Biomedical subjects

Eric J Wagner

Publications and source records attributed to Eric J Wagner.

15 recordsLinked to original sources

PubMed2006

Nuclear autoantigenic sperm protein (NASP), a linker histone chaperone that is required for cell proliferation.

A multichaperone nucleosome-remodeling complex that contains the H1 linker histone chaperone nuclear autoantigenic sperm protein (NASP) has recently been described. Linker histones (H1) are required for the proper completion of normal development, and NASP transports H1 histones into nuclei and exchanges H1 histones with DNA. Consequently, we investigated whether NASP is required for normal cell cycle progression and development. We now report that without sufficient NASP, HeLa cells and U2OS cells are unable to replicate their DNA and progress through the cell cycle and that the NASP(-/-) null mutation causes embryonic lethality. Although the null mutation NASP(-/-) caused embryonic lethality, null embryos survive until the blastocyst stage, which may be explained by the presence of stored NASP protein in the cytoplasm of oocytes. We conclude from this study that NASP and therefore the linker histones are key players in the assembly of chromatin after DNA replication.

Animals↗

PubMed2006

Conserved zinc fingers mediate multiple functions of ZFP100, a U7snRNP associated protein.

Formation of the 3' end of replication-dependent histone mRNAs is most robust during S phase and is mediated by both the stem-loop binding protein (SLBP) and the U7 snRNP. We previously identified a 100-kDa zinc finger protein (ZFP100) as a component of U7 snRNP that interacts with the SLBP/pre-mRNA complex. Here, we show that myc- or GFP-tagged ZFP100 overexpressed after transfection is concentrated in Cajal bodies (CBs), and unlike components of the spliceosomal snRNPs, photobleaching experiments demonstrate that ZFP100 is stably associated with CBs. Of the 18 zinc fingers contained within ZFP100, the region encompassing fingers 2-6 is sufficient to maintain CB localization. Zn fingers 5-10 are required for maximal binding of ZFP100 to a 20-amino-acid region of Lsm11, a U7 snRNP core protein. Expression of ZFP100 stimulates histone mRNA processing in vivo, assayed by activation of a reporter gene that encodes a GFP mRNA ending in a histone 3' end. Importantly, the domain that is required for CB localization and Lsm11 binding is also sufficient to stimulate histone pre-mRNA processing in vivo. Comparisons with other mammalian ZFP100 orthologs show that the central Zn fingers sufficient for in vivo activity are most highly conserved, whereas the number and sequence of the Zn fingers in the N- and C-terminal domains vary.

Amino Acid Sequence↗

PubMed2006

ZFP100, a component of the active U7 snRNP limiting for histone pre-mRNA processing, is required for entry into S phase.

Metazoan replication-dependent histone mRNAs are the only eukaryotic mRNAs that are not polyadenylated. The cleavage of histone pre-mRNA to form the unique 3' end requires the U7 snRNP and the stem-loop binding protein (SLBP) that binds the 3' end of histone mRNA. U7 snRNP contains three novel proteins, Lsm10 and Lsm11, which are part of the core U7 Sm complex, and ZFP100, a Zn finger protein that helps stabilize binding of the U7 snRNP to the histone pre-mRNA by interacting with the SLBP/pre-mRNA complex. Using a reporter gene that encodes a green fluorescent protein mRNA ending in a histone 3' end and mimics histone gene expression, we demonstrate that ZFP100 is the limiting factor for histone pre-mRNA processing in vivo. The overexpression of Lsm10 and Lsm11 increases the cellular levels of U7 snRNP but has no effect on histone pre-mRNA processing, while increasing the amount of ZFP100 increases histone pre-mRNA processing but has no effect on U7 snRNP levels. We also show that knocking down the known components of U7 snRNP by RNA interference results in a reduction in cell growth and an unsuspected cell cycle arrest in early G(1), suggesting that active U7 snRNP is necessary to allow progression through G(1) phase to S phase.

Animals↗

PubMed2005

Nuclear import of the stem-loop binding protein and localization during the cell cycle.

A key factor involved in the processing of histone pre-mRNAs in the nucleus and translation of mature histone mRNAs in the cytoplasm is the stem-loop binding protein (SLBP). In this work, we have investigated SLBP nuclear transport and subcellular localization during the cell cycle. SLBP is predominantly nuclear under steady-state conditions and localizes to the cytoplasm during S phase when histone mRNAs accumulate. Consistently, SLBP mutants that are defective in histone mRNA binding remain nuclear. As assayed in heterokaryons, export of SLBP from the nucleus is dependent on histone mRNA binding, demonstrating that SLBP on its own does not possess any nuclear export signals. We find that SLBP interacts with the import receptors Impalpha/Impbeta and Transportin-SR2. Moreover, complexes formed between SLBP and the two import receptors are disrupted by RanGTP. We have further shown that SLBP is imported by both receptors in vitro. Three sequences in SLBP required for Impalpha/Impbeta binding were identified. Simultaneous mutation of all three sequences was necessary to abolish SLBP nuclear localization in vivo. In contrast, we were unable to identify an in vivo role for Transportin-SR2 in SLBP nuclear localization. Thus, only the Impalpha/Impbeta pathway contributes to SLBP nuclear import in HeLa cells.

Alanine↗

PubMed2005

Characterization of the intronic splicing silencers flanking FGFR2 exon IIIb.

The cell type-specific alternative splicing of FGFR2 pre-mRNA results in the mutually exclusive use of exons IIIb and IIIc, which leads to critically important differences in receptor function. The choice of exon IIIc in mesenchymal cells involves activation of this exon and repression of exon IIIb. This repression is mediated by the function of upstream and downstream intronic splicing silencers (UISS and DISS). Here we present a detailed characterization of the determinants of silencing function within UISS and DISS. We used a systematic mutational analysis, introducing deletions and substitutions to define discrete elements within these two silencers of exon IIIb. We show that UISS requires polypyrimidine tract-binding protein (PTB)-binding sites, which define the UISS1 sub-element, and an eight nucleotide sequence 5'-GCAGCACC-3' (UISS2) that is also required. Even though UISS2 does not bind PTB, the full UISS can be replaced with a synthetic silencer designed to provide optimal PTB binding. DISS is composed of a 5'-conserved sub-element (5'-CE) and two regions that contain multiple PTB sites and are functionally redundant (DISS1 and DISS2). DISS1 and DISS2 are separated by the activator sequence IAS2, and together these opposing elements form the intronic control element. Deletion of DISS in the FGFR2 exon IIIb context resulted in the near full inclusion of exon IIIb, and insertion of this silencer downstream of a heterologous exon with a weak 5' splice site was capable of repressing exon inclusion. Extensive deletion analysis demonstrated that the majority of silencing activity could be mapped to the conserved octamer CUCGGUGC within the 5'CE. Replacement of 5'CE and DISS1 with PTB-binding elements failed to restore repression of exon IIIb. We tested the importance of the relative position of the silencers and of the subelements within each silencer. Whereas UISS1, UISS2, DISS1, and DISS2 appear somewhat malleable, the 5'CE is rigid in terms of relative position and redundancy. Our data defined elements of function within the ISSs flanking exon IIIb and suggested that silencing of this exon is mediated by multiple trans-acting factors.

Alternative Splicing↗

PubMed2005

The polypyrimidine tract binding protein is required for efficient picornavirus gene expression and propagation.

Mammalian host factors required for efficient viral gene expression and propagation have been often recalcitrant to genetic analysis. A case in point is the function of cellular factors that trans-activate internal ribosomal entry site (IRES)-driven translation, which is operative in many positive-stranded RNA viruses, including all picornaviruses. These IRES trans-acting factors have been elegantly studied in vitro, but their in vivo importance for viral gene expression and propagation has not been widely confirmed experimentally. Here we use RNA interference to deplete mammalian cells of one such factor, the polypyrimidine tract binding protein, and test its requirement in picornavirus gene expression and propagation. Depletion of the polypyrimidine tract binding protein resulted in a marked delay of particle propagation and significantly decreased synthesis and accumulation of viral proteins of poliovirus and encephalomyocarditis virus. These effects could be partially restored by expression of an RNA interference-resistant exogenous polypyrimidine tract binding protein. These data indicate a critical role for the polypyrimidine tract binding protein in picornavirus gene expression and strongly suggest a requirement for efficient IRES-dependent translation.

DNA-Binding Proteins↗

PubMed2005

A CPSF-73 homologue is required for cell cycle progression but not cell growth and interacts with a protein having features of CPSF-100.

Formation of the mature 3' ends of the vast majority of cellular mRNAs occurs through cleavage and polyadenylation and requires a cleavage and polyadenylation specificity factor (CPSF) containing, among other proteins, CPSF-73 and CPSF-100. These two proteins belong to a superfamily of zinc-dependent beta-lactamase fold proteins with catalytic specificity for a wide range of substrates including nucleic acids. CPSF-73 contains a zinc-binding histidine motif involved in catalysis in other members of the beta-lactamase superfamily, whereas CPSF-100 has substitutions within the histidine motif and thus is unlikely to be catalytically active. Here we describe two previously unknown human proteins, designated RC-68 and RC-74, which are related to CPSF-73 and CPSF-100 and which form a complex in HeLa and mouse cells. RC-68 contains the intact histidine motif, and hence it might be a functional counterpart of CPSF-73, whereas RC-74 lacks this motif, thus resembling CPSF-100. In HeLa cells RC-68 is present in both the cytoplasm and the nucleus whereas RC-74 is exclusively nuclear. RC-74 does not interact with CPSF-73, and neither RC-68 nor RC-74 is found in a complex with CPSF-160, indicating that these two proteins form a separate entity independent of the CPSF complex and are likely involved in a pre-mRNA processing event other than cleavage and polyadenylation of the vast majority of cellular pre-mRNAs. RNA interference-mediated depletion of RC-68 arrests HeLa cells early in G(1) phase, but surprisingly the arrested cells continue growing and reach the size typical of G(2) cells. RC-68 is highly conserved from plants to humans and may function in conjunction with RC-74 in the 3' end processing of a distinct subset of cellular pre-mRNAs encoding proteins required for G(1) progression and entry into S phase.

Amino Acid Sequence↗

PubMed2004

Autoregulation of polypyrimidine tract binding protein by alternative splicing leading to nonsense-mediated decay.

Polypyrimdine tract binding protein (PTB) is a regulator of alternative splicing, mRNA 3' end formation, mRNA stability and localization, and IRES-mediated translation. Transient overexpression of PTB can influence alternative splicing, sometimes resulting in nonphysiological splicing patterns. Here, we show that alternative skipping of PTB exon 11 leads to an mRNA that is removed by NMD and that this pathway consumes at least 20% of the PTB mRNA in HeLa cells. We also show that exon 11 skipping is itself promoted by PTB in a negative feedback loop. This autoregulation may serve both to prevent disruptively high levels of PTB expression and to restore nuclear levels when PTB is mobilized to the cytoplasm. Our findings suggest that alternative splicing can act not only to generate protein isoform diversity but also to quantitatively control gene expression and complement recent bioinformatic analyses, indicating a high prevalence of human alternative splicing leading to NMD.

Alternative Splicing↗

PubMed2004

Imaging alternative splicing in living cells.

We have developed an in vivo reporter of alternative splicing decisions that allows for the determination of FGF-R2 splicing patterns without the destruction of cells. This method has broad applications, including the study of other alternatively spliced genes in tissue culture and in whole animals, and may be useful in creating imaging markers for the study of tumor progression and metastasis. In this chapter, the authors present one example of this method using fluorescence reporters. As with any new assay, a series of experiments were performed to validate the method. This chapter documents some of these experiments.

Alternative Splicing↗

PubMed2003

Physical and chemical effects on viability of the Myxobolus cerebralis triactinomyxon.

Various chemical and physical methods for destroying the triactinomyxon (TAM) stage of the myxozoan parasite Myxobolus cerebralis were tested. The fluorescent stains propidium iodide and fluorescein diacetate were used as indicators of viability. Physical variables tested included freezing, drying, high temperature, sonication, and pressure of 6.2 x 10(7) Pa (9000 psi). Chemicals evaluated included chlorine bleach, povidone-iodine, and hydrogen peroxide. Freezing or drying for 1 h was effective in killing TAMs, but pressure was not. Temperatures above 75 degrees C for at least 5 min were also effective. Sonication with a laboratory instrument cleaner for 10 to 13 min killed and ruptured TAMs, resulting in <1.9% recovery. However, among the surviving TAMs, 39 to 58% were still viable. Chlorine concentrations of 130 ppm for 10 min were also effective at temperatures ranging from ice-water to room temperature and total hardness ranging from 10 to 500 mg l(-1). Lethal concentrations of hydrogen peroxide and povidone-iodine (10% solution) were quite high: 10% for 10 min, and 50% (5000 ppm active iodine) for 60 min, respectively. The stain results indicating TAM death were verified in 2 tests in which rainbow trout Oncorhynchus mykiss were exposed to TAMs that had been either frozen for 1 h or treated with 66 ppm chlorine as sodium hypochlorite for 1 min. None of the fish exposed to the treated TAMs became infected. These results should provide disinfection guidelines to prevent transfer of M. cerebralis TAMs to uninfected areas and provide information on the risks of parasite transfer under various treatment scenarios.

Animals↗

PubMed2003

A stem structure in fibroblast growth factor receptor 2 transcripts mediates cell-type-specific splicing by approximating intronic control elements.

Alternative splicing of fibroblast growth factor receptor 2 (FGFR2) occurs in a cell-type-specific manner with the mutually exclusive use of exon IIIb or exon IIIc. Specific inclusion of exon IIIb is observed in epithelial cells, whereas exon IIIc inclusion is seen in mesenchymal cells. Epithelium-specific activation of exon IIIb and repression of exon IIIc are coordinately regulated by intronic activating sequence 2 (IAS2) and intronic splicing activator and repressor (ISAR) elements in FGFR2 pre-mRNA. Previously, it has been suggested that IAS2 and a 20-nucleotide core sequence of ISAR form a stem structure that allows for the proper regulation of FGFR2 alternative splicing. Replacement of IAS2 and the ISAR core with random sequences capable of stem formation resulted in the proper activation of exon IIIb and repression of exon IIIc in epithelial cells. Given the high degree of phylogenetic conservation of the IAS2-ISAR core structure and the fact that unrelated stem-forming sequences could functionally substitute for IAS2 and ISAR elements, we postulated that the stem structure facilitated the approximation of intronic control elements. Indeed, deletion of the entire stem-loop region and juxtaposition of sequences immediately upstream of IAS2 with sequences immediately downstream of the ISAR core maintained proper cell-type-specific inclusion of exon IIIb. These data demonstrate that IAS2 and the ISAR core are dispensable for the cell-type-specific activation of exon IIIb; thus, the major, if not the sole, role of the IAS2-ISAR stem in exon IIIb activation is to approximate sequences upstream of IAS2 with sequences downstream of the ISAR core. The downstream sequence is very likely a highly conserved GCAUG element, which we show was required for efficient exon IIIb activation.

Alternative Splicing↗

PubMed2003

Of urchins and men: evolution of an alternative splicing unit in fibroblast growth factor receptor genes.

Alternative splicing of mammalian transcripts, which yields many diverse protein products from one gene, is the rule and not the exception. Although the mechanisms that govern alternative splicing are being unraveled, little is known about the evolution of this critical engine of proteome diversity. Here we present a phylogenetic analysis from a sea urchin to humans of the alternative splicing unit encoding the third Ig domain of fibroblast growth factor receptors. The remarkable conservation of intronic control elements, both in structure and function, indicates that the mechanisms that regulate this alternative splicing unit evolved over 600 million years ago.

Alternative Splicing↗

PubMed2003

Quantification of alternatively spliced FGFR2 RNAs using the RNA invasive cleavage assay.

The regulated splicing of fibroblast growth factor receptor-2 (FGFR2) transcripts leads to tissue-specific expression of distinct receptor isoforms. These isoforms contain two different versions of the ligand binding Ig-like domain III, which are encoded by exon IIIb or exon IIIc. The mutually exclusive use of exon IIIb and exon IIIc can be recapitulated in tissue culture using DT3 and AT3 rat prostate carcinoma cells. We used this well-characterized system to evaluate the precision and accuracy of the RNA invasive cleavage assay to specifically measure FGFR2 alternative splicing outcomes. Experiments presented here demonstrated that the RNA invasive cleavage assay could specifically detect isoforms with discrimination levels that ranged from 1 in 5 x 10(3) to 1 in 10(5). Moreover the assay could detect close to 0.01 amole of FGFR2 RNAs. The assay detected the expected levels of transcripts containing either exon IIIb or IIIc, but, surprisingly, it detected high levels of IIIb-IIIc double inclusion transcripts. This finding, which has important implications for the role of exon silencing and of mRNA surveillance mechanisms, had been missed by RT-PCR. Additionally, we used the RNA invasive cleavage assay to demonstrate a novel function for the regulatory element IAS2 in repressing exon IIIc inclusion. We also show here that purification of RNA is not necessary for the invasive cleavage assay, because crude cell lysates could be used to accurately measure alternative transcripts. The data presented here indicate that the RNA invasive cleavage assay is an important addition to the repertoire of techniques available for the study of alternative splicing.

Alternative Splicing↗

PubMed2002

Both natural and designed micro RNAs can inhibit the expression of cognate mRNAs when expressed in human cells.

Animal cells have recently been shown to express a range of approximately 22 nucleotide noncoding RNAs termed micro RNAs (miRNAs). Here, we show that the human mir-30 miRNA can be excised from irrelevant, endogenously transcribed mRNAs encompassing the predicted 71 nucleotide mir-30 precursor. Expression of the mir-30 miRNA specifically blocked the translation in human cells of an mRNA containing artificial mir-30 target sites. Similarly, designed miRNAs were also excised from transcripts encompassing artificial miRNA precursors and could inhibit the expression of mRNAs containing a complementary target site. These data indicate that novel miRNAs can be readily produced in vivo and can be designed to specifically inactivate the expression of selected target genes in human cells.

Animals↗

PubMed2002

RNAi-mediated PTB depletion leads to enhanced exon definition.

Mutually exclusive use of exons IIIb or IIIc in FGF-R2 transcripts requires the silencing of exon IIIb. This repression is mediated by silencer elements upstream and downstream of the exon. Both silencers bind the polypyrimidine tract binding protein (PTB) and PTB binding sites within these elements are required for efficient silencing of exon IIIb. Recruitment of MS2-PTB fusion proteins upstream or downstream of exon IIIb causes repression of this exon. Depletion of endogenous PTB using RNAi increases exon IIIb inclusion in transcripts derived from minigenes and from the endogenous FGF-R2 gene. These data demonstrate that PTB is a negative regulator of exon definition in vivo.

Blotting, Western↗