participation of m6A in decreasement of RPS15AP12 lncRNA stability. Luciferase reporter assays were performed to verify that RPS15AP12 enhances RPS15A expression by competitively binding to miR‐96‐3p. Western blot and phosphorylation assays were performed to investigate the impairment of RPS15AP12 towards the sensors of MAVS (RIG‐I and MDA5), and downstream p‐TBK1 and p‐IRF3. Finally, ELISA assays were performed to explore the regulatory role of RPS15AP12 in IFN‐β expression.ResultsM6A is distributed across over a thousand pseudogenes, and hypomethylation leads to their upregulation in HGSOC. We identified a processed pseudogene, RPS15AP12, upregulated by FTO‐mediated m6A demethylation. RPS15AP12 enhances the growth ability and metastatic capabilities of ovarian cancer (OC) cells via functioning as a competitive endogenous RNA (ceRNA) for its host gene, RPS15A, through the sequestration of miR‐96‐3p. Importantly, the deletion of RPS15AP12 diminishes the expression of RPS15A, leading to the upregulation of anti‐tumour immune responses by activating RIG‐I and MDA5 and downstream p‐TBK1 and p‐IRF3 as well as IFN‐β levels.ConclusionOur findings expand the understanding of m6A‐modulated pseudogenes in tumour growth and anti‐tumour innate immunity in OC.Key Points Genome‐wide profiling reveals the redistribution of m6A modification on pseudogene‐derived lncRNAs and m6A redistribution‐relevant dysregulation of pseudogenes in HGSOC. RPS15AP12, as a representative processed pseudogene, is up‐regulated by FTO‐mediated demethylation and acts as a miRNA sponge to promote RPS15A expression via competitively binding to miR‐96‐3p. RPS15AP12/RPS15A axis inhibits MAVS sensors (RIG‐I and MDA5) and downstream IFN‐β levels in ovarian cancer.

m6A-driven NAT10 translation facilitates fatty acid metabolic rewiring to suppress ferroptosis and promote ovarian tumorigenesis through enhancing ACOT7 mRNA acetylation

RNA epigenetic modifications have been implicated in cancer progression. However, the interplay between distinct RNA modifications and its role in cancer metabolism remain largely unexplored. Our study demonstrates that N-acetyltransferase 10 (NAT10) is notably upregulated in ovarian cancer (OC), correlating with poor patient prognosis. IGF2BP1 enhances the translation of NAT10 mRNA in an m

IGF2BP2 binding to CPSF6 facilitates m6A‐mediated alternative polyadenylation of PUM2 and promotes malignant progression in ovarian cancer

AbstractBackgroundN6‐methyladenosine (m6A) and alternative polyadenylation (APA) are common posttranscriptional regulatory mechanisms in eukaryotes. However, the m6A‐dependent mechanism of APA regulation in ovarian cancer (OC) is still unclear.MethodsThe correlation between m6A and APA was analyzed by using RNA methylation sequencing of OC cells and single‐cell sequencing of clinical samples from public databases. To explore the core regulatory factors that served as a bridge between m6A and APA, we employed RNA pull‐down with biotin‐labelled m6A, immunoprecipitation, mass spectrometry, western blot, protein purification and GST pull‐down assays. Furthermore, the important target genes were screened by PAS‐seq, eCLIP‐seq, RIP‐seq and meRIP‐seq, and verified by RT‐qPCR, 3′RACE, RNA stability, and dual luciferase reporter assays. Multiple phenotypic experiments were conducted to evaluate the function of the IGF2BP2‐PUM2 axis in vitro and in vivo.ResultsThis study found that the m6A was correlated with the APA and affected the 3′end processing in OC. The APA regulator CPSF6 tended to bind the m6A‐modified transcripts in OC cells. Mechanistically, we demonstrated that the m6A reader IGF2BP2 KH1‐4 domains could directly bind to the CPSF6‐RS domain to regulate the 3′end processing of OC. Furthermore, sequencing revealed that the m6A was highly enriched in the 3′UTR near the proximal polyadenylation signal (PAS), which promotes the use of proximal PAS and leads to 3′UTR shortening. PUM2 was carried m6A and recognized by IGF2BP2, and CPSF6 was recruited at the proximal polyadenylation signal (pPAS) to generate the short‐3′UTR transcript. The short PUM2 transcript was more stable than the long transcript, which promoted the malignant progression of OC.ConclusionsWe revealed a novel mechanism in which the m6A could regulate the APA processing of pre‐mRNAs by crosstalk of IGF2BP2 and CPSF6. This study provides a potential strategy for the effective treatment of OC.Highlights The interaction between m6A and APA is mediated by the m6A regulator IGF2BP2 and the APA factor CPSF6. The transcripts harboring m6A modification tend to use the proximal polyadenylation signal (PAS) in ovarian cancer (OC). PUM2 promotes the malignant progression of OC through its m6A methylation and APA processing.