While searching for a human homolog of the S.cerevisiae splicing factor PRP18, we found a polypeptide that reacted strongly with antibodies against PRP18. We purified this polypeptide from HeLa cells using a Western blot assay, and named it p54nrb (for nuclear RNA-binding protein, 54 kDa). cDNAs encoding p54nrb were cloned with probes derived from partial sequence of the purified protein. These cDNAs have identical coding sequences but differ as a result of alternative splicing in the 5' untranslated region. The cDNAs encode a 471 aa polypeptide that contains two RNA recognition motifs (RRMs). Human p54nrb has no homology to yeast PRP18, except for a common epitope, but is instead 71% identical to human splicing factor PSF within a 320 aa region that includes both RRMs. In addition, both p54nrb and PSF are rich in Pro and Gln residues outside the main homology region. The Drosophila puff-specific protein BJ6, one of three products encoded by the alternatively spliced no-on-transient A gene (nonA), which is required for normal vision and courtship song, is 42% identical to p54nrb in the same 320 aa region. The striking homology between p54nrb, PSF, and NONA/BJ6 defines a novel phylogenetically conserved protein segment, termed DBHS domain (for Drosophila behavior, human splicing), which may be involved in regulating diverse pathways at the level of pre-mRNA splicing.
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...
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