No 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 conserved CDC5 family of Myb-related proteins performs an essential function in cell cycle control at G2͞M. Although c-Myb and many Myb-related proteins act as transcription factors, herein, we implicate CDC5 proteins in pre-mRNA splicing. Mammalian CDC5 colocalizes with pre-mRNA splicing factors in the nuclei of mammalian cells, associates with core components of the splicing machinery in nuclear extracts, and interacts with the spliceosome throughout the splicing reaction in vitro. Furthermore, genetic depletion of the homolog of CDC5 in Saccharomyces cerevisiae, CEF1, blocks the first step of pre-mRNA processing in vivo. These data provide evidence that eukaryotic cells require CDC5 proteins for pre-mRNA splicing. The Schizosaccharomyces pombe cdc5-120 mutant was isolated in a screen for mutants defective in cell cycle progression (1). At the restrictive temperature, cdc5-120 cells arrest growth in G 2 (1, 2), indicating that cdc5 ϩ function is required for G 2 ͞M progression. CDC5 has been conserved throughout evolution, and related genes have been cloned from Saccharomyces cerevisiae (termed CEF1; ref.3), Arabidopsis thaliana (4), Drosophila melanogaster (3), Caenorhabditis elegans (3), Xenopus laevis (5), and Homo sapiens (3, 6, 7). We conclude that these proteins are conserved functionally, because D. melanogaster and human CDC5 (hCDC5) complement the cdc5-120 mutant, S. cerevisiae CEF1 is essential during G 2 ͞M in this evolutionarily distinct yeast (3), and overexpression of dominant negative forms of hCDC5 slows G 2 progression in mammalian cells (8).In their N termini, CDC5 proteins are highly related to the DNA-binding domain of human c-Myb (2, 3, 9). Whereas human c-Myb contains three Myb repeats, Ϸ50-amino acid motifs with characteristic spacing of tryptophan residues (9), CDC5 proteins contain two Myb repeats (R1 and R2) followed by a Myb-likerepeat (MLR3) that contains some, but not all, of the hallmarks of a typical Myb repeat (3). Based on their homologies to c-Myb, CDC5 proteins were hypothesized to carry out their essential function in cell cycle control through transcriptional regulation, a notion supported by the following observations: (i) the Myb repeats of S. pombe cdc5p fused to glutathione S-transferasebound DNA cellulose (2); (ii) the Myb repeats of A. thaliana cdc5p selected a specific DNA sequence in a cyclic amplification and selection of targets protocol (4); and (iii) the C terminus of hCDC5 fused to the GAL4-DNA binding domain activated transcription in a reporter assay (8). To date, however, no downstream transcriptional targets for any of the CDC5 proteins have been identified. hCDC5 was identified recently in a biochemical purification of the mammalian spliceosome assembled in vitro (10), indicating that CDC5 proteins may be involved in pre-mRNA splicing rather than transcriptional regulation. Herein, we extend this observation by showing that mammalian CDC5 colocalizes with splicing factors in the nuclei of mammalian cells, coimmunoprecipitates with core components of th...

A series of in vitro protein-RNA binding studies using purified native (C1)3C2 and (A2)3B1 tetramers, total soluble heterogeneous nuclear ribonucleoprotein (hnRNP), and pre-mRNA molecules differing in length and sequence have revealed that a single C-protein tetramer has an RNA site size of230 to 240 nucleotides (nt). Two tetramers bind twice this RNA length, and three tetramers fold monoparticle lengths of RNA (700 nt assembly the 40S hnRNP core particle, and they provide insight into the mechanism through which the core proteins package 700-nt increments of RNA. These findings also demonstrate that unless excluded by other factors, the C proteins are likely to be located along the length of nascent transcripts.Whether released from isolated nuclei by sonic disruption or by low-salt extraction, the majority of the pre-mRNA molecules remain dispersed in solution following chromatin removal by brief centrifugation. Under conditions of minimal nuclease activity, 70 to 95% of this RNA is recovered in large ribonucleoprotein (RNP) complexes which sediment from 30S to more than 200S (26, 39, 52). Electron micrographs of the faster-sedimenting complexes reveal 20-to 25-nm particles arranged either as an array of polyparticles (29,36,42,43,52,53,61) or as clusters of particles when nuclease activity is aggressively inhibited (56). Upon mild nuclease activity the polyparticle complexes are lost and the cleaved RNA (mostly 500-to 1,000-nucleotide [nt] fragments) is recovered in 20-to 25-nm 30S-40S monoparticles (heterogeneous nuclear RNP [hnRNP] particles or ribonucleosomes) (3, 10, 18, 56, 64). Monoparticles purified from HeLa nuclei via glycerol gradients are primarily composed of six abundant nuclear proteins (the core particle proteins) (10, 28, 54, 65), which exist as three heterotypic tetramers, (A1)3B2, (A2)3B1, and (C1)3C2 (4, 7, 39). The (A2)3B1 and (C1)3C2 tetramers have been isolated and partially characterized (4, 7). The (A1)3B2 tetramer has not been isolated, but its existence is inferred from chemical cross-linking studies which reveal that Al exists in monoparticles as homotrimers (34, 41) and in a 3:1 ratio with B2. Like the histones (reviewed in reference 63), the core particle proteins are transcribed from multigene families (13,20,50) (11,17,35,38,46,65).Isolated 40S monoparticles completely dissociate upon RNA digestion with nuclease (22, 64). Spontaneous reassembly occurs in vitro upon the addition of 700 ± 20 nt of exogenous RNA or single-stranded DNA (22). Multiples of this length support the spontaneous in vitro assembly of dimers, trimers, and polyparticle complexes (22, 39). Reconstituted particles possess the same sedimentation coefficient, protein stoichiometry, chemical and UV cross-linking properties, pattern of salt-induced protein dissociation, nuclease sensitivity, and ultrastructural morphology as native hnRNP (22,27,64). Most of the noncore proteins present in initial hnRNP preparations do not quantitatively reconstitute with the core particle proteins (22, 64), a finding which indicat...

The major nuclear ribonucleoproteins (RNPs) involved in pre-mRNA processing are classified in broad terms either as small nuclear RNPs (snRNPs), which are major participants in the splicing reaction, or heterogeneous nuclear RNPs (hnRNPs), which traditionally have been thought to function in general pre-mRNA packaging. We obtained antibodies that recognize these two classes of RNP in Drosophila melanogaster. Using a sequential immunostaining technique to compare directly the distribution of these RNPs on Drosophila polytene chromosomes, we found that the two patterns were very similar qualitatively but not quantitatively, arguing for the independent deposition of the two RNP types and supporting a role for hnRNP proteins, but not snRNPs, in general transcript packaging.Both heterogeneous nuclear ribonucleoproteins (hnRNPs; reviewed in refs. 1 and 2) and small nuclear ribonucleoproteins (snRNPs; reviewed in ref.3) are deposited cotranscriptionally on eukaryotic RNA polymerase II transcripts (4-8). Whereas the major basic hnRNP proteins have been considered traditionally to function in general pre-mRNA packaging (2, 9), they have been proposed recently to be specific splicing cofactors or to be preferentially associated with splice junction sequences (10-15). snRNPs are major participants in the splicing reaction (3) but have been implicated recently in general packaging as part of a previously assembled unitary processing complex also containing hnRNPs (5, 6). The various proposals predict different amounts and ratios of the two protein types on nuclear pre-mRNA molecules at chromosomal sites of transcription, which is the issue we have addressed by sequential immunostaining.The core hnRNP proteins (A, B, and C proteins of 32-45 kDa) were originally identified as the major proteins that are associated with newly synthesized pre-mRNA (in the form of 30-50S RNP particles) when it is extracted from nuclei (reviewed in refs. 1 and 2). This observation, together with their nuclear abundance, their ability to bind single-stranded nucleic acids regardless of sequence, and their helixdestabilizing properties, led to the notion that these core hnRNP proteins are involved in general pre-mRNA packaging, much as the histones are involved in the general packaging of DNA (1, 2). However, more recent investigations of hnRNP proteins, using in vitro splicing or in vitro RNA binding studies, have suggested that these proteins play a role in the splicing reaction (10-12), that they bind with high affinity to sequences at 3' splice sites (13,14), and that they are dependent on snRNPs for acquisition of a crosslinkable association with RNA (13). These in vitro studies have led to a reappraisal of the independent structural role of hnRNP proteins in pre-mRNA packaging towards a view that they are a few of the many required cofactors for splicing. The simplest version of this view would predict a constant stoichiometry of snRNPs and the core hnRNP proteins on pre-mRNA, in amounts that correlate with the number of splicing sign...

No abstract

The M2 pyruvate kinase (PKM2) isoform is upregulated in most cancers and plays a crucial role in regulation of the Warburg effect, which is characterized by the preference for aerobic glycolysis over oxidative phosphorylation for energy metabolism. PKM2 is an alternative-splice isoform of the PKM gene and is a potential therapeutic target. Antisense oligonucleotides (ASO) that switch PKM splicing from the cancer-associated PKM2 to the PKM1 isoform have been shown to induce apoptosis in cultured glioblastoma cells when delivered by lipofection. Here, we explore the potential of ASO-based PKM splice switching as a targeted therapy for liver cancer. A more potent lead constrained-ethyl (cEt)/DNA ASO induced PKM splice switching and inhibited the growth of cultured hepatocellular carcinoma (HCC) cells. This PKM isoform switch increased pyruvate-kinase activity and altered glucose metabolism. In an orthotopic HCC xenograft mouse model, the lead ASO and a second ASO targeting a nonoverlapping site inhibited tumor growth. Finally, in a genetic HCC mouse model, a surrogate mouse-specific ASO induced Pkm splice switching and inhibited tumorigenesis, without observable toxicity. These results lay the groundwork for a potential ASO-based splicing therapy for HCC. Significance: Antisense oligonucleotides are used to induce a change in PKM isoform usage in hepatocellular carcinoma, reversing the Warburg effect and inhibiting tumorigenesis.

We have determined the DNA sequence surrounding the transcription terminator following rpoC, the gene that codes for the beta' subunit of RNA polymerase in E. coli K12. The 2044 bp sequence obtained contains the distal 335 codons of rpoC followed by a 212 bp non-coding region and a second open reading frame (ORFa) of 179 codons. The final 181 nucleotides of the sequence form the 5' end of a third open reading frame (ORFb). The in vivo 3' end of the rpoC mRNA was located by analysis of RNA/DNA hybrids cleaved with nuclease S1 (S1 mapping). These results indicated that the major transcription termination of the rplJL-rpoBC transcription unit occurs a short distance past the translation stop codon for rpoC. Four regions of symmetry, suggesting secondary structure in the mRNA, were found in the DNA sequence near the rpoC translation termination codon. The last of these hairpin structures is similar to other rho-independent transcription terminators and its 3' end coincides with the end of the rpoC mRNA as predicted by S1-mapping. Inspection of the open reading frames indicates that rpoC uses a high percentage of codons that are recognized by the major tRNA species of E. coli while ORFa and ORFb contain many codons recognized by minor tRNA species. ORFa specifies a very basic peptide.