Epigenetic regulation of co-transcriptional R-loops via IGF2BPs drives SEMA3F-mediated prostate cancer growth retardation and docetaxel chemosensitivity enhancement

doi:10.21203/rs.3.rs-3111467/v1

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Epigenetic regulation of co-transcriptional R-loops via IGF2BPs drives SEMA3F-mediated prostate cancer growth retardation and docetaxel chemosensitivity enhancement

https://doi.org/10.21203/rs.3.rs-3111467/v1

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R-loops are prevalent three-stranded nucleic acid structures, comprising a DNA-RNA hybrid and a displaced single-stranded DNA, that frequently form during transcription and may be attributed to genomic stability and gene expression regulation. It was recently discovered that RNA modification contributes to maintain the stability of R-loops such as N6-methyladenosine (m⁶A). Yet, m⁶A-modified R-loops in regulating gene transcription remains poorly understood. Here, we demonstrated that insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) recognize R-loops in an m⁶A-dependent way. Consequently, IGF2BPs overexpression leads to increased overall R-loop levels, cell migration inhibition, cell growth retardation and docetaxel chemosensitivity in prostate cancer (PCa) via precluding the binding of DNA methyltransferase 1(DNMT1) to semaphorin 3F (SEMA3F) promoters. Moreover, the K homology (KH) domains of IGF2BPs are required for their recognition of m⁶A-containing R-loops and are required for tumor suppressor functions. Our findings point to a distinct R-loop resolution pathway mediated by IGF2BPs, emphasizing the functional importance of IGF2BPs as epigenetic R-loop readers in transcriptional genetic regulation and cancer biology.

R-loop

M6A

IGF2BP proteins

prostate cancer

R-loops have typically been recognized as harmful consequences of transcription that contribute to genomic instability over the last decade[1]. Broadly, R-loops are divided into two categories: physiological and pathological[2]. Physiological R loops often emerge as a result of a pre-programmed process involving particular elements, whereas pathological R-loops occur mistakenly and abruptly. R-loops have lately been highlighted in biomedical research as being important in cellular processes such as gene control and homologous recombination repair[3–6]. Based on the accumulated evidence for the abundance of physiological R-loops in gene promoters and termination regions, it appears that the presence of physiological R-loops may contribute to gene expression regulation. In mammalian cells, R-loops have been linked to the unmethylated state of CpG islands presenting at promoters of numerous genes via preventing the binding of DNA methyltransferase (DNMTs) and attracting ten-eleven translocation (TET) DNA demethylases [7, 8].

As the most abundant RNA modification, N6-methyladenosine (M⁶A) directs a broad range of crucial post-transcriptional cellular processes[9–11]. A growing body of evidence has uncovered m⁶A's novel role in R-loop development and resolution[12–15]. Depletion of METTL3 lead to the reduction of R-loop accumulation around transcription end sites (TESs) and enhancement of read-through in m⁶A-containing transcripts [16]. In addition, the m⁶A modification of R-loops can be recognized by YTHDF2, leading to the elimination of RNA–DNA hybrid [13]. The m⁶A modification, as a whole, is critical for the regulation of physiological R-loops.

A group of proteins known as IGF2BPs are involved in regulating RNA processing, such as mRNA translation and stabilization. Recent studies indicated that IGF2BPs contain RNA recognition motifs (RRMs) and KH domains. Moreover, the KH domains are assigned to the recognition of m⁶A modification and are essential for their biological functions[17]. A similar conclusion was reached in our previous studies[18] Although the mass spectrometry results demonstrated IGF2BP1/3 can be the specific regulator of R-loops [19, 20], the co-transcriptional roles of IGF2BPs are not yet unknown. Here, our research revealed the new functional role of IGF2BPs as R-loop readers, which stabilize m⁶A-modified R-loops in the gene promoters. Moreover, our findings demonstrated the new crosstalk between m⁶A RNA methylation and DNA methylation during R-loop metabolism.

Cell lines and transfection

RWPE-1, DU-145, PC-3, 22Rv1, LNCaP, C4-2B, VCaP and HEK293T cell lines were cultured according to the ENCODE cell culture standards. IGF2BPs KO and RBM15 KO cells were generated using the CRISPR-Cas9 technology following the manufacturer’s instructions. The guided RNAs (sgRNAs) were cloned into pLentiCRSPIR V2 (Genscript, USA). Transfections were carried out with Polyplus (Franch) for plasmids and siRNAs. The sgRNA sequences used are listed in Supplementary Table S1.

Clinical Tissue Samples

Thirty-six paired prostate cancer tissues and adjacent tissues were obtained from patients. The clinical tissues were all collected at the First Affiliated Hospital, School of Medicine, Zhejiang University after the approval of the Ethics Committee of Zhejiang University and informed consent. Clinicopathological characteristics of the PCa patients are listed in Supplementary Table S2.

Antibodies, siRNAs, plasmids and RNA oligos

Antibodies used in this study were: anti-Myc tag (ab32, Abcam), anti-IGF2BP1 (ab290736, Abcam), anti-IGF2BP2 (ab128175, Abcam), anti-IGF2BP3 (ab177477, Abcam), anti-RBM15 (10587-1-AP, Proteintech), anti-DNMT1 (ab92314), anti-SEMA3F (SAB2107196, Sigma), anti-S9.6 (MABE1095, Sigma), anti-5-mC (ab214727, Abcam), anti-m⁶A (SAB5600251, Sigma). siRNAs against DNMT1 used were purchased from Ribobio Co.,Ltd (Guangzhou, China). pcDNA3-based vectors encoding wild-type, RRM domain mutant, KH domain mutant Myc-tagged IGF2BP1, IGF2BP2, IGF2BP3 were produced by Shanghai Yoche Biotechnology Co.,Ltd (Shanghai, China). The plasmids encoding RBM15 and SEMA3F (M35-FLAG-RBM15, M35-FLAG-SEMA3F) were obtained from Guangzhou FulenGen Co., Ltd (Guangzhou, China). DNA and RNA oligos synthesized by TsingKe Biotech Co., Ltd (Beijing, China) are listed in Supplementary Table S1.

Lentiviruses transfection

The lentiviruses vectors, pReceiver-Lv242-FLAG, encoding IGF2BP1, IGF2BP2, IGF2BP3 and RBM1 5 were obtained from Guangzhou FulenGen Co., Ltd (Guangzhou, China). Lentivirus infection was performed following the manufacturer’s manuals.

Immunoprecipitation and immunobloting

After extracting proteins from cultured cells with a modified buffer, immunoprecipitation and following immunoblotting were performed with antibodies as described previously (25). Briefly, 5 µg corresponding antibodies were added to PC-3 cell lysates and incubated at room temperature, followed by incubation with Protein A/G Magnetic beads (MCE) at 4°C overnight. After washing with modified wash buffer, protein complexes were eluted by elution buffer and analyzed by immunoblotting and LC-MS/MS.

S9.6 IP

Protein co-immunoprecipitation with S9.6 antibody was performed from cultured PC-3 and DU-145 cells as previously described[21].

In vitro RNA-protein pulldown assays

3’-biotin-labeled ssRNA (5'-CGUCUCGGACUCGGACUGCU-3') and complementary ssDNA (5’-AGCAGTCCGAGTCCGAGACG-3’) were synthesized from TsingKe Biotech Co., Ltd (Beijing, China). We generated m⁶A ssRNA by substituting all two adenines with m⁶A-modified adenines. The ssRNA with or without m⁶A modification was annealed with complementary ssDNA in a 1:1 ratio. As directed by the manufacturer’s instructions, proteins in cell lysate were pulled down with the ssRNA and DNA:RNA hybrids using the Pierce magnetic RNA-Protein pull-down kit (20164, ThermoFisher). The final elution and supernatant were analyzed by western blot and LC-MS/MS.

LC-MS/MS

Elution samples were prepared for LC-MS/MS analysis as described previously[21]. After in-gel digestion, the peptides were dissolved on an EASY-nLC 1000 UPLC system according to the manufacturer’s protocol. And then peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The final MS/MS data were processed using Proteome Discoverer 2.4.

Dot-blot

Total genomic DNA from prostate cells were isolated according to standard procedures. The same amount of DNA from divergent groups was diluted to the same concentration with NaOH/TE solution and denatured at 99°C. Afterward, DNA was dot-blotted on Hybond N + membrane (GE health) via a dot-blot apparatus (Bio-rad), and linked by a UV crosslink at 1500 µJ. The membrane was stained by 0.1% methylene blue (Sigma-Aldrich). Primary antibody was diluted 1:1000 in universal antibody diluent (NCM Biotech). After incubation at 4°C, the membrane was washed twice gently in 0.02% TBST for 10 min. Secondary antibody was diluted 1:5000 in 5% BSA in 0.02% TBST and then washed three times.

Immunofluorescence

Cells for immunofluorescence were fixed in ice-cold methanol for 15 min at -20°C and further treated with 0.1% Triton X-100 for 30 min. Cells were then blocked by 5% BSA for 30 minutes at room temperature. Primary antibodies were diluted in universal antibody diluent (NCM Biotech) and incubated with prostate cells overnight at 4°C. The cells were then washed three times with 0.02% TBST, and were incubated with secondary antibodies for 50 min at room temperature followed by wash with 0.02% TBST twice. DAPI was incubated with cells at room temperature for 5 min.

DRIP-seq and m⁶A DIP

Genomic DNA was isolated from prostate cells by Proteinase K and RNase A treatment in lysis buffer, followed by phenol-chloroform extraction and ethanol precipitation. The DNA was fragmented to 200–500 bp by sonication. 20 µg of genomic DNA was used for each immunoprecipitation. S9.6 DRIP was performed as described previously using S9.6 antibody[22]. The DNA libraries were sequenced on the Illumina sequencing platform by Genedenovo Biotechnology Co., Ltd (Guangzhou, China). M⁶A DIP was carried out as described in a previously published study[13] using anti-m⁶A antibody.

RNA-seq

Total RNA was isolated from different groups of PC-3 cells by standard protocol. RNA-seq libraries were constructed using standard Illumina RNA-seq protocols by Genedenovo Biotechnology Co., Ltd (Guangzhou, China).

DNMT1 ChIP

DU-145 and PC-3 cells cultured in 10 cm dishes were fixed with 1% paraformaldehyde for 15 min at room temperature. The ChIP assay was carried out using the ChIP kit (Merck and Millipore). Briefly, cells were lysed using lysis buffer on ice. Nuclei was collected and fragmented by ultrasound. The chromatin was isolated and added to DNMT1 beads in ChIP buffer. After incubation overnight, the beads were washed and eluted. The eluted chromatin was combined and crosslinks reversed, followed by DNA purification by PCR purification columns (Thermo Fisher). The eluted ChIP-DNA was used for RT-PCR analysis. All the primers used are shown in Supplementary Table S1.

DNMT1 activity assay

The DNMT1 activity assay (Abcam) was used to quantify DNMT1 binding activity. The procedures to measure the DNMT1 activity were directed by manufacturer’s manual.

MethylationEPIC(850K) BeadChip

The MethylationEPIC BeadChip experiments and data analysis of the PCa samples were conducted by OE Biotechnology Co.Ltd. (Shanghai, China). DNA concentration and integrity were assessed by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. DNA was bisulfite treated using the Zymo Research EZ DNA methylaiton-Glod Kits (Zymo Research, Irvine, CA, USA). Bisulfite-converted DNA was analysed on an Illumina Infinium MethylationEPIC(850K) BeadChip (Illumina). Finally, Illumina iSCAN was used to scan the chip to get the Idat files.

Cell migration and proliferation assays

All specific procedures were performed as previously described[18].

Animal models and in vivo imaging

Subcutaneous transplantation models were prepared as described previously[23]. All the animal studies and protocols followed the institutional guidelines of the First Affiliated Hospital, School of Medicine, Zhejiang University.

RNA isolation and quantitative PCR

Total RNA was extracted from prostate cell lines and PCa tissues by RNAiso plus (Takara, Japan) and analyzed by RT-qPCR as previously described [24]. All the primers used in the study are shown in Supplementary Table S1.

Statistics

Data are presented as mean ± SD. Differences between two groups were evaluated using the two-tailed, unpaired t test. Survival curves were constructed using the Kaplan-Meier method and analyzed by the log-rank test. All statistical analysis was performed using the GraphPad Prism 9.0 software. Statistical significance was defined as P value of < 0.05.

1. R-loops are reduced in PCa and recruit IGF2BP proteins

We employed the well-established monoclonal antibody S9.6 to analyze R-loops in PCa tissues and cell lines to see if overall R-loop levels are altered in prostate cancer. The findings demonstrated that R-loop abundance was higher in surrounding tissues than in carcinomas (Fig. 1A). R-loop levels in prostate cancer cell lines were also compared to RWPE-1 cells (Fig S1A). PC-3 cells were treated to S9.6 IP to investigate how R-loops are dynamically regulated in cells, and the pull-down elution fractions were examined by mass spectrometry. S9.6 IP elution proteins are engaged in various RNA processing processes, including mRNA splicing, degradation, and stabilization, which is consistent with previous findings (Fig S1B). IGF2BP family are known m⁶A readers which play a crucial role in controlling the fate of methylated mRNA. Furthermore, m⁶A is critical for R-loop resolution in recent studies[13, 15]. Therefore, we investigated the role of IGF2BP family in R-loop resolution. We first confirmed that IGF2BP1/2/3 physically binds R-loops using S9.6 IP assays (Fig. 1C).

Given the finding that IGF2BPs binds R-loop, we examined their colocalization via immunofluorescence assays. In PC-3 cells, IGF2BP1/2/3 spontaneously formed foci that overlapped with R-loop foci (Fig. 1D). Mutual co-immunoprecipitation(Co-IP) experiments were performed to assess the interactions among IGF2BPs (Fig. 1E, S1C). To further determine the domains involved in the IGF2BPs interactions, serial deletion mutants of IGF2BPs were constructed (Fig S1D). Co-IP with the deletion mutants of IGF2BPs indicated that KH domains were crucial for the interactions of IGF2BP proteins (Fig. 1F). Taken together, these data indicated that IGF2BP proteins could be direct R-loop binding proteins.

2. IGF2BPs preferentially bind DNA:RNA hybrids containing m⁶A-modified RNA and overexpression of IGF2BPs causes R-loop accumulation

Since IGF2BPs are known m⁶A readers in mammalian cells[25], we anticipated that IGF2BPs could bind m⁶A-modified DNA:RNA hybrids. We initially employed negative control RNA, biotin-labeled DNA:RNA hybrids with or without two m⁶A sites for pull-down (Fig. 2A). The 20-bp DNA:RNA hybrids containing m⁶As captured IGF2BPs in cell lysates more efficiently than negative control RNA and DNA:RNA hybrids without m⁶A modification (Fig. 2A). Then, methylated single-stranded RNA bait (ss-m⁶A) and DNA:RNA hybrids with or without m⁶A were used for RNA pull-down, followed by mass spectrometry analysis. All three IGF2BP proteins were identified by MS and confirmed to selectively bind the methylated DNA:RNA hybrids with a 1.96–5.80 fold higher affinity than the unmethylated probes and 0.14–0.53 fold higher than methylated single-stranded RNA bait (Fig. 2B). Thus, IGF2BP proteins preferentially bind the DNA:RNA hybrids containing m⁶A-modified RNA (Fig. 2C). In addition, subsequent immunofluorescence staining showed the enrichment of m⁶A signal in R-loops (Fig. 2D). Given that IGF2BPs interacts each other and binds R-loops, we hypothesized that IGF2BPs would influence R-loop levels. The overall R-loop levels markedly increased in IGF2BPs OE cells (Fig. 2E), which can be impaired by m⁶A demethylation (Fig. 2F). Interestingly, the R-loop accumulation induced by IGF2BPs overexpression was not visibly affected by another IGF2BPs knockout (Fig. 2G). Taken together, overexpression of IGF2BPs causes R-loop accumulation in prostate cancer cells in an m⁶A-dependent way.

3. The KH domains of IGF2BPs are essential for R-loop accumulation

All of the IGF2BP proteins were structurally composed of similar functional domains, including four K homology (KH) domains and two RNA recognition motif domains (RRM). Studies indicated that KH domains are essential for the binding of IGF2BPs to m⁶A-modified mRNAs [17]. The RRM domains and KH domains of the IGF2BP mutants were truncated as previously described 9. In order to prevent the potential consequences of endogenous IGF2BP proteins, PC-3 cells with IGF2BPs CRISPR KO were generated (Fig. 3A). Only truncating the KH1-2 domains marginally inhibited the association of IGF2BPs with ss-m⁶A probes and m⁶A-RNA:DNA hybrid probes, whereas the KH3-4 and KH1-4 alterations virtually removed the connection (Fig. 3B). Similarly, KH3-4 and KH1-4 alterations almost impaired the upregulation of R-loop levels (Fig. 3C). Notably, KH3-4 and KH1-4 mutations also affected the colocalization of IGF2BPs with R-loops in the nucleus (Fig. 3D). Collectively, these findings demonstrated the crucial roles of KH domains in IGF2BPs binding of ^m6A-modified DNA:RNA hybrids.

4. Overexpression of IGF2BPs globally upregulates target gene expression and R-loop levels in promoter regions

Following that, we performed RNA-seq on individual IGF2BPs overexpression and control PC-3 cells. The similarity between each IGF2BPs overexpression sample was shown using principal component analysis (PCA) (Fig.S1E). Further DRIP-seq investigation revealed that R-loops accumulate when IGF2BPs were overexpressed (Fig. 4A). According to the combined data, IGF2BPs overexpression groups show higher levels of R-loop signals throughout promoter regions than the control group (Fig. 4B, S1F). Individual published IGF2BPs CLIP-seq targets overlapped with 57.2%, 70.3%, and 71.2% of them, respectively (Fig.S1G). In general, the levels of most transcripts were considerably increased in each IGF2BPs overexpression team (88.5%, 51.9%, and 82.8%), whereas R-loop signals accumulated in the promoter regions (Fig. 4C). Combined analysis suggested the weak positive correlation between R-loop density and transcript levels (r = 0.0942, 0.0963, 0.0564), and five putative targets were shared by the three IGF2BP proteins (Fig. 4C). Among them, SEMA3F contains one m⁶A peak in the R-loop region, as discovered by m⁶A-DIP in one published study[13]. Furthermore, we found the promoter region of SEMA3F is significant GC rich and revealed high GC skewing (Fig. 4D). Previous research has consistently shown that a G-rich non-templated DNA strand favors R-loop formation.

We then investigated whether IGF2BPs had a direct effect on R-loop metabolism in the SEMA3F locus of PC-3 cells. Notably, overexpression of IGF2BPs increased R-loop formation at the SEMA3F promoter. Furthermore, subsequent investigations of the SEMA3F, which yielded a substantial promoter IGF2BPs-RIP signal, revealed a high positional overlap of SEMA3F in DRIP-seq assays (Fig. 4E). The R-loop level and expression of SEMA3F was confirmed by qPCR to be markedly upregulated upon IGF2BPs overexpression (Fig. 4F, S1H). Similar findings were obtained in the western blot experiments, which can be partially rescued by overexpression of RNaseH1 (Fig. S2A). All IGF2BPs knockouts decreased SEMA3F expression in PC-3 cells (Fig. S2B). Then we interrogated TCGA database to determine the correlation between the mRNA expression of SEMA3F and IGF2BPs. And findings revealed a positive correlation between all IGF2BPs and SEMA3F in PCa tissues (Fig. S2C). The correlated regulation of R-loop levels and gene expression via IGF2BP proteins revealed that IGF2BPs are responsible for the R-loops and expression of target genes.

5. IGF2BPs upregulate SEMA3F expression via repelling DNMT1

Specifically, we investigated how gene expression is affected by R-loops. Recent study has shown that R-loop abundance is connected with de novo methylation and is responsible for DNA methylation prevention[6]. Nevertheless, it is unknown whether R-loops induced by IGF2BPs alter DNA methylation patterns in prostate cancer. Consequently, we questioned if R-loops may influence target promoter methylation and expression in PCa. Initially, we compared promoter methylation status in SEMA3F high expression and low expression PCa patients. We discovered considerably higher methylation levels in SEMA3F promoter areas of low expression team compared to high expression as found by MEXPRESS database (Fig. 5A). We further analyzed the correlation between SEMA3F expression and expression of major DNA methyltransferases (DNMTs). SEMA3F mRNA abundance was all negatively linked with DNMTs, as expected (Fig. 5B). To further investigate the relationship between DNA methylation and SEMA3F expression, we treated PCa cells with 5-Aza-C, a DNMTs inhibitor. The western blot experiment revealed enhanced SEMA3F expression in 5-Aza-C treated group (Fig. 5C). As the predominant DNA methyltransferases, silencing DNMT1 also resulted in upregulated SEMA3F expression (Fig. 5D, S2H). After that, chromatin immunoprecipitation indicated that DNMT1 binds directly to the promoter of SEMA3F, which is severely suppressed by IGF2BPs overexpression (Fig. 5E).

Previous research has documented that R-loop has substantially lower DNMT1 activity than ssDNA. IGF2BPs consistently inhibited DNMT1 activity, which can be restored by KH1-4 mutants (Fig. 5F, S2G). With that confirmation, we compared SEMA3F promoter methylation in IGF2BPs overexpression PC-3 cells to control, and discovered that IGF2BPs overexpression reduced methylation levels at the SEMA3F promoter (Fig. 5G).

Moreover, overexpression of IGF2BPs altered global DNA methylation patterns, which were accompanied by hypomethylation of CpG islands (CGIs), CGI shelves and shores (Fig. 5H, S2D). Importantly, IGF2BPs overexpression resulted in a significant decrease in global DNA methylation by dot-blot, which could be partially restored by KH3-4 and KH1-4 mutants (Fig. S2E). These findings suggested that IGF2BPs facilitated target transcription by avoiding DNA methylation.

6. RBM15 induces m⁶A RNA methylation of R-loops

METTL3 is crucial for the m⁶A methylation of co-transcriptional R-loops[16]. Nevertheless, our analyses discovered approximately 1300 proteins involved in R-loop metabolism except METTL3. As a result, we investigated if other enzymes were involved in m⁶A methylation of R-loops in PCa cells. We searched that the location of RBM15/15B proteins is believed to influence m⁶A modification sites according to the previous studies, and RBM15 was included in the proteins identified by LC-MS/MS in Fig. 1B. Since RBM15 binds METTL3, we investigated its activity at R-loops and its function in m⁶A-modified R-loops. We initially performed co-immunoprecipitation with the S9.6 antibody in PC-3 cells, and the results revealed the direct interactions between R-loops and RBM15 (Fig. 6A). Then we observed that endogenous RBM15 was colocalized with R-loops and m⁶A modification (Fig. 6B). Next we assessed whether RBM15 was involved in m⁶A modification of R-loops by dot-blot. RBM15 Knockout significantly reduced R-loop levels and m⁶A modification, whereas RBM15 overexpression markedly upregulated R-loop levels and m⁶A modification (Fig. 6C-6E). SEMA3F was further studied to corroborate the regulation in m⁶A modification of R-loops. SRAMP was used to predict the detail m⁶A sites in SEMA3F promoter region (Fig. S3A). When RBM15 expression was upregulated, the methylation level of the m⁶A site at SEMA3F promoter altered considerably via MeDIP-PCR (Fig. 6F). Moreover, the expression of RBM15 mRNA markedly positively correlated with SEMA3F across PCa samples (Fig. S3B). Consistently, SEMA3F expression was upregulated when RBM15 was overexpression (Fig. 6G). After that, we asked whether IGF2BPs promote R-loops formation and targets transcription in an m⁶A-dependent manner. We measured R-loop levels in IGF2BPs overexpression and control groups after RBM15 knockout-induced m⁶A RNA demethylation, and no significant difference was found between these groups (Fig. 6I). Notably, similar results were observed in western blot assay to assess the expression of SEMA3F in RBM15 KO cells (Fig. 6I, S2C). Together, these data suggested that RBM15 was responsible for the m⁶A methylation of R-loops in prostate cancer.

7. RBM15 and IGF2BPs play tumor suppressor roles in prostate cancer

A thorough analysis of the GEO database revealed that all three IGF2BPs are frequently downregulated in prostate cancer (Fig. S2). The expression of IGF2BPs in PCa tissues and cell lines was then assessed. In comparison to controls, the expression of all IGF2BP proteins varied in PCa tissues and cell lines (Fig. 7A-7D). Similar outcomes were seen with the RBM15 protein (Fig. 7D, S3C). Given the similar expression patterns and effects on R-loop metabolism, we assume IGF2BPs and RBM15 could function as tumor suppressor in prostate cancer. Specifically, overexpression of individual IGF2BPs and RBM15 all markedly inhibited cell migration and proliferation viabilities in vitro and in vivo (Fig. 7E-7J, 8A-8C). Notably, KH3-4 and KH1-4 mutants could partially repair the inhibition of cell proliferation and migration capacities caused by IGF2BPs overexpression (Fig. 8A, S3D). Additionally, the inhibition of proliferation and migration viabilities of IGF2BPs overexpression cells could be partially reversed by RBM15 KO and forced expression of RNaseH1 (Fig. 8E, S3E-S3F, S4A), suggesting the suppressor role of IGF2BPs relies on the m⁶A modification and R-loop regulation.

Meanwhile, as IGF2BPs’ common target, SEMA3F was likewise downregulated in PCa tissues and cell lines (Fig. 8F, 8G, S4B). Remarkably as in PCa, individuals with low SEMA3F expression showed a significantly worse prognosis (Fig. 8H). Decreased proliferation and migration viabilities were also observed in SEMA3F overexpression cells as we expect (Fig. 8I, 8J). As shown recently, SEMA3s showed the special ability to activate Hippo pathway and attenuate tumorigenesis, angiogenesis and tissue growth. We observed similar results in SEMA3F and RBM15 overexpression PC-3 and DU-145 cells compared to control (Fig. S4C, S4D). Previous studies have indicated potential associations between Hippo pathway and docetaxel chemosensitivity[26–28], and we observed significant changes of docetaxel chemosensitivity in IGF2BPs and SEMA3F overexpression groups compared to controls (Fig. 8K, 8L). Altogether, our data showed that RBM15 and IGF2BPs play tumor suppressor roles via SEMA3F-mediated regulation of Hippo pathway in prostate cancer. Further evaluation of the mechanism for the RBM15/IGF2BPs/SEMA3F axis may provide a new strategy for prostate cancer.

The characterization of the IGF2BP proteins as direct m⁶A readers has provided novel insights into our understanding of effects of m⁶A on post-transcriptional regulation. However, the role of IGF2BPs on transcription is poorly understood. R-loops have initially been considered as by-products during transcription without any functional role. However, multiple of recent studies have revealed that they may have an effect on transcriptional regulation [6]. Here we demonstrated that IGF2BPs are extremely efficient in binding and regulating m⁶A-RNA methylated R-loops in prostate cancer. Surprisingly, we found that RBM15 was also important for R-loop recognition and metabolism, and recruits METTL3 to the R-loop for m⁶A RNA methylation. As expect, RBM15 knockout not only results in downregulation of m⁶A RNA methylation but also diminishes the interactions between IGF2BPs and R-loop. Furthermore, RBM15 depletion also impairs the biological function of IGF2BPs in prostate cancer.

IGF2BP proteins localized in the cytosol and the nucleus, while most studies focus on the cytosolic function[29-32]. Our previous study also revealed that IGF2BPs enhance the mRNA stability in the renal cancer[18]. Here, our finding illustrates for the first time that IGF2BPs play a crucial role in the nucleus. And the direct binding of IGF2BPs to m⁶A-modifed R-loops, as same as m⁶A RNAs, relies on their KH domains. Our studies demonstrated that KH domains, especially KH3-4 domain, are crucial for the regulation of R-loops and biological function of IGF2BPs. Notably, KH domain is a conserved RNA recognition element in other proteins such as HNRNPK, FMR1. It will be interesting that other proteins contain KH domain play a similar role in R-loop regulation.

Our results also illustrate the importance of IGF2BPs-meditated R-loops in gene regulation. IGF2BPs-meditated R-loops are commonly positioned at promoter region in PCa. IGF2BP proteins shared more than 50% RNA targets, but our data indicated only a small part (4%-10%) of R-loops are shared by the three IGF2BP proteins. Additional researches will be required to further explore the relationship between IGF2BP proteins in R-loop regulation. Nevertheless, all IGF2BP proteins-mediated R-loops could protect SEMA3F promoter from DNA methylation via repelling DNMT1, as noted in another study (Christopher et al., 2018).

Previous studies revealed the opposite role of IGF2BPs versus YTHDF2 on mRNA stabilization[17]. Similarly, our data showed that IGF2BPs are more likely to promote co-transcriptional R-loop formation while YTHDF2 eliminates m⁶A-containing R-loops. However, published ENCODE PAR-CLIP data demonstrated approximately 1% of IGF2BP binding sites being shared by YTHDF2, suggesting that R-loop metabolism and reader binding preferences may be attributed to the different m⁶A sites.

We demonstrated that IGF2BPs could regulate R-loop metabolism in an m⁶A dependent way. In the nucleus, IGF2BP proteins prefers to bind the methylated DNA:RNA hybrids rather than ssRNA. Moreover, KH domains are crucial for the binding of IGF2BPs and R-loops. In addition, RBM15 is responsible for the m6A modification of R-loops. More importantly, our study provides a novel RBM15/IGF2BPs/DNMT1 trans-omics regulation m⁶A axis, indicating the new crosstalk between RNA m⁶A methylation and DNA methylation in prostate cancer. Simultaneously, as IGF2BPs co-target, SEMA3F was correlated with the survival of PCa patients, suggesting it might be a promising biomarker and predicting the risk of prostate cancer in the future.

m⁶A	N6-methyladenosine
IGF2BPs	insulin-like growth factor 2 mRNA-binding proteins
PCa	prostate cancer
TESs	transcription end sites
ssRNA	single strand RNA
ssDNA	single strand DNA
Co-IP	co-immunoprecipitation
PCA	principal component analysis

Acknowledgments

We are grateful for the supports from Jie Fang in all animal experiments.

Author contributions

BL, J Luo, and XZ designed the study. YY carried out the experiments and drafted the manuscript. YT, YW, JY, ZL,DC and XM performed data analysis. LX, XW, J Li, and JF helped in the experimental design and manuscript writing. XZ and LX revised the manuscript. All authors have seen and approved the final version of the manuscript.

Funding

This work was supported by grants from the National Natural Science Foundation of China (81972374, 82072848, 82103501, 82273132), the Natural Science Foundation of Zhejiang Province (LY21H160038, LQ21H160026), Zhejiang Province Medical and Health Scientific Research Project (2022RC019) and the Scientific Research Fund of the Health Commission of Zhejiang Province (2022499171).

Availability of data and materials

The RNA-seq and DRIP-seq data used and analyzed during the study are available within the manuscript and its additional files.

Ethical approval and consent to participate

Written informed consent was gained from all participants based on the guidelines of the Declaration of Helsinki. The collection of samples and operation of animals in our study were evaluated and approved by the Medical Ethics Committee of the First Affiliated Hospital of Zhejiang University.

Consent for publication

All patients involved in our study obtained written consent for publication.

Competing interests

The authors declare no potential conflicts of interest.

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No competing interests reported.

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Epigenetic regulation of co-transcriptional R-loops via IGF2BPs drives SEMA3F-mediated prostate cancer growth retardation and docetaxel chemosensitivity enhancement

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Version 1

Abstract

Figures

Introduction

Methods

Cell lines and transfection

Clinical Tissue Samples

Antibodies, siRNAs, plasmids and RNA oligos

Lentiviruses transfection

Immunoprecipitation and immunobloting

S9.6 IP

In vitro RNA-protein pulldown assays

LC-MS/MS

Dot-blot

Immunofluorescence

DRIP-seq and m6A DIP

RNA-seq

DNMT1 ChIP

DNMT1 activity assay

MethylationEPIC(850K) BeadChip

Cell migration and proliferation assays

Animal models and in vivo imaging

RNA isolation and quantitative PCR

Statistics

Results

1. R-loops are reduced in PCa and recruit IGF2BP proteins

2. IGF2BPs preferentially bind DNA:RNA hybrids containing m6A-modified RNA and overexpression of IGF2BPs causes R-loop accumulation

3. The KH domains of IGF2BPs are essential for R-loop accumulation

4. Overexpression of IGF2BPs globally upregulates target gene expression and R-loop levels in promoter regions

5. IGF2BPs upregulate SEMA3F expression via repelling DNMT1

6. RBM15 induces m6A RNA methylation of R-loops

7. RBM15 and IGF2BPs play tumor suppressor roles in prostate cancer

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

DRIP-seq and m⁶A DIP

2. IGF2BPs preferentially bind DNA:RNA hybrids containing m⁶A-modified RNA and overexpression of IGF2BPs causes R-loop accumulation

6. RBM15 induces m⁶A RNA methylation of R-loops