A nuclear function for an oncogenic microRNA as a modulator of snRNA and splicing

Abstract

Background miRNAs are regulatory transcripts established as repressors of mRNA stability and translation that have been functionally implicated in carcinogenesis. miR-10b is one of the key onco-miRs associated with multiple forms of cancer. Malignant gliomas exhibit particularly striking dependence on miR-10b. However, despite the therapeutic potential of miR-10b targeting, this miRNA’s poorly investigated and largely unconventional properties hamper the clinical translation. Methods We utilized Covalent Ligation of Endogenous Argonaute-bound RNAs and their high-throughput RNA sequencing to identify miR-10b interactome and a combination of biochemical and imaging approaches for target validation. They included Crosslinking and RNA immunoprecipitation with spliceosomal proteins, a combination of miRNA FISH with protein immunofluorescence in glioma cells and patient-derived tumors, native Northern blotting, and the transcriptome-wide analysis of alternative splicing. Results We demonstrate that miR-10b binds to U6 snRNA, a core component of the spliceosomal machinery. We provide evidence of the direct binding between miR-10b and U6, in situ imaging of miR-10b and U6 co-localization in glioma cells and tumors, and biochemical co-isolation of miR-10b with the components of the spliceosome. We further demonstrate that miR-10b modulates U6 N-6-adenosine methylation and pseudouridylation, U6 binding to splicing factors SART3 and PRPF8, and regulates U6 stability, conformation, and levels. These effects on U6 result in global splicing alterations, exemplified by the altered ratio of the isoforms of a small GTPase CDC42, reduced overall CDC42 levels, and downstream CDC42 -mediated effects on cell viability. Conclusions We identified U6 snRNA, the key RNA component of the spliceosome, as the top miR-10b target in glioblastoma. We, therefore, present an unexpected intersection of the miRNA and splicing machineries and a new nuclear function for a major cancer-associated miRNA.

Secreted PGK1 and IGFBP2 contribute to the bystander effect of miR-10b gene editing in glioma

“…In silico analysis of PGK1 and IGFBP2 mRNAs identified both canonical (seed-containing) and non-canonical miR-10b-binding sites in the PGK1 and IGFBP2 3′ UTRs and additional sites in the IGFBP2 ORF and 5′ UTR ( Table S1 ). 20 As miR-10b has an established non-canonical binding activity, 7 , 21 we considered multiple putative binding sites and tested them for the direct binding and regulation by miR-10b using PGK1 and IGFBP2 fragments subcloned into psiCheck2 luciferase reporter vectors. Two reporters have been constructed for PGK1 and four reporters for IGFBP2 (corresponding to three alternative mRNA variants and their shared sequence).…”

“…However, the mechanisms mediating the effects of both PGK1 and IGFBP2 on GSCs require further investigation. Of note, the incomplete rescue provided by PGK1 and IGFBP2 inhibitors, as well as established functions of miR-10b in controlling diverse signaling pathways in glioma, 5 , 6 , 7 , 10 , 21 , 50 suggest that additional factors released by miR-10b-edited cells may contribute to the observed bystander mechanism. Overall, our findings demonstrate that miR-10b editing reduces the growth of glioma cells in both autonomous and non-autonomous ways, further supporting its potential for GBM therapy development.…”

Emerging roles of noncoding RNAs in human cancers

“…SnRNA can also regulate the occurrence of cancer. For example, U6snRNA is a key component of spliceosome RNA and the primary target of miR-10b in glioblastoma, which can regulate its development [132]. SnoRNA can compete with U11snRNA RNP, thereby altering the splicing of mRNA encoding E2F transcription factor (E2F7), which can lead to head and neck cancer and retinal cancer [51].…”

Metabolic Rewiring in Glioblastoma Cancer: EGFR, IDH and Beyond

“…Recent studies revealed that approximately 351 miRNAs are dysregulated in GBM (68), and some of them have been associated with acquired malignant phenotypes such as tumor growth, proliferation, invasion, and angiogenesis. Among others, miRNA-21, miRNA-10b, miRNA-7, miRNA-100, miRNA-296, miRNA-210-3p, miRNA-128, and miRNA-221 are the prominent candidates that have been studied and investigated for miRNA-based therapeutic strategies in GBM (68)(69)(70)(71)(72). Alongside miRNAs, long ncRNAs (lncRNAs) have also been observed to control many cellular processes in glioma cells predominantly associated with GBM and its malignancy (68,73,74).…”