Ring finger protein 144A (RNF144A), a poorly characterized member of the RING-in-between-RING family of E3 ubiquitin ligases, is an emerging tumor suppressor, but its underlying mechanism remains largely elusive. To address this issue, we used Affymetrix GeneChip Human Transcriptome Array 2.0 to profile gene expression in MDA-MB-231 cells stably expressing empty vector pCDH and Flag-RNF144A, and found that 128 genes were differentially expressed between pCDH- and RNF144A-expressing cells with fold change over 1.5. We further demonstrated that RNF144A negatively regulated the protein and mRNA levels of glial maturation factor γ (GMFG). Mechanistical investigations revealed that transcription factor YY1 transcriptionally activated GMFG expression, and RNF144A interacted with YY1 and promoted its ubiquitination-dependent degradation, thus blocking YY1-induced GMFG expression. Functional rescue assays showed that ectopic expression of RNF144A suppressed the proliferative, migratory, and invasive potential of breast cancer cells, and the noted effects were partially restored by re-expression of GMFG in RNF144A-overexpressing breast cancer cells. Collectively, these findings reveal that RNF144A negatively regulates GMFG expression by targeting YY1 for proteasomal degradation, thus inhibiting the proliferation, migration, and invasion of breast cancer cells.

RNF144A is involved in protein ubiquitination and functions as an ubiquitin-protein ligase (E3) via its RING finger domain (RNF144A RING). RNF144A is associated with degradation of heat-shock protein family A member 2 (HSPA2), which leads to the suppression of breast cancer cell proliferation. In this study, the solution structure of RNF144A RING was determined using nuclear magnetic resonance. Moreover, using a metallochromic indicator, we spectrophotometrically determined the stoichiometry of zinc ions and elucidated that RNF144A RING binds two zinc atoms. This structural analysis provided the position and range of the active site of RNF144A RING at the atomic level, which contributes to the creation of artificial RING fingers having the specific ubiquitin-conjugating enzyme (E2)-binding capability.

is widely used in the laboratory as an infection model for the research on pathogenesis and host defense against gram-positive intracellular bacteria. Macroautophagy (called simply "autophagy" hereafter), is important in the host defense against pathogens, such as bacteria, viruses, and parasites. BECN1 plays a pivotal role in the initiation of autophagy and accumulating evidence indicates that post-translational modifications of BECN1 provide multiple strategies for autophagy regulation. In this study, we demonstrated that the RING1-IBR-RING2 (RBR) family member RNF144A (ring finger protein 144A), which was induced by infection, promoted infection in an autophagy-dependent but STING1-independent pattern. deficiency in mice protected mice from infection with inhibited innate immune responses. Interestingly, RNF144A decreased -induced autophagosome accumulation. Mechanistic investigation indicated that RNF144A interacted with BECN1 and promoted its K48-linked ubiquitination, leading to the subsequent proteasome-dependent degradation of BECN1 and reduced autophagosome accumulation. Further study demonstrated that RNF144A promoted the ubiquitination of BECN1 at K117 and K427, and these two ubiquitination sites were essential to the role of BECN1 in autophagy and Lm infection. Thus, our findings suggested a new regulator in intracellular bacterial infection and autophagy, which may contribute to our understanding of host defense against intracellular bacterial infection via autophagy.: ATG3: autophagy related 3; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG10: autophagy related 10; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; Baf A: bafilomycin A; BECN1: beclin 1; BMDC: bone marrow-derived dendritic cell; BMDM: bone marrow-derived macrophage; CFUs: colony-forming units; CHX: cycloheximide; CQ: chloroquine; CXCL10/IP-10: C-X-C motif chemokine ligand 10; EBSS: Earle's balanced salt solution; ELISA: enzyme-linked immunosorbent assay; IFIT1/ISG56: interferon induced protein with tetratricopeptide repeats 1; IFNB/IFN-β: interferon beta; IL6: interleukin 6; IRF3, interferon regulatory factor 3; Lm: ; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MOI: multiplicity of infection; PLA: proximity ligation assay; PMA: phorbol myristate acetate; PMA-THP1, PMA-differentiated THP1; PMs: peritoneal macrophages; PTMs: posttranslational modifications; RBR: RING1-IBR-RING2; RNF144A: ring finger protein 144A; STING1, stimulator of interferon response cGAMP interactor 1; TBK1, TANK binding kinase 1; TNF/TNF-α: tumor necrosis factor.