Innate immune responses in NF-kappaB-repressing factor-deficient mice.

NF-kappaB-repressing factor (NRF) is a transcriptional silencer protein that specifically counteracts the basal activity of several NF-kappaB-dependent promoters by direct binding to specific neighboring DNA sequences. In cell culture experiments, the reduction of NRF mRNA leads to a derepression of beta interferon, interleukin-8, and inducible nitric oxide synthase transcription. The X chromosome-located single-copy NRF gene is ubiquitously expressed and encodes a protein of 690 amino acids. The N-terminal part contains a nuclear localization signal, the DNA-binding domain, and the NF-kappaB-repressing domain, while the C-terminal part is responsible for double-stranded RNA binding and nucleolar localization. To study the function of NRF in a systemic context, transgenic mice lacking the NRF gene were created. Against predictions from in vitro experiments, mice with a deletion of the NRF gene are viable and have a phenotype that is indistinguishable from wild-type mice, even after challenge with different pathogens. The data hint towards an unexpected functional redundancy of NRF.

Nucleolar localization and mobility analysis of the NF-kappaB repressing factor NRF.

NF-kappaB plays a central role in mediating pathogen and cytokine-stimulated gene transcription. NF-kappaB repressing factor (NRF) has been shown to interact with specific negative regulatory DNA elements (NRE) to mediate transcriptional repression by inhibition of the NF-kappaB activity at certain promoters. mRNA ablation experiments demonstrated that the trans-acting NRF protein is involved in constitutive but not post-stimulated silencing of IFN-beta, IL-8 and iNOS genes by binding to cis-acting NRE elements in their promoters. We have examined the subcellular localization and mobility of the NRF protein. Since neither tagging nor overexpression perturbs NRF localization the GFP-tagged protein was used for detailed localization and mobility studies. Owing to an N-terminal nuclear localization sequence, all NRF fragments that contain this signal show a constitutive nuclear accumulation. C-terminal NRF fragments also localize to the nucleus although no canonical NLS motifs were detected. Full-length NRF is highly enriched in nucleoli and only a small fraction of NRF is found in the nucleoplasm and cytoplasm. This relationship was found to be independent of the protein expression rate. FRAP analysis proved to be a sensitive method to determine protein mobility and made it possible to differentiate between the NRF protein fragments. Nucleolar localization correlated inversely with mobility. The data demonstrate that a series of neighboring fragments in a large central domain of the protein contribute to the strong nucleolar affinity. These properties were not altered by viral infection or LPS treatment. Several sequence motifs for RNA binding were predicted by computer-mediated databank searches. We found that NRF binds to double stranded RNA (dsRNA). This property mapped to several NRF fragments which correlate with the nucleolar affinity domain. Since treatment with actinomycin D releases NRF from nucleoli the identified RNA binding motifs might act as nucleolar localization signals.