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Topoisomerase IIalpha interacts with numerous nuclear factors, through which it is engaged in diverse nuclear events such as DNA replication, transcription and the formation or maintenance of heterochromatin. We previously reported that topoisomerase IIalpha interacts with RNA helicase A (RHA), consistent with a recent view that topoisomerases and helicases function together. Intrigued by our observation that the RHA-topoisomerase IIalpha interaction is sensitive to ribonuclease A, we explored whether the RHA-topoisomerase IIalpha interaction can be recapitulated in vitro using purified proteins and a synthetic RNA. This work led us to an unexpected finding that an RNA-binding activity is intrinsically associated with topoisomerase IIalpha. Topoisomerase IIalpha stably interacted with RNA harboring a 3'-hydroxyl group but not with RNA possessing a 3'-phosphate group. When measured in decatenation and relaxation assays, RNA binding influenced the catalytic function of topoisomerase IIalpha to regulate DNA topology. We discuss a possible interaction of topoisomerase IIalpha with the poly(A) tail and G/U-rich 3'-untranslated region (3'-UTR) of mRNA as a key step in transcription termination.

Ribonucleoprotein complexes (RNPs) perform a multitude of functions in the cell. Elucidating the composition of such complexes and unraveling their many interactions are current challenges in molecular biology. To stabilize complexes formed in cells and to preclude reassortment of their components during isolation, we employ chemical crosslinking of the RNA and protein moieties. Here we describe the identification of cellular RNAs bound to nuclear factor 90 (NF90), the founder member of a family of ubiquitous double-stranded RNA-binding proteins. Crosslinked RNA-NF90 complexes were immunoprecipitated from stable cell lines containing epitope-tagged NF90 protein isoforms. The bound RNA was released and identified through RNase H digestion and by various gene amplification techniques. We appraise the methods used by altering crosslinking conditions, and the binding profiles of different NF90 protein isoforms in synchronized and asynchronous cells are compared. This study discovers two novel RNA species and establishes NF90 as a multiclass RNA-binding protein, capable of binding representatives of all three classes of RNA.

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.

Members of the nuclear factor 90 (NF90) family of double-stranded RNA (dsRNA)-binding proteins have been implicated in several biological processes including the regulation of gene expression. cDNA sequences predict that the proteins have a functional nuclear localization signal and two dsRNA-binding motifs (dsRBMs), and are identical at their N termini. Isoforms are predicted to diverge at their C termini as well as by the insertion of four amino acid residues (NVKQ) between the two dsRBMs. In this study, we verified the expression of four of the isoforms by cDNA cloning and mass spectrometric analysis of proteins isolated from human cells. Cell fractionation studies showed that NF90 and its heteromeric partner, NF45, are predominantly nuclear and largely chromatin-associated. The C-terminally extended NF90 species, NF110, are almost exclusively chromatin-bound. Both NF110 isoforms are more active than NF90 isoforms in stimulating transcription from the proliferating cell nuclear antigen reporter in a transient expression system. NF110b, which carries the NVKQ insert, was identified as the strongest activator. It stimulated transcription of some, but not all, promoters in a fashion that suggested that it functions in concert with other transcription factors. Finally, we demonstrate that NF110b associates with the dsRBM-containing transcriptional co-activator, RNA helicase A, independently of RNA binding.