The Intracisternal A-Particle Proximal Enhancer-Binding Protein Activates Transcription and Is Identical to the RNA- and DNA-Binding Protein p54^nrb/NonO

Abstract

The long terminal repeats of murine intracisternal A particles (IAPs) contain an IAP proximal enhancer (IPE) element that is inactive in murine F9 embryonal carcinoma cells and active in the parietal endoderm cell line PYS-2. The element binds efficiently to a 60-kDa IPE-binding protein (IPEB) present in PYS-2 cells but poorly to F9 proteins, suggesting a role for IPEB in regulating IAP expression. We have purified calf thymus IPEB, which binds to the IPE and transactivates a reporter gene in HeLa cell extracts. Based on the peptide sequence of the purified calf IPEB, we have cloned a 420-bp cDNA and showed that the encoded protein is the homolog of human p54 nrb and mouse NonO, which are characterized by the presence of two RNA recognition motifs. We show that p54 nrb is an IPE-binding transcription activator with its DNA-binding and activation domains in the N-and C-terminal halves, respectively. The activation domain of p54 nrb is active in HeLa, PYS-2, and F9 cells, whereas p54 nrb as a whole molecule is active in HeLa and PYS-2 cells but not in F9 cells. Thus, the lack of activity of p54 nrb in F9 cells is due to an ineffective DNA-binding domain. We demonstrate that p54 nrb also binds to a pre-mRNA. Based on the close sequence relatedness of this protein to PSF, which is required for pre-mRNA splicing in vitro, we discuss the possibility that p54 nrb has dual roles in transcription and splicing.Murine intracisternal A particles (IAPs) are defective endogenous retroviruses encoded by proviral elements that are reiterated 2,000 times and dispersed throughout the genome. IAPs are constitutively expressed at high levels in many mouse tumors and at basal levels in most normal adult mouse tissues (reviewed in references 21 and 22). In tumor cells where IAPs are actively expressed, these elements transpose and act as insertional mutagens via integration of actively synthesizing extrachromosomal viral DNA to new sites in the host genome. Such transpositions induce aberrant expression of target genes, contributing to augmented growth autonomy of the host cell and thus to the process of neoplastic transformation (21,22,25). Several germ line insertions contributing to mutagenesis have also been found (7,22). Thus, investigation of the mechanism of IAP transcriptional activation should ultimately lead to an understanding of the role of endogenous retroviruses in transposition-induced mutagenesis.IAP proviral elements contain in their long terminal repeats (LTRs) all of the cis-acting elements required for the enhancement of IAP gene transcription (8,23,26). The cis elements and their interacting transcription factors identified so far include the Enh1 and Enh2 elements located between nucleotides (nt) Ϫ164 and Ϫ110, which bind to EBP-80 of murine myeloma cells (13,14), and sites located at the 5Ј end of the LTR between nt Ϫ210 and Ϫ168, which bind to 28-and 46-kDa proteins of murine PCC3 embryonal carcinoma (EC) cells (40). In our analyses of the mechanism by which IAP expression becomes activated, we have found that...

The Bacterial Histone-like Protein HU Specifically Recognizes Similar Structures in All Nucleic Acids

“…Interestingly, in parallel to this work it was shown that HBsu, the HU protein of Bacillus subtilis, specifically binds the Alu domain of a small cytoplasmic RNA (scRNA), a homologue of mammalian signal recognition particle RNA (24). In the eukaryotic field, a growing body of evidence shows that a number of proteins including transcriptional factors containing zinc finger or RNA recognition motifs are able to bind specifically to both DNA and RNA (25)(26)(27)(28)(29)(30)(31)(32)(33).…”

PSF and p54nrb bind a conserved stem in U5 snRNA

“…PSF and p54 nrb associate with U4/U6+U5 tri-snRNP and splicing complexes+ Two hundred-microliter splicing reactions using AdML pre-mRNA were separated on 10%-30% sucrose gradients+ RNAs from each fraction (numbered) were recovered by phenol/CHCl 3 extraction and separated on 8 M urea-15% PAGE+ Splicing products and snRNAs were visualized by phosphorimager analysis (A) and silver staining (B), respectively+ Splicing RNAs and snRNAs are indicated on the right+ The position of U4/U6+U5 tri-snRNP is shown+ C: Proteins from each fraction above (except fractions 8 and 10) were analyzed by western blot analysis using different antibodies as indicated+ We have demonstrated that PSF interacts with p54 nrb and that both of these proteins, individually and in combination, select the same optimal RNA-binding sequence from random pools of RNA+ PSF and p54 nrb share 71% identity over a 320-amino-acid region encompassing their RRMs (Dong et al+, 1993) and multiple functions have been ascribed to each+ PSF copurifies with U4/U6+U5 tri-snRNP preparations (Teigelkamp et al+, 1997) and a variety of biochemical experiments indicate that PSF plays an important role in pre-mRNA splicing (Patton et al+, 1993;Gozani et al+, 1994)+ In addition, roles for PSF in transcription, topoisomerase activity, nuclear RNA retention, and DNA recombination have also been postulated (Straub et al+, 1998;Akhmedov & Lopez, 2000;Straub et al+, 2000;Urban et al+, 2000;Mathur et al+, 2001;Zhang & Carmichael, 2001;Sewer et al+, 2002)+ Similarly, multiple functions involving both RNA and DNA binding have been proposed for p54 nrb (Zhang et al+, 1993;Basu et al+, 1997;Straub et al+, 1998Straub et al+, , 2000Zhang & Carmichael, 2001;Sewer et al+, 2002)+ Several lines of evidence indicate that p54 nrb is also involved in splicing+ First, p54 nrb was originally isolated in screens designed to identify proteins that cross-react with antibodies against Prp18, a yeast second-step splicing factor associated with U4/ U6+U5 tri-snRNP and U5 snRNP (Vijayraghavan & Abelson, 1990;Dong et al+, 1993;Horowitz & Abelson, 1993)+ Second, GFP fusions and antibodies raised against p54 nrb have been used to demonstrate localization to nuclei in a speckled pattern albeit somewhat more diffuse than some splicing factors (not shown)+ Third, overexpression of Spi-1/PU+1, an Ets-related transcription factor, blocks p54 nrb RNA binding, which correlates with an effect on in vitro splicing (Hallier et al+, 1996)+ Last, in this study, PSF and p54 nrb were found to cosediment with splicing complexes (Fig+ 6)+ As to how these two proteins might function in splicing, early experiments showed that immunodeplet...…”

PRCC, the commonest TFE3 fusion partner in papillary renal carcinoma is associated with pre-mRNA splicing factors

“…This striking degree of homology suggested that NonO was also a splicing factor and indeed it has been shown that its depletion from nuclear extract inhibits b-globin splicing in vitro (Hallier et al, 1998). NonO may have a role in transcriptional activation (Basu et al, 1997) and was also shown to bind PU.1 an ETS family protein that can alter the splicing activity of NonO in vitro (Hallier et al, 1996).…”