Nuclear Factor 45 (NF45) Is a Regulatory Subunit of Complexes with NF90/110 Involved in Mitotic Control

Abstract

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 ...

Characterization of proteins associating with 5’ terminus of PGHS-1 mRNA

“…However, validation of LC-MS results by immunoprecipitation experiment demonstrates the association between nucleolin, serpin B1, NF45 and NF90 with PGHS-1 mRNA. Previous studies have listed nucleolin, NF90 and NF45 as members of the same protein complex in HeLa S3 and HEK 293 cells [39]. Our data do not Brought to you by | MIT Libraries Authenticated Download Date | 5/10/18 1:41 AM allow for concluding if one or more complexes are formed between the PGHS-1 mRNA sequence and the identified proteins or whether these proteins bind individually to PGHS-1 mNRA.…”

“…These effects of NF90 are consistent with an earlier report showing that the alternatively spliced form of NF90, TCP80, suppresses the translation of beta-glucosidase mRNA [43] suggesting a broad role of NF90 in translational repression. More recently, experiments aiming at depleting NF45 or NF90 in HeLa cells resulted in a mitotic defect resulting in an accumulation of multinucleated cells [39]. Interestingly, megakaryocytes, used in our study, are notorious for their polyploidy state during their differentiation process, with ploidity sometimes reaching 64 [44].…”

“…It is suggested that NF45 acts as a regulator of NF90 activity [28]. Originally identified as DNA-binding proteins [26,36,37], NF90 and NF45 do not contain a recognized sequence-specific DNA-binding domain and the complex does not appear to interact with DNA directly [26,39]. Nevertheless, NF45 and NF90 were shown to bind to dsRNA as well as to proteins [26,39].…”

“…Originally identified as DNA-binding proteins [26,36,37], NF90 and NF45 do not contain a recognized sequence-specific DNA-binding domain and the complex does not appear to interact with DNA directly [26,39]. Nevertheless, NF45 and NF90 were shown to bind to dsRNA as well as to proteins [26,39]. In neuronal cells, the NF45/DRBP76 heterodimer complex can inhibit translational initiation by binding to the 5'UTR of human rhinovirus type 2 that encoding the internal ribosome entry site (IRES) [40].…”

“…The absence of nucleolin protein in the immunoprecipitate obtained with anti-NF45 antibody could indicate an indirect or a weak interaction between those two proteins. Data from other laboratories indicate that NF45 associates with nucleolin through NF90, which is a common complex partner of NF45 and also binds to nucleolin [39]. Dissecting the nature of complex composition and association between RNA binding proteins requires extensive experimentations.…”