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.
Niedick
Ina
I
Department of Gene Regulation and Differentiation, German Research Center for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany.
Froese
Natali
N
Oumard
André
A
Mueller
Peter P
PP
Nourbakhsh
Mahtab
M
Hauser
Hansjörg
H
Köster
Mario
M
D000595:Amino Acid Sequence; D002466:Cell Nucleolus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D012330:RNA, Double-Stranded / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D016328:NF-kappa B; C111027:NKRF protein, human; D012330:RNA, Double-Stranded; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins