Nuclear factor 90 (NF90) is a member of an expanding family of double-stranded (ds) RNA-binding proteins thought to be involved in gene expression. Originally identified in complex with nuclear factor 45 (NF45) as a sequence-specific DNA-binding protein, NF90 contains two double stranded RNA-binding motifs (dsRBMs) and interacts with highly structured RNAs as well as the dsRNA-activated protein kinase, PKR. In this report, we characterize the biochemical interactions between these two dsRBM containing proteins. NF90 binds to PKR through two independent mechanisms: an RNAindependent interaction occurs between the N terminus of NF90 and the C-terminal region of PKR, and an RNAdependent interaction is mediated by the dsRBMs of the two proteins. Co-immunoprecipitation analysis demonstrates that NF90, NF45, and PKR form a complex in both nuclear and cytosolic extracts, and both proteins serve as substrates for PKR in vitro. NF90 is phosphorylated by PKR in its RNA-binding domain, and this reaction is partially blocked by the NF90 N-terminal region. The C-terminal region also inhibits PKR function, probably through competitive binding to dsRNA. A model for NF90-PKR interactions is proposed. Nuclear factor 90 (NF90)1 is a cellular protein initially purified in complex with nuclear factor 45 (NF45) through its ability to bind to the antigen response recognition element in the interleukin-2 promoter (1). Independently, our laboratory identified NF90 as a cellular protein that interacts with VA RNA II , a structured adenoviral RNA, and with double-stranded RNA (dsRNA) (2). NF90 is one of the most abundant dsRNAbinding proteins in human cells, interacting preferentially with highly structured RNAs, in the order dsRNA Ͼ VA RNA II Ͼ VA RNA I Ͼ single stranded RNA (ssRNA). NF90 and NF45 have also been purified from human placental extracts in association with the protein synthesis eukaryotic initiation factor 2 and the catalytic subunit of the DNA-activated protein kinase DNA-PK (3). NF90 stabilizes the interactions between the catalytic subunit of DNA-PK and its regulatory heterodimeric DNA-binding subunits, Ku, in vitro, and it serves as a substrate for the activated DNA-PK in vitro.NF90 contains no recognized DNA binding motifs; rather it contains two dsRNA-binding motifs (dsRBMs) which lie downstream of a bipartite nuclear localization signal.2 Several homologues of NF90 have since been identified, including Xenopus 4F.1 (4), Spnr (5), and ILF3 (6) in mouse, and p74 in rat (7). All of these proteins share homology at their N terminus to a mouse protein, Zfr, a protein of unknown function which has a conserved homologue in Drosophila (8) (Fig. 1A). Downstream of this Zfr homology domain is a region of homology which these proteins share with NF45, which we term the NF45 homology domain.Several NF90 homologues and variants have been identified in human cells, and recent work from Duchange and colleagues (9) has helped clarify the relationships of several of these proteins. Alternatively spliced variants of the protein exist...

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Bovine papillomavirus type 1 (BPV-1) encodes two regulatory proteins, E1 and E2, that are essential for viral replication and transcription. E1, an ATP-dependent helicase, binds to the viral ori and is essential for viral replication, while the viral transcriptional activator, E2, plays cis-dominant roles in both viral replication and transcription. At low reporter concentrations, E1 stimulates E2 enhancer function, while at high reporter concentrations, repression results. An analysis of cis requirements revealed that neither replication nor specific E1-binding sites are required for the initiators' effect on E2 transactivator function. Though no dependence on E1-binding sites was found, analysis of E1 DNA binding and ATPase mutants revealed that both domains are required for E1 modulation of E2. Through the use of E2 fusion-gene constructs we showed that a heterologous DNA-binding domain could be substituted for the E2 DNA-binding domain and this recombinant protein remained responsive to E1. Furthermore, E1 could rescue activation domain mutants of E2 defective for transactivation. These data suggest that E1 stimulation of E2 involves interactions between E1 and the E2 activation domain on DNA. We speculate that E1 may allosterically interact with the E2 activation domain, perhaps stabilizing a particular structure, which increases the enhancer function of E2.