An alternative miRISC targeting a coding mutation site inFOXL2links to granulosa cell tumor

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 induces 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 mouse model of AGCT, the inversely regulated variant FOXL2 abundance with the miR-1236 levels was 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 noncanonical miRISC. 4 small set of miRNAs (Azuma-Mukai, Oguri et al., 2008). AGO3 is also associated with slicer activity (Park, Phan et al., 2017). 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 comprised of 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 (Schmidt, Ovitt et al., 2004, Uhlenhaut, Jakob 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, Kobel et al., 2009). However, the etiological nature of the 402C>G mutation remains largely unknown. Previously, we showed that the FOXL2 protein acted as a tumor suppressor in granulosa cells, whereas C134W FOXL2 did not, due to serine 33 hyperphosphorylation by GSK3β, leading to accelerated MDM2-mediated ubiquitination and proteasomal degradation , Kim, Yoon et al., 2011.However, a relatively moderate change in FOXL2 protein stability by the C134W mutation does not appear to wholly account for haploinsufficiency of FOXL2 , Kim et al., 2011.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. 5 RESULTS Allelic imbalance of FOXL2 transcripts in AGCT samplesTo study allelic imbalance of heterozygous FOXL2 mRNAs, we analyzed the rela...