Die Rolle der langen nicht-kodierenden RNA lincRNA-LUCAT1 im Mausmodell des systemischen Lupus erythematodes
Zusammenfassung der Projektergebnisse
Long non-coding RNAs (lncRNAs) are RNA molecules larger than 200 nucleotides without any proteincoding capacity. Although it is assumed that the number of lncRNAs in the human genome is at least comparable to the number of protein-coding genes, only a low number of lncRNAs have been functionally characterized. In recent years however, it became clear that lncRNAs have important cellular and regulatory functions. The role of lncRNAs in immune responses and inflammatory pathways is of particular interest as the immune system is tightly regulated and, if thrown out of balance, can lead to autoimmune disorder or insufficient clearance of pathogens. Here, we identified lncRNA LUCAT1 as novel regulator of innate immune responses in human myeloid cells. LUCAT1 is located in the nucleus of myeloid cells such as macrophages (MF), dendritic cells (DCs) or monocytes and rapidly upregulated after stimulation with microbe-associated patterns (MAMPs) such as lipopolysaccharide (LPS) or viral infection. In absence of LUCAT1, immune cells secrete larger amounts of cytokines and type I interferons (IFNs) whereas increased levels LUCAT1 restrain the immune response. This led to the assumption that LUCAT1 acts as a negative feedback inhibitor after immune stimulation and infection. One aim of this study was to characterize an orthologue of LUCAT1 in a murine model of systemic lupus erythematosus (SLE). Unfortunately, in contrast to LUCAT1 in humans, immune cells from murine LUCAT1 (mLUCAT1)-deficient mice did not show any phenotypic alterations compared to wildtype mice as shown by various in vitro and in vivo assays. We found that mLUCAT1 has much lower expression levels at basal state and is not inducible stimulation with LPS or NRF2 activation. Based on these findings we assume that mLUCAT1 is non-functional. This finding is not uncommon in lncRNA biology as many lncRNAs are species-specific. Thus, we focused on the functional and molecular characterization human LUCAT1. In this study, we characterized the regulation of human LUCAT1 as well as the molecular mechanism of LUCAT1-dependent negative feedback inhibition of IFN and inflammatory responses in myeloid cells. Induction of LUCAT1 is mainly dependent on the transcription factors NF-kB and NRF2. A synergistic induction of LUCAT1 can be achieved by simultaneous activation of both transcription factors e.g., through stimulation with LPS (NF-kB activation) and itaconate or an KEAP1 inhibitor (NRF2 activation). Furthermore, we utilized long-read Nanopore sequencing of stimulated DCs and MF to identify the major isoform of LUCAT1. This isoform is a 7.3 kb long transcript that has not been annotated previously. To get better insight into the molecular mechanism of immune regulation through LUCAT1, Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) was performed. Using this method, we were able to efficiently pull-down LUCAT1 from LPS-stimulated, crosslinked cells and to identify LUCAT1 binding partners. These proteins are mainly nuclear-localized, RNA-processing proteins such as hnRNPs (C, M, A2B1, U), DHX9, DDX5 as well as DNA-PK. We hypothesize that LUCAT1 is involved in regulation of RNA processing events and analyzed alterations in LUCAT1-deficient cells regarding RNA splicing events using RNA-seq as well as RNA stability using Actinomycin D treatment. mRNA of the anti-inflammatory nuclear transcription factor NR4A2 showed a higher rate of alternative splicing events and was less stable in LUCAT1-KO cells which finally lead to lower levels and a delayed induction of NR4A2. These findings indicate that regulation of NR4A2 through LUCAT1 plays a major role in negative feedback inhibition that is induced by LUCAT1 after stimulation.
Projektbezogene Publikationen (Auswahl)
- (2020) Long Non-coding RNAs in Antiviral Immunity. Semin. Cell Dev. Biol. S1084-9521(19)30228-9
Vierbuchen T & Fitzgerald KA
(Siehe online unter https://doi.org/10.1016/j.semcdb.2020.06.009) - (2020) The long non-coding RNA LUCAT1 is a negative feedback regulator of Interferon response in humans. Nat. Commun. 11(1):6348
Agarwal S, Vierbuchen T, …, Ricci E, Fitzgerald KA
(Siehe online unter https://doi.org/10.1038/s41467-020-20165-5)
