Nuclear factor 90 (NF90) and its C-terminally extended isoform, NF110, have been isolated as DNA-and RNA-binding proteins together with the less-studied protein NF45. These complexes have been implicated in gene regulation, but little is known about their cellular roles and whether they are redundant or functionally distinct. We show that heterodimeric core complexes, NF90-NF45 and NF110-NF45, exist within larger complexes that are more labile and contain multiple NF90/110 isoforms and additional proteins. Depletion of the NF45 subunit by RNA interference is accompanied by a dramatic decrease in the levels of NF90 and NF110. Reciprocally, depletion of NF90 but not of NF110 greatly reduces the level of NF45. Coregulation of NF90 and NF45 is a posttranscriptional phenomenon, resulting from protein destabilization in the absence of partners. Depletion of NF90-NF45 complexes retards cell growth by inhibition of DNA synthesis. Giant multinucleated cells containing nuclei attached by constrictions accumulate when either NF45 or NF90, but not NF110, is depleted. This study identified NF45 as an unstable regulatory subunit of NF90-NF45 complexes and uncovered their critical role in normal cell division. Furthermore, the study revealed that NF90 is functionally distinct from NF110 and is more important for cell growth.Human nuclear factor 90 (NF90) and nuclear factor 45 (NF45) were originally purified as a sequence-specific DNA binding complex regulating the interleukin-2 (IL-2) promoter (10, 17). NF90 is the founder member of a family of proteins generated from differentially spliced transcripts of the ILF3 gene (12). NF90 and NF110, which differ at their C termini, are the two most prominent ILF3 isoforms in cells (12,33,42,55). Both have been repeatedly isolated in diverse studies and have been given a variety of names. For example, MPP4 (M-phase phosphoprotein 4) is similar, if not identical, to NF90 and is phosphorylated during M phase (23), and closely related proteins 4F.1 and 4F.2 were characterized in Xenopus as doublestranded RNA (dsRNA)-binding proteins (3). NF90 is also known as DRBP76, NFAR1, and TCP80 (34, 43, 55), and NF110 is also known as ILF3, NFAR2, TCP110, and CBTF 122 (4,43,53,55). Underlining the importance of these proteins, knockout of the mouse ILF3 gene led to muscle degeneration, respiratory failure, and death soon after birth (44).NF90 and NF110 contain two dsRNA binding motifs (dsRBMs) which are responsible for their ability to interact with structured RNA. They also have an RGG domain that is capable of nucleic acid binding, and NF110 has an additional GQSY region that can interact with nucleic acids. Although characterized as DNA-binding proteins (17,36,40,41), NF90 and NF45 do not contain a recognized sequence-specific DNAbinding domain and the complex containing NF90 and NF45 does not appear to interact with DNA directly. NF90 and NF45 have been purified in complexes containing the Ku proteins and DNA-protein kinase (PK), as well as eukaryotic initiation factor 2 (eIF2), and it is likely ...

Upon DNA damage, p53-binding protein 1 (53BP1) relocalizes to sites of DNA double-strand breaks and forms discrete nuclear foci, suggesting its role in DNA damage responses. We show that 53BP1 changed its localization from the detergent soluble to insoluble fraction after treatment of cells with x-ray, but not with ultraviolet or hydroxyurea. Either DNase or phosphatase treatment of the insoluble fraction released 53BP1 into the soluble fraction, showing that 53BP1 binds to chromatin in a phosphorylation-dependent manner after X-irradiation of cells. 53BP1 was retained at discrete nuclear foci in X-irradiated cells even after detergent extraction of cells, showing that the chromatin binding of 53BP1 occurs at sites of DNA double-strand breaks. The minimal domain for focus formation was identified by immunofluorescence staining of cells ectopically expressed with 53BP1 deletion mutants. This domain consisted of conserved Tudor and Myb motifs. The Tudor plus Myb domain possessed chromatin binding activity in vivo and bound directly to both double-stranded and singlestranded DNA in vitro. This domain also stimulated endjoining by DNA ligase IV/Xrcc4, but not by T4 DNA ligase in vitro. We conclude that 53BP1 has the potential to participate directly in the repair of DNA double-strand breaks.

HIV exploits cellular proteins during its replicative cycle and latent infection. The positive transcription elongation factor, P-TEFb, is a key cellular transcription factor critical for these viral processes and is a drug target. During viral replication P-TEFb is recruited via interactions of its cyclin T1 subunit with the HIV Tat protein and TAR element. Through RNA silencing and over-expression experiments we discovered that nuclear factor 90 (NF90), a cellular RNA binding protein, regulates P-TEFb expression. NF90 depletion reduced cyclin T1 protein levels by inhibiting translation initiation. Regulation was mediated by the 3′UTR of cyclin T1 mRNA independently of microRNAs. Cyclin T1 induction is involved in the escape of HIV-1 from latency. We show that the activation of viral replication by phorbol ester in latently infected monocytic cells requires the posttranscriptional induction of NF90 and cyclin T1, implicating NF90 in protein kinase C (PKC) signaling pathways. This investigation reveals a novel mechanism of cyclin T1 regulation and establishes NF90 as a regulator of HIV-1 replication during both productive infection and induction from latency.

Nuclear factor 90 (NF90) and its C‐terminally extended isoform, NF110, belong to a family of nucleic acid binding proteins. NF90/110 are isolated with the less‐studied protein NF45, as complexes interacting with highly‐structured and double‐stranded RNAs. They have been implicated in the regulation of cellular and viral genes at several levels but it remains unknown whether NF90 and NF110 form functionally distinct complexes. We studied the composition of the complexes in vivo and used siRNA strategies to elucidate their function in cells.NF90 and NF110 form heterodimeric core complexes with NF45, and large in vivo complexes in which multiple NF90/110 isoforms interact with each other. NF45 knock‐down by RNA interference led to decreased expression of NF90 and NF110. This co‐regulation is at the protein level: the stability of NF90 family proteins is dependent on binding to their NF45 partner. Reciprocally, NF90 knock‐down reduced the expression of NF45 as a result of its accelerated degradation. Cell growth is retarded and giant multi‐nucleated cells accumulate when the expression of NF45 or NF90, but not NF110, is reduced in HeLa cells. The defect leaves nuclei attached by constrictions. Thus, NF90 family members form complexes inside cells whose stability is dependent on their binding to NF45. NF90 and NF45 are essential for cell growth and their depletion results in defects in nuclei division and cytokinesis.Support: NIH AI034552