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A 36-base hairpin within lncRNA DRAIC interacts with the IKKα coiled-coil domain, weakening the IKK complex and inhibiting NF-kappaB activation

ABSTRACT: Long noncoding RNAs have diverse functions, but not enough is known about the molecular mechanisms underlying their cellular functions. The 1.7 kb long down-regulated non-coding RNA in cancers (DRAIC) slows tumor growth and inhibits the activation of the transcription factor NF-kappaB through interactions with IKK subunits. Starting with SHAPE-MaP-based structure-function studies of DRAIC we have now identified the minimal regions of DRAIC and IKK involved in the interactions and the anti-cancer function of DRAIC. A hairpin structure comprised of bases 705-722/741-758 (referred to as A+B) within DRAIC is responsible for inhibiting NF-kappaB activity. A+B binds specifically with the IKKα coiled-coil domain with a KD of ~1-5 nM. The coiled-coil domain of IKKα (the kinase subunit) and NEMO (the substrate recognition subunit,) mediate the dimerization of the subunits in the functional kinase complex. Consistent with its interaction with the coiled-coil domain of IKKα, A+B weakens the interaction between NEMO and the IKKα coiled-coil domain in an in vitro complex formation assay. The 36 nt A+B RNA was sufficient and necessary for DRAIC to inhibit tumor cell migration, invasion, and clonal growth in multiple cancer cell-lines. Therefore, this minimal hairpin fragment of the growth-suppressive DRAIC lncRNA inhibits NF-kappaB activation by interacting with high affinity with the coiled-coil domain of IKKα, weakening the interaction between the kinase subunit and the substrate recognition subunit of IKK, thereby inhibiting IB phosphorylation and NF-kappaB activation, and suppressing tumor phenotypes.

ORGANISM(S): Homo sapiens

PROVIDER: GSE279192 | GEO | 2025/07/01

REPOSITORIES: GEO

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Project description:Classical Hodgkin lymphoma (cHL) is one of the most common malignant lymphomas. It is characterized by the presence of rare Hodgkin and Reed/Sternberg (HRS) cells embedded in an extensive inflammatory infiltrate. Constitutive activation of nuclear factor-kappaB (NF-kappaB) in HRS cells which transcriptionally regulates expression of multiple anti-apoptotic factors and pro-inflammatory cytokines plays a central role in the pathogenesis of cHL (1, 2). In non-stimulated condition, NF-kappaB proteins are rendered inactive by binding to their inhibitors (IkappaB s), which sequester them in the cytoplasm. Stimulation of multiple receptors activates the IkappaB kinase (IKK) complex that phosphorylates IkappaB at two specific serine residues, followed by its ubiquitination and proteasomal degradation, thereby releasing NF-kappaB proteins and allowing their nuclear translocation (3). Recently, two studies provided further insights into the molecular mechanisms of IKK activation upon TNF stimulation (4, 5). Activation of the IKK complex and subsequent NF-kappaB activation requires Lys63 polyubiquitination of RIP1, a kinase which is recruited to the receptor upon TNF stimulation. IKK-ï§ï (NEMO), the regulatory subunit of the IKK complex, specifically recognizes these Lys63-linked polyubiquitins attached to RIP1 and thereby activates IKK and NF-kappaB (4, 5). A20 is an ubiquitin-modifying enzyme that inhibits NF-kappaB activation in succession of tumor necrosis factor (TNF) receptor and Toll-like receptor induced signals (6-8). This enzyme removes Lys63 linked ubiquitin chains from RIP1 and adds Lys48 polyubiquitins to RIP1, thereby targeting this factor for proteasomal degradation, thus explaining the molecular mechanism of NF-kappaB inhibition by A20 (6). A20 likely inhibits NF-kappaB acitivity also by additional means, including interaction with TRAF1 and TRAF2 (9). SNP 6.0 array (Affymetrix) analyses were performed according to the manufacturer's directions on DNA extracted from three Hodgkin cell lines (L1236, HDLM-2, U-HO1), HapMap samples included in the Genotyping Console Software 3.0 were used as references.

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A 36-base hairpin within lncRNA DRAIC interacts with the IKKα coiled-coil domain, weakening the IKK complex and inhibiting NF-kappaB activation

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