RNA binding and phosphorylation determine the intracellular distribution of nuclear factors 90 and 110

Members of the nuclear factor 90 (NF90) family of human double-stranded RNA (dsRNA) binding proteins are phosphorylated and translocate into the cytoplasm with the onset of mitosis. We investigated the mechanism of translocation for NF90 and NF110, its larger splice variant. During interphase, NF90 is predominantly nuclear, NF110 is exclusively nuclear, and both are bound to RNA. About half of the NF90 is tethered in the nucleus by RNA bound to the protein's dsRNA-binding motifs. The nuclear localization of NF110 is also dependent on RNA binding but is independent of these motifs, and is governed by contacts made to the protein's unique C terminus. During mitosis, about half of the cytoplasmic NF90 becomes dissociated from RNA, but phosphorylation does not impair the binding affinity of either NF90 or NF110 for dsRNA. We conclude that NF90 and NF110 engage RNA differentially and translocate from the nucleus to the cytoplasm in mitosis because phosphorylation disturbs their interactions with other nuclear proteins.     

Nuclear factor 90 is a substrate and regulator of the eukaryotic initiation factor 2 kinase double-stranded RNA-activated protein kinase.

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 RNA-independent interaction occurs between the N terminus of NF90 and the C-terminal region of PKR, and an RNA-dependent 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 factors are involved in hepatitis C virus RNA replication

Unraveling the molecular basis of the life cycle of hepatitis C virus (HCV), a prevalent agent of human liver disease, entails the identification of cell-encoded factors that participate in the replication of the viral RNA genome. This study provides evidence that the so-called NF/NFAR proteins, namely, NF90/NFAR-1, NF110/NFAR-2, NF45, and RNA helicase A (RHA), which mostly belong to the dsRBM protein family, are involved in the HCV RNA replication process. NF/NFAR proteins were shown to specifically bind to replication signals in the HCV genomic 5' and 3' termini and to promote the formation of a looplike structure of the viral RNA. In cells containing replicating HCV RNA, the generally nuclear NF/NFAR proteins accumulate in the cytoplasmic viral replication complexes, and the prototype NFAR protein, NF90/NFAR-1, stably interacts with a viral protein. HCV replication was inhibited in cells where RNAi depleted RHA from the cytoplasm. Likewise, HCV replication was hindered in cells that contained another NF/NFAR protein recruiting virus. The recruitment of NF/NFAR proteins by HCV is assumed to serve two major purposes: to support 5'-3' interactions of the viral RNA for the coordination of viral protein and RNA synthesis and to weaken host-defense mechanisms.     

Autoantibodies define a family of proteins with conserved double-stranded RNA-binding domains as well as DNA binding activity

Cellular responses to viral infection are signaled by double-stranded (ds) RNA, which is not found in substantial amounts in uninfected cells. Although cellular dsRNA-binding proteins have been described, their characterization is incomplete. We show that dsRNA-binding proteins are prominent autoantigens. Sera from B6 and B10.S mice with pristane-induced lupus and human autoimmune sera immunoprecipitated a novel set of 130-, 110-, 90-, 80-, and 45-kDa proteins. The proteins were all major cellular poly(IC)-binding factors. N-terminal amino acid sequences of p110 and p90 were identical and matched nuclear factor (NF) 90 and M phase phosphoprotein 4. p45 and p90 were identified as the NF45.NF90 complex, which binds the interleukin-2 promoter as well as certain highly structured viral RNAs. NF90.NF45 and M phase phosphoprotein 4 belong to a large group of proteins with conserved dsRNA-binding motifs. Besides binding dsRNA, NF90.NF45, p110, and p130 had single-stranded and dsDNA binding activity. Some sera contained autoantibodies whose binding was inhibited by poly(IC) but not single-stranded DNA or vice versa, suggesting that the DNA- and RNA-binding sites are different. These autoantibodies will be useful probes of the function of dsRNA-binding proteins. Their interaction with dsRNA, an immunological adjuvant, also could promote autoimmunity.     

Organization of gene structure and expression of nuclear factor 1 family in rat.

The nuclear factor 1 (NF1) family proteins are encoded by four different genes (Nfia, Nfib, Nfic and Nfix) and regulate gene expression and DNA replication. All four genes bear many splicing isoforms, but the biological function of each of them awaits further characterization. We have previously isolated several splicing variant cDNAs derived from four NF1 genes of rat, and elucidated the structure of the rat Nfia gene. In this study, we determined the genomic organization and nucleotide sequences of the exon/intron boundaries of the rat Nfib, Nfic and Nfix genes in silico. We also constructed plasmids including entire open reading frames (ORFs) of NF1 isoforms and verified the expression of them in vitro. This information is made available for the production of NF1 knockout animals and expression of NF1 isoforms in vivo to elucidate the physiological function of NF1 proteins and to reveal the functional differences between NF1 splicing variants.     

Analysis of Xenopus dsRNA adenosine deaminase cDNAs reveals similarities to DNA methyltransferases.

We isolated two similar, but distinct, cDNA classes that encode Xenopus double-stranded RNA (dsRNA) adenosine deaminase. The longest, full-length open reading frame (ORF) predicts a 1,270-amino acid protein of 138,754 Da that is similar in size and about 50% identical to proteins encoded by mammalian cDNAs, yet larger than the 120-kDa protein purified from Xenopus eggs. Alignments of the Xenopus and mammalian ORFs show N-terminal heterogeneity, three conserved dsRNA binding motifs (dsRBMs), and strongly conserved carboxyl termini. Consistent with the observation of two cDNA classes, northern analyses of Xenopus oocyte poly A+ RNA show at least three mRNA species. Multiple nuclear polyadenylation hexamers and putative cytoplasmic polyadenylation elements were found in the 3'' UTRs of cDNAs corresponding to the largest mRNA. In vitro translation experiments show that the cDNAs encode active deaminases and that the entire N-terminus and first dsRBM are dispensable for deaminase activity. Importantly, an analysis of the C-termini of five known dsRNA adenosine deaminases, and two putative deaminases, reveals motifs that are strikingly similar to the conserved motifs of the DNA-(adenine-N6alpha)-aminomethyltransferases and the DNA-(cytosine-5)-methyltransferases.     

Characterization of adjacent E-box and nuclear factor 1-like DNA binding sequence in the human CYP1A2 promoter

To better understand the molecular mechanisms of cytochrome P450 1A2 (CYP1A2) regulation, we have characterized a region of the promoter (+3 to -176) that contains a single E-box and an adjacent nuclear factor 1 (NF1)-like DNA binding site. The E-box was shown to specifically bind nuclear proteins that were recognized by antibodies against upstream stimulatory factor (USF) 1 and 2. Comparison of NF1 binding proteins in HepG2 cells and primary cultures of rat hepatocytes revealed different patterns of DNA-protein complexes, all of which were recognized by a general NF1 antibody. Mutations of the E-box resulted in substantial reduction of promoter activity in either primary hepatocytes or HepG2 cells regardless of the presence in the reporter constructs of other CYP1A2 regulatory elements, such as the hepatic nuclear factor 1 (HNF-1) binding site. In contrast, reporter gene activity of the promoter construct harboring the mutated NF1-like binding site was affected by upstream sequences when transfected into HepG2 cells, but not in primary hepatocytes. We conclude that both USF proteins and different isoforms of NF1 contribute to the constitutive expression of CYP1A2.