Background:The role of AIF redox activity in mitochondrial respiration and redox metabolism was unknown. Results: AIF has rotenone-sensitive NADH:ubiquinone oxidoreductase activity when reconstituted with bacterial or mitochondrial membranes. Conclusion: AIF is a previously unidentified mammalian NDH-2-type enzyme that could contribute to NADH oxidation in cells. Significance: The catalytic function of AIF as an NADH:UQ oxidoreductase was uncovered.
Increased osteoclastogenesis is responsible for osteolysis that is a severe consequence of inflammatory diseases associated with bone destruction, such as rheumatoid arthritis (RA) and periodontitis. The mechanisms that limit osteoclastogenesis under inflammatory conditions are largely unknown. We previously identified transcription factor RBP-J as a key negative regulator that restrains TNF-α induced osteoclastogenesis and inflammatory bone resorption. In this study, we tested whether RBP-J suppresses inflammatory osteoclastogenesis by regulating expression of microRNAs (miRNAs) important for this process. Using high throughput sequencing of miRNAs (miRNA-seq), we obtained the first genome-wide profile of miRNA expression induced by TNF-α in mouse bone marrow derived macrophages (BMMs)/osteoclast precursors during inflammatory osteoclastogenesis. We furthermore identified miR-182 as a novel miRNA that promotes inflammatory osteoclastogenesis driven by TNF-α and whose expression is suppressed by RBP-J. Downregulation of miR-182 dramatically suppressed the enhanced osteoclastogenesis program induced by TNF-α in RBP-J-deficient cells. Complementary loss and gain of function approaches showed that miR-182 is a positive regulator of osteoclastogenic transcription factors NFATc1 and Blimp1. Moreover, we identified that direct miR-182 targets Foxo3 and Maml1 play important inhibitory roles in TNF-α mediated osteoclastogenesis. Thus, RBP-J-regulated miR-182 promotes TNF-α induced osteoclastogenesis via inhibition of Foxo3 and Maml1. Suppression of miR-182 by RBP-J serves as an important mechanism that restrains TNF-α induced osteoclastogenesis. Our results provide a novel miRNA mediated mechanism by which RBP-J inhibits osteoclastogenesis and suggest that targeting of the newly described RBP-J-miR-182-Foxo3/Maml1 axis may represent an effective therapeutic approach to suppress inflammatory osteoclastogenesis and bone resorption.
NORAD is a conserved long noncoding RNA (lncRNA) that is required for genome stability in mammals. NORAD acts as a negative regulator of PUMILIO (PUM) proteins in the cytoplasm, and we previously showed that loss of NORAD or PUM hyperactivity results in genome instability and premature aging in mice (Kopp et al., 2019). Recently, however, it was reported that NORAD regulates genome stability through an interaction with the RNA binding protein RBMX in the nucleus. Here, we addressed the contributions of NORAD:PUM and NORAD:RBMX interactions to genome maintenance by this lncRNA in human cells. Extensive RNA FISH and fractionation experiments established that NORAD localizes predominantly to the cytoplasm with or without DNA damage. Moreover, genetic rescue experiments demonstrated that PUM binding is required for maintenance of genomic stability by NORAD whereas binding of RBMX is dispensable for this function. These data provide an important foundation for further mechanistic dissection of the NORAD-PUMILIO axis in genome maintenance.
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