An alternative miRISC targets a cancer‐associated coding sequence mutation in FOXL2
2020; Springer Nature; Volume: 39; Issue: 24 Linguagem: Inglês
10.15252/embj.2020104719
ISSN1460-2075
AutoresEunkyoung Shin, Hanyong Jin, Dae‐Shik Suh, Yongyang Luo, Hye‐Jeong Ha, Tae Heon Kim, Yoonsoo Hahn, Seogang Hyun, Kangseok Lee, Jeehyeon Bae,
Tópico(s)Immune Cell Function and Interaction
ResumoArticle20 November 2020Open Access Source DataTransparent process An alternative miRISC targets a cancer-associated coding sequence mutation in FOXL2 Eunkyoung Shin Eunkyoung Shin orcid.org/0000-0002-3295-9000 School of Pharmacy, Chung-Ang University, Seoul, Korea These authors contributed equally to this work Search for more papers by this author Hanyong Jin Hanyong Jin orcid.org/0000-0002-5850-0791 Department of Life Science, Chung-Ang University, Seoul, Korea These authors contributed equally to this work Search for more papers by this author Dae-Shik Suh Dae-Shik Suh Department of Obstetrics and Gynecology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea These authors contributed equally to this work Search for more papers by this author Yongyang Luo Yongyang Luo orcid.org/0000-0001-5577-8977 School of Pharmacy, Chung-Ang University, Seoul, Korea Search for more papers by this author Hye-Jeong Ha Hye-Jeong Ha School of Pharmacy, Chung-Ang University, Seoul, Korea Search for more papers by this author Tae Heon Kim Tae Heon Kim Department of Pathology, Bundang CHA Hospital, CHA University, Seongnam, Korea Search for more papers by this author Yoonsoo Hahn Yoonsoo Hahn orcid.org/0000-0003-4273-9842 Department of Life Science, Chung-Ang University, Seoul, Korea Search for more papers by this author Seogang Hyun Corresponding Author Seogang Hyun [email protected] orcid.org/0000-0003-2008-0506 Department of Life Science, Chung-Ang University, Seoul, Korea Search for more papers by this author Kangseok Lee Corresponding Author Kangseok Lee [email protected] orcid.org/0000-0002-0060-6884 Department of Life Science, Chung-Ang University, Seoul, Korea Search for more papers by this author Jeehyeon Bae Corresponding Author Jeehyeon Bae [email protected] orcid.org/0000-0003-1995-1378 School of Pharmacy, Chung-Ang University, Seoul, Korea Search for more papers by this author Eunkyoung Shin Eunkyoung Shin orcid.org/0000-0002-3295-9000 School of Pharmacy, Chung-Ang University, Seoul, Korea These authors contributed equally to this work Search for more papers by this author Hanyong Jin Hanyong Jin orcid.org/0000-0002-5850-0791 Department of Life Science, Chung-Ang University, Seoul, Korea These authors contributed equally to this work Search for more papers by this author Dae-Shik Suh Dae-Shik Suh Department of Obstetrics and Gynecology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea These authors contributed equally to this work Search for more papers by this author Yongyang Luo Yongyang Luo orcid.org/0000-0001-5577-8977 School of Pharmacy, Chung-Ang University, Seoul, Korea Search for more papers by this author Hye-Jeong Ha Hye-Jeong Ha School of Pharmacy, Chung-Ang University, Seoul, Korea Search for more papers by this author Tae Heon Kim Tae Heon Kim Department of Pathology, Bundang CHA Hospital, CHA University, Seongnam, Korea Search for more papers by this author Yoonsoo Hahn Yoonsoo Hahn orcid.org/0000-0003-4273-9842 Department of Life Science, Chung-Ang University, Seoul, Korea Search for more papers by this author Seogang Hyun Corresponding Author Seogang Hyun [email protected] orcid.org/0000-0003-2008-0506 Department of Life Science, Chung-Ang University, Seoul, Korea Search for more papers by this author Kangseok Lee Corresponding Author Kangseok Lee [email protected] orcid.org/0000-0002-0060-6884 Department of Life Science, Chung-Ang University, Seoul, Korea Search for more papers by this author Jeehyeon Bae Corresponding Author Jeehyeon Bae [email protected] orcid.org/0000-0003-1995-1378 School of Pharmacy, Chung-Ang University, Seoul, Korea Search for more papers by this author Author Information Eunkyoung Shin1, Hanyong Jin2, Dae-Shik Suh3, Yongyang Luo1, Hye-Jeong Ha1, Tae Heon Kim4, Yoonsoo Hahn2, Seogang Hyun *,2, Kangseok Lee *,2 and Jeehyeon Bae *,1 1School of Pharmacy, Chung-Ang University, Seoul, Korea 2Department of Life Science, Chung-Ang University, Seoul, Korea 3Department of Obstetrics and Gynecology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea 4Department of Pathology, Bundang CHA Hospital, CHA University, Seongnam, Korea *Corresponding author. Tel: +82 2 8205805; E-mail: [email protected] *Corresponding author. Tel: +82 2 8205241; E-mail: [email protected] *Corresponding author. Tel: +82 2 8205604; E-mail: [email protected] The EMBO Journal (2020)39:e104719https://doi.org/10.15252/embj.2020104719 Correction(s) for this article An alternative miRISC targets a cancer-associated coding sequence mutation in FOXL216 August 2021 Correction added on 2 August 2021, after first online publication: panel A was corrected, see the associated corrigendum at https://doi.org/10.15252/embj.2021108163. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Recent evidence suggests that animal microRNAs (miRNAs) can target coding sequences (CDSs); however, the pathophysiological importance of such targeting remains unknown. Here, we show that a somatic heterozygous missense mutation (c.402C>G; p.C134W) in FOXL2, a feature shared by virtually all adult-type granulosa cell tumors (AGCTs), introduces a target site for miR-1236, which causes haploinsufficiency of the tumor-suppressor FOXL2. This miR-1236-mediated selective degradation of the variant FOXL2 mRNA is preferentially conducted by a distinct miRNA-loaded RNA-induced silencing complex (miRISC) directed by the Argonaute3 (AGO3) and DHX9 proteins. In both patients and a mouse model of AGCT, abundance of the inversely regulated variant FOXL2 with miR-1236 levels is highly correlated with malignant features of AGCT. Our study provides a molecular basis for understanding the conserved FOXL2 CDS mutation-mediated etiology of AGCT, revealing the existence of a previously unidentified mechanism of miRNA-targeting disease-associated mutations in the CDS by forming a non-canonical miRISC. SYNOPSIS An alternative miRNA-loaded RNA-induced silencing complex (miRISC) comprising AGO3, DHX9 and miR-1236 targets a conserved coding sequence mutation site in the tumor suppressor FOXL2, which is found in nearly all patients that have adult-type granulosa cell tumors. This leads to specific decay of the mutated FOXL2 mRNA. Patients with an adult-type granulosa cell tumor that contains a somatic missense mutation of FOXL2 (c.402C> G; p.C134W) exhibit an allelic imbalance of heterozygous FOXL2 mRNAs. miR-1236 causes the decay of the mutated variant FOXL2 mRNA, but not the wild-type FOXL2 mRNA, by targeting the 402C> G mutation site in the coding sequence. A non-canonical miRISC complex comprising AGO3 and DHX9 preferentially targets mutated FOXL2 mRNA variant in the coding region. miR-1236 acts as an oncomiR for adult-type granulosa cell tumor development by causing haploinsufficiency of the tumor suppressor FOXL2. Introduction MicroRNAs (miRNAs) are endogenous, noncoding RNAs of ~22 nucleotides (nt) in length that suppress the stability or translational efficiency of mRNAs. Conventionally, miRNAs are known to target sequences in the 3′-untranslated regions (UTRs) of mRNAs. However, recent multiple high-throughput sequencing and proteomic analyses suggest that miRNAs can also bind sites within mRNA coding sequences (CDSs; Chi et al, 2009; Hafner et al, 2010; Schnall-Levin et al, 2010; Hausser et al, 2013; Xue et al, 2013; Broughton et al, 2016), and the intracellular effects of miRNAs targeting CDS sites have been proposed (Forman et al, 2008; Tay et al, 2008; Elcheva et al, 2009; Schnall-Levin et al, 2011; Zhang et al, 2018). However, the physiological relevance and the pathological consequences of miRNA binding to CDSs remain unclear. Argonaute (AGO) clade proteins are essential components of miRNA-loaded RNA-induced silencing complexes (miRISCs) that select target mRNAs by directly interacting with mature miRNAs (Hock & Meister, 2008; Czech & Hannon, 2011). In mammals, four AGO paralogs (AGO1–4) are involved in miRNA pathways (Hock & Meister, 2008; Czech & Hannon, 2011), and they share ~80% amino acid-sequence identity (Sasaki et al, 2003). AGO2 has been described as a specialized AGO that possesses slicer activity, enabling cleavage of target mRNAs by miRNAs and small-interfering RNAs (siRNAs; Liu et al, 2004; Meister et al, 2004). However, previous data suggested that all mammalian AGOs may serve overlapping and distinct functions in miRNA-mediated regulation (Su et al, 2009). High-throughput pyrosequencing data showed that the profiles of miRNAs associated with AGO2 and AGO3 largely overlap, but preferential associations with AGO2 or AGO3 also occur for a small set of miRNAs (Azuma-Mukai et al, 2008). However, the functional significances of mammalian AGO1, AGO3, and AGO4 in miRNA activity are poorly understood. Conclusive evidence demonstrating clear pathophysiological consequences elicited by miRNA binding to the CDSs of disease-associated gene loci is lacking. Here, we investigated whether miRNA binding to the CDS of FOXL2 contributes to adult-type granulosa cell tumor (AGCT) development. GCTs are malignant ovarian cancers comprising AGCTs and juvenile GCTs (JGCTs) (Schumer & Cannistra, 2003). FOXL2 is evolutionarily conserved and encodes a forkhead-domain transcription factor essential for the ovary development and function (Crisponi et al, 2001; Schmidt et al, 2004; Uhlenhaut et al, 2009). A highly prevalent heterozygous somatic missense mutation (c.402C>G; p.C134W) in FOXL2 is exclusively found in > 97% of patients with ACGT and is considered the main cause of AGCT (Shah et al, 2009). However, the etiological nature of the 402C>G mutation remains largely unknown. Previously, we showed that the FOXL2 protein acts as a tumor suppressor and directs DNA double-strand repair pathways in granulosa cells, whereas C134W FOXL2 was defective due to accelerated MDM2-mediated ubiquitination and proteasomal degradation (Kim et al, 2011; Kim et al, 2014; Jin et al, 2020). However, a relatively moderate change in FOXL2 protein stability by the C134W mutation does not appear to wholly account for haploinsufficiency of the mutant FOXL2 (Kim et al, 2011; Kim et al, 2014). Here, we identified allelic imbalance in FOXL2 mRNAs in patients with AGCT arising from recognition of the 402C>G locus as a target site of miR-1236 that drives degradation of this variant FOXL2 mRNA, which explains the etiology of this conserved mutation in AGCTs. Results Allelic imbalance of FOXL2 transcripts in AGCT samples To study allelic imbalance of heterozygous FOXL2 mRNAs, we analyzed the relative levels of wild-type (WT) and variant FOXL2 (402C>G) mRNAs from complementary DNA (cDNA) samples from the individual AGCT tissues by high-throughput ultra-deep RNA sequencing. Ultra-deep RNA sequencing analysis of AGCT tissues showed that decreased proportion of variant FOXL2 mRNA level compared with WT FOXL2 mRNA in 20 AGCT patients with an average ratio of 62:38 for WT to 402C>G (P = 0.004), with a variable relative abundance, was observed among samples (Appendix Fig S1A). A previous study, which identified this conserved mutation, reported relative abundance of WT versus variant FOXL2 mRNA levels in four AGCT patients, where no uniformed trend was observed (Shah et al, 2009). For these reasons, we recruited additional AGCT patients from two independent hospitals and performed high-throughput pyrosequencing analysis (n = 46) that enables amplification and detection of both alleles from the same pyrosequencing reaction using common primers designed to bind FOXL2 cDNA. As shown in Fig 1A and Appendix Fig S1B, the relative abundance of FOXL2 mRNA analyzed by pyrosequencing was 72:28 for WT:402C>G in 46 AGCTs including 20 corresponding AGCTs analyzed for RNA sequencing presented in Appendix Fig S1A. In addition, allele-specific real-time and semi-quantitative RT–PCR analyses of 46 AGCTs were performed using primers presented in Appendix Fig S1C, and we observed consistent lower steady-state levels of FOXL2 variant mRNA compared with WT FOXL2 mRNA (Appendix Fig S1D and E). For these analyses, we used paired genomic DNA (gDNA) levels of both alleles for the normalization of data, where the gDNA levels of both alleles were similar in all AGCTs (Appendix Fig S1F). These results indicate that contamination of non-cancerous stromal cells in preparation of total RNA from AGCT tissues for these analyses was minimal. Figure 1. Allelic imbalance of heterozygous FOXL2 transcripts in AGCT cells § Bar graph and box-and-whisker plots are presented, which show the allelic proportions of WT FOXL2 mRNA and 402C> G FOXL2 mRNA in AGCT tissues from 46 patients analyzed by pyrosequencing. The box plot represents the minimum value, first quartile, median, third quartile, and maximum value of a data set. X-axis indicates mRNAs of WT FOXL2 and 402C>G FOXL2. The whiskers extend to the most extreme data points not considered outliers, and the outliers are represented as dots. Comparisons between groups were performed using Student’s t-test, and P values are presented. The relative abundances of WT and variant FOXL2 mRNA were analyzed in KGN and COV434 cells by pyrosequencing (left graph), allele-specific RT–PCR (middle graph), and real-time RT–PCR (right graph). gDNA was detected as a positive control. The relative abundances of the variant FOXL2 mRNA were normalized to that of WT mRNA (set to 1). FOXL2 mRNA levels detected by real-time RT–PCR were normalized to matching gDNA levels. The pyrosequencing data are presented from two independent experiments. The allele-specific semi-quantitative and real-time RT–PCR data are presented as the mean ± SEM from three independent experiments. The P values were analyzed by unpaired, two-tailed Student’s t-test (***P < 0.001). n.d. not detected. RNA-decay rates of WT and 402C>G FOXL2 mRNAs in KGN cells were determined after treatment with 5 µg/ml ActD for the indicated times. The estimated half-lives of each transcript are presented. The data are presented as the mean ± SEM from three independent experiments. Source data are available online for this figure. Source Data for Figure 1 [embj2020104719-sup-0002-SDataFig1.zip] Download figure Download PowerPoint We obtained analogous results using AGCT-derived KGN cells by pyrosequencing, allele-specific semi-quantitative RT–PCR, and allele-specific real-time RT–PCR, which are heterozygous for the 402C>G mutation (Fig 1B). The relative abundance of variant FOXL2 mRNA was ~22% of WT FOXL2 mRNA levels, while the gDNA levels of both alleles were similar in KGN cells (Fig 1B). When COV434, a cell line derived from JGCTs lacking the 402C>G mutation (Jamieson et al, 2010), was tested as a control using the mutant allele-specific primer, no amplicons containing the 402C>G mutation were detected (Fig 1B). We also observed a comparable allelic imbalance by allele-specific real-time RT–PCR on full-length FOXL2 mRNA generated by the cap analysis of gene expression (CAGE) method from KGN cells (Appendix Fig S1G). Next, we tested whether the decreased steady-state levels of the variant FOXL2 mRNA resulted from alterations in mRNA stability. WT and variant FOXL2 mRNA abundances were measured in KGN cells at several time points using allele-specific real-time RT–PCR after adding actinomycin D (ActD), which blocks transcription. The level of variant FOXL2 mRNA decreased more rapidly than that of WT mRNA (t1/2 = 3.68 h for variant FOXL2 versus t1/2 = 15.43 h for WT), indicating that the lower steady-state level of the variant FOXL2 mRNA resulted from decreased mRNA stability (Fig 1C). Variant FOXL2 mRNA instability was unlikely due to its altered secondary structure, as the 402C>G mutation was not predicted to affect the mRNA structure, based on M-fold analysis (http://www.bioinfo.rpi.edu/applications/mfold; Zuker, 2003; Appendix Fig S2A and B). Selective degradation of the variant FOXL2 mRNA by miR-1236-3p Next, we determined whether selective degradation of the variant FOXL2 mRNA was due to miRNAs targeting the mutation site in the CDS. By analyzing the variant FOXL2 mRNA sequence with an miRNA-prediction tool (https://genie.weizmann.ac.il/pubs/mir07/index.html; Kertesz et al, 2007), we identified miRNAs predicted to bind the sequence surrounding the mutation (Appendix Table S1). We selected five miRNAs predicted to preferentially bind the mutant allele over the WT allele for further analysis. Using DNA oligonucleotides complementary to these miRNAs (anti-miRNAs), which effectively inhibited the respective miRNAs based on upregulation of their known target mRNAs (Appendix Fig S3A), we found that anti-miR-1236 specifically increased the variant FOXL2 mRNA-expression level without affecting that of WT FOXL2 in KGN cells (Fig 2A). The remaining four anti-miRNAs did not affect the mRNA levels of WT FOXL2 or the variant (Fig 2A). Consistent with this effect at the mRNA level, anti-miR-1236 also increased FOXL2 protein expression (Fig 2B). Conversely, transfection of an miR-1236 RNA mimic decreased FOXL2 protein expression (Fig 2C). In contrast to our observations with KGN cells, transfecting miR-1236 into COV434 cells (which lack the 402C>G mutation) did not affect FOXL2 protein expression (Fig 2C). To further validate the specificity of miR-1236 on the FOXL2 variant, we cotransfected a WT- or variant FOXL2-expression plasmid together with miR-1236 RNA into 293T cells expressing minimal WT FOXL2 (but not the variant), and changes in FOXL2 expression were monitored by Western blotting. The miR-1236 mimic specifically decreased the variant FOXL2 level without affecting that of WT FOXL2 (Appendix Fig S3B). The effective depletion or overexpression of miR-1236 in cells transfected with anti-miRNAs or mimic oligonucleotides, respectively, was confirmed with a TaqMan® microRNA assay for miR-1236 (Appendix Fig S3C). Figure 2. Selective downregulation of the variant FOXL2 mRNA allele by miR-1236 A, B. Changes in WT and variant FOXL2 mRNA expression were assessed by RT–PCR (top) and real-time RT–PCR (bottom) (A) or by Western blot analysis (B) after transfecting KGN cells with anti-miRNAs for 48 h. C. FOXL2 protein levels after transfection of control or miR-1236 were assessed in KGN and COV434 cells. D. The mRNA levels of WT and variant FOXL2 in control, miR-1236−/+, and miR-1236−/− KGN cells, with or without miR-1236 transfection, were determined by RT–PCR (top) or real-time RT–PCR (bottom). E. FOXL2 protein expression in control, miR-1236−/+, and miR-1236−/− KGN cells and two independent miR-1236−/− (#1 and #2) COV434 cell lines were determined by Western blotting. Data information: Representative gel images are also shown. All quantified results (mean ± SEM) are from at least three independent experiments. Different letters (P < 0.0001; Student–Newman–Keuls test) or asterisks (*P < 0.05; Student’s t-test) denote significant differences. Source data are available online for this figure. Source Data for Figure 2 [embj2020104719-sup-0003-SDataFig2.zip] Download figure Download PowerPoint Moreover, we generated miR-1236-knockout (KO) KGN and COV434 cell lines using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-nickase-based system (Appendix Fig S4A–H) that is known to exert minimal off-target effects (Ran et al, 2013). Using extracted total RNA from the KO cells, we performed TaqMan® microRNA analyses and confirmed miR-1236 depletion (Appendix Fig S4B and F). Northern blotting showed that miR-1236 was detectable in control KGN cells, but not in miR-1236−/− cells (Appendix Fig S4I). Then, we evaluated WT and variant FOXL2 mRNA levels in the KO cells. Variant FOXL2 mRNA expression reverted to the level of WT mRNA when both alleles (miR-1236−/−) were excised and was partially restored when a single allele (miR-1236−/+) of miR-1236 was disrupted, without affecting WT FOXL2 mRNA expression (Fig 2D). Re-introducing miR-1236 mimic via transfection in miR-1236-KO cells downregulated variant FOXL2 mRNA expression without altering WT FOXL2 expression (Fig 2D), demonstrating that restoration of variant FOXL2 mRNA level resulted from miR-1236 KO. The observation that known miR-1236 target genes, AFP and ZEB1 (Wang et al, 2014; Gao et al, 2015), were upregulated in these KO cells also suggested that efficient and effective excision of miR-1236 occurred (Appendix Fig S4J and K). Consistently, elevated FOXL2 protein expression was confirmed in miR-1236-KO KGN cells, whereas no change in FOXL2 expression occurred in miR-1236-KO COV434 cells (Fig 2E). These in vivo genome-editing results further corroborated miR-1236 as an endogenous functional miRNA that selectively acted on variant FOXL2 mRNA. Allele-specific downregulation of the FOXL2 mRNA variant with miR-1236 via targeting the 402C>G mutation site Reporter constructs were developed to confirm that miR-1236 targets the 402C>G locus in the variant FOXL2 mRNA CDS. A 231-bp DNA fragment containing the 402C>G locus of the FOXL2 variant or the corresponding fragment of WT FOXL2 was inserted, in-frame, into the CDS of the luciferase gene (Fig 3A). miR-1236 transfection decreased the activity of the luciferase reporter harboring the 402C>G FOXL2 variant sequence by 70%, without affecting the activity of the luciferase reporter containing the WT FOXL2 sequence (Fig 3B). Similarly, we observed allele-specific repression when the 231-bp FOXL2 fragments were inserted in the 3′-UTR of the luciferase reporter gene (Fig 3C and D). The specificity of the interaction between miR-1236 and the variant locus in FOXL2 mRNA was further tested using two miR-1236 mutants. The miR-1236-G mutant shifted the 7-mer seed match from the 402C>G FOXL2 mRNA to the WT FOXL2 mRNA (Fig 3E), whereas the seed sequence of the miR-1236-U mutant did not match either 402C>G or WT FOXL2 mRNA (Fig 3F). Notably, the selectivity of the miR-1236-G mutant was reversed, as it repressed the WT reporter without affecting the 402C>G mutant reporter (Fig 3E). In contrast, the miR-1236-U mutant did not suppress the 402C>G mutant or WT FOXL2 reporter (Fig 3F). Figure 3. Allele-specific downregulation of FOXL2 mRNA by miR-1236, which targets 402C>G A. Schematic representation of the luciferase reporter constructs used to assay miR-1236 activity against a CDS target site in FOXL2 mRNA. The 231-bp human FOXL2 segments harboring either the C402 (WT) or the G402 (mutant) nucleotide were inserted in-frame into the CDS of the luciferase gene in the pGL3 control vector. B. Luciferase activity of the reporter constructs shown in (A) was measured in KGN cells after transfection with an miR-1236 mimic for 48 h. The black arrow indicates the position of 402C>G mutation site. C. A schematic diagram of the luciferase reporter constructs generated by inserting the predicted miR-1236-target sequences of WT and 402C>G FOXL2 mRNAs in the 3′-UTR of luciferase. D. Luciferase activities were measured in KGN cells, using the reporter constructs shown in (C), after transfection with a control miRNA or an miR-1236 mimic for 48 h. E, F. miR-1236 mutants, in which the C that pairs with G402 of the FOXL2 mutant was substituted with either G (miR-1236-G) (E) or U (miR-1236-U) (F), were cotransfected into KGN cells with one of the reporter constructs described above. Luciferase activities were subsequently determined. Arrows indicate the mismatched sites. G. In vitro annealing kinetics of miR-1236 with 230 nt-long transcripts of WT or variant FOXL2. 32P-labeled miR-1236 (0.5 nM) was incubated with increasing concentrations of synthetic FOXL2 transcripts (0, 2.5, 12.5, 25, or 50 nM). FOXL2 mRNA–miR-1236 complexes were resolved on a 6% native gel and detected by autoradiography (left). The predicted Kds for the WT and 402C>G FOXL2 transcripts are presented in the right graph. Data information: The data are expressed as the mean ± SEM from three independent experiments, performed in triplicate. The P values were analyzed by unpaired, two-tailed Student’s t-test (***P < 0.001). Source data are available online for this figure. Source Data for Figure 3 [embj2020104719-sup-0004-SDataFig3.zip] Download figure Download PowerPoint In addition, the preferential binding of miR-1236 to 402C>G over the WT FOXL2 transcript was confirmed by performing in vitro-binding assays. Synthetic 230-nt transcripts of WT or 402C>G FOXL2 mRNA were incubated with radiolabeled miR-1236, and RNA-duplex formation was monitored. miR-1236 preferentially duplexed with the 402C>G FOXL2 transcript, with predicted dissociation constants (Kd) of 1,589 and 194 nM for the WT and 402C>G FOXL2 transcripts, respectively (Fig 3G). Thus, these data indicate that the 402C>G locus was critical for distinguishing the effects of miR-1236 on FOXL2 expression. AGO3 as the major miRISC component for miR-1236-mediated FOXL2 variant mRNA degradation Because AGO2 is known to act primarily on the 3′-UTR of target mRNAs (Hafner et al, 2010) and a recent study demonstrated that AGO3 is also associated with slicer activity (Park et al, 2017), we investigated the possibility that AGO3 can regulate RISC activity against the variant FOXL2 mRNA by recognizing the mutated site in its CDS. Each AGO was knocked down using siRNAs, and changes in the levels of WT and variant FOXL2 mRNAs were examined in KGN cells. Of interest, we found that AGO3 knockdown preferentially increased mRNA expression of the FOXL2 variant without affecting that of WT FOXL2 (Fig 4A). In contrast, AGO2 knockdown increased both the WT and variant mRNAs (Fig 4A), indicating that WT FOXL2 mRNA is degraded by AGO2-mediated miRNAs targeting the 3′-UTR, as previously described (Dai et al, 2013; Luo et al, 2015; Wang et al, 2015). Depletion of either AGO1 or AGO4 did not affect the levels of the WT and variant FOXL2 mRNAs (Fig 4A). Consistent with the effects on the mRNA levels, increased FOXL2 protein levels were observed in KGN cells following the depletion of either AGO2 or AGO3 (Fig 4B). In addition, we determined whether the slicer activity of AGO3 promoted the decay of the variant FOXL2 using a slicer-incompetent E638A mutant of AGO3 (Park et al, 2017). AGO3 knockdown consistently elevated the level of variant FOXL2 mRNA and transfecting WT-AGO3 in AGO3-depleted cells effectively restored the level of variant FOXL2 mRNA to that in cells without any treatment (Appendix Fig S5A). Transfected E638A-AGO3 also decreased the expression of variant FOXL2 mRNA to a similar extent as transfected WT-AGO3 (Appendix Fig S5A). Consistently, the transfected WT-AGO3 or E638A mutant-AGO3 similarly decreased the level of FOXL2 protein (Appendix Fig S5B). Thus, these results indicate that AGO3-mediated destabilization of the variant FOXL2 mRNA does not require its slicer activity. Based on this result, we speculate that formation of the miR-1236-AGO3-RISC on the variant FOXL2 mRNA may induce translational repression, which leads to rapid degradation of the variant FOXL2 mRNA via pathways that do not involve the slicer activity of Ago3 (e.g., decapping and exosome activities). Figure 4. Identification of AGO3 as the major miRISC regulator for variant FOXL2 mRNA degradation A, B. Changes in WT and variant FOXL2 mRNA-expression levels were assessed by real-time RT–PCR (A) or Western blot analysis (B) after transfecting KGN cells with siRNAs against AGO mRNAs for 48 h. The data (mean ± SEM) are from three independent experiments, performed in triplicate. C. The mRNA levels of WT (left) and variant FOXL2 (right) were determined in KGN cells by real-time RT–PCR, after transfecting a control miRNA or miR-1236. The data (mean ± SEM) are from three independent experiments, performed in triplicate. D. The mRNA levels of WT and the variant FOXL2 in control (left) and miR-1236−/− KGN cells (right) after transfecting siRNAs against AGO mRNAs were determined by allele-specific real-time RT–PCR. The data (mean ± SEM) are from three independent experiments, performed in triplicate. E. 293T cells were transfected with an miR-1236 mimic (50 nM) for 24 h, followed by cotransfection with expression vectors encoding FLAG/HA-tagged variants of the indicated human AGOs and pGL3c-CDS-MT for 24 h. The empty p3XFLAG-CMV-10 vector was used as control. Co-immunoprecipitated mRNAs were reverse transcribed, and the cDNA products were used for allele-specific real-time PCR analysis of the FOXL2 variant and GAPDH mRNAs (top). The level of variant FOXL2 mRNA immunoprecipitated using FLAG-tagged AGO proteins was normalized using the level of GAPDH mRNA from the same lysates. The immunoprecipitated-AGO proteins were detected by Western blotting (bottom). The data (mean ± SEM) are from three independent experiments. F. In vivo association of AGO3-mediated miRISC formation with FOXL2 mRNAs is shown. Following transfection of a control miRNA or the miR-1236-G mutant into KGN cells, AGO3-mediated RISC-associated RNAs were isolated by immunoprecipitation with an anti-AGO3 antibody. IgG was used as a control. The co-immunoprecipitated mRNAs were reverse transcribed using a FOXL2-430-R primer binding downstream of the 402C>G site. The cDNA products were used for FOXL2 allele-specific PCR analysis with the FOXL2-279F primer (Appendix Fig S1), and a representative result (top left) is shown. Quantitative real-time RT–PCR results
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