Xin Luo

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.

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.

CSTF3 contributes to platinum resistance in ovarian cancer through alternative polyadenylation of lncRNA NEAT1 and generating the short isoform NEAT1_1

AbstractPlatinum-based chemotherapy is the standard postoperative adjuvant treatment for ovarian cancer (OC). Despite the initial response to chemotherapy, 85% of advanced OC patients will have recurrent disease. Relapsed disease and platinum resistance are the major causes of death in OC patients. In this study, we compared the global regulation of alternative polyadenylation (APA) in platinum-resistant and platinum-sensitive tissues of OC patients by analyzing a set of single-cell RNA sequencing (scRNA-seq) data from public databases and found that platinum-resistant patients exhibited global 3’ untranslated region (UTR) shortening due to the different usage of polyadenylation sites (PASs). The APA regulator CSTF3 was the most significantly upregulated gene in epithelial cells of platinum-resistant OC. CSTF3 knockdown increased the sensitivity of OC cells to platinum. The lncRNA NEAT1 has two isoforms, short (NEAT1_1) and long (NEAT1_2) transcript, because of the APA processing in 3’UTR. We found that CSTF3 knockdown reduced the usage of NEAT1 proximal PAS to lengthen the transcript and facilitate the expression of NEAT1_2. Downregulation of the expression of NEAT1 (NEAT1_1/_2), but not only NEAT1_2, also increased the sensitivity of OC cells to platinum. Overexpressed NEAT1_1 reversed the platinum resistance of OC cells after knocking down CSTF3 expression. Furthermore, downregulated expression of CSTF3 and NEAT1_1, rather than NEAT1_2, was positively correlated with inactivation of the PI3K/AKT/mTOR pathway in OC cells. Together, our findings revealed a novel mechanism of APA regulation in platinum-resistant OC. CSTF3 directly bound downstream of the NEAT1 proximal PAS to generate the short isoform NEAT1_1 and was conducive to platinum resistance, which provides a potential biomarker and therapeutic strategy for platinum-resistant OC patients.