Role of hnRNP A1 in NF-κB transcriptional activation

Abstract

The correct temporal and spatial expression of genetic material governs the phenotypic differences that characterise the multitude of cells observed in higher eukaryotic organisms. Gene expression is controlled at many levels. For most genes the primary control point is the regulation of transcriptional initiation. NF-κB is a ubiquitously expressed transcription factor that is retained cytoplasmically in an inactive form by the inhibitor protein IκBα. Following cell stimulation, by a variety of different inducers including UV irradiation, cytokines and bacterial or viral products, IκBα is degraded. Active NF-κB then translocates to the nucleus where it activates transcription in a protein synthesis independent manner. To identify proteins involved in the activity of the transcription factor NF-κB, proteins bound to the IκBα inhibitor were isolated by immuno affinity chromatography. The data clearly demonstrated the association of IκBα with both forms of hnRNP A1 in vivo. In vitro experiments with bacterially produced recombinant proteins indicated that the interaction between hnRNPAl and IκBα was direct and was not mediated by a bridging protein present in the human cell extracts. Gel electrophoresis DNA binding experiments indicated that the ability of IkBcc to inhibit the DNA binding activity of NF-κB p65 was not influenced by hnRNP Al. Sites of interaction between hnRNP Al and IκBα were determined by deletion analysis and revealed that the acidic carboxy terminus of IkBcx (residues 265-303) was the minimal region required for binding to hnRNP Al. A single RNA binding domain linked to part of the RGG box (residues 95-207) was the minimal region of hnRNP Al required for interaction with IκBα. Immunofluoresence experiments demonstrated that hnRNPAl, in response to actinomycin D treatment, partially delocalises from the nucleus to the cytoplasm. Under these conditions IκBα wt also delocalised from the nucleus to the cytoplasm although the carboxy terminally truncated form of IκBα was not delocalised in response to actinomycin D treatment . Functional consequences of the interaction between IκBα and hnRNPAl were investigated in vivo using a mouse erythroleukaemia cell line (CB3) which lacks endogenous hnRNPAl (Ben-David et al., 1992). Cells lacking hnRNP A1 are defective in NF-κB dependent transcriptional activation but the defect in these cells is restored by ectopic expression of hnRNP Al. Thus in addition to regulating mRNA processing and transport, hnRNP Al may also contribute to the control of NF-κB dependent transcription.