Biomedical subjects

Michael Antoniou

Publications and source records attributed to Michael Antoniou.

15 recordsLinked to original sources

PubMed2006

An interaction between U2AF 65 and CF I(m) links the splicing and 3' end processing machineries.

The protein factor U2 snRNP Auxiliary Factor (U2AF) 65 is an essential component required for splicing and involved in the coupling of splicing and 3' end processing of vertebrate pre-mRNAs. Here we have addressed the mechanisms by which U2AF 65 stimulates pre-mRNA 3' end processing. We identify an arginine/serine-rich region of U2AF 65 that mediates an interaction with an RS-like alternating charge domain of the 59 kDa subunit of the human cleavage factor I (CF I(m)), an essential 3' processing factor that functions at an early step in the recognition of the 3' end processing signal. Tethered functional analysis shows that the U2AF 65/CF I(m) 59 interaction stimulates in vitro 3' end cleavage and polyadenylation. These results therefore uncover a direct role of the U2AF 65/CF I(m) 59 interaction in the functional coordination of splicing and 3' end processing.

3' Untranslated Regions↗

PubMed2006

Abundance of the largest subunit of RNA polymerase II in the nucleus is regulated by nucleo-cytoplasmic shuttling.

Eukaryotic RNA polymerase II is a complex enzyme composed of 12 distinct subunits that is present in cells in low abundance. Transcription of mRNA by RNA polymerase II involves a phosphorylation/dephosphorylation cycle of the carboxyl-terminal domain (CTD) of the enzyme's largest subunit. We have generated stable murine cell lines expressing an alpha-amanitin-resistant form of the largest subunit of RNA polymerase II (RNA Pol II LS). These cells maintained transcriptional activity in the presence of alpha-amanitin, indicating that the exogenous protein was functional. We observed that over-expressed RNA Pol II LS was predominantly hypophosphorylated, soluble and accumulated in the cytoplasm in a CRM1-dependent manner. Our results further showed that the transcriptionally active form of RNA Pol II LS containing phosphoserine in position 2 of the CTD repeats was restricted to the nucleus and its levels remained remarkably constant. We propose that nucleo-cytoplasmic shuttling of RNA Pol II LS may provide a mechanism to control the pool of RNA polymerase subunits that is accessible for assembly of a functional enzyme in the nucleus.

Active Transport, Cell Nucleus↗

PubMed2006

The human desmin locus: gene organization and LCR-mediated transcriptional control.

Locus control regions (LCRs) are defined by their ability to confer reproducible physiological levels of transgene expression in mice and therefore thought to possess the ability to generate dominantly a transcriptionally active chromatin structure. We report the first characterization of a muscle-cell-specific LCR, which is linked to the human desmin gene (DES). The DES LCR consists of five regions of muscle-specific DNase I hypersensitivity (HS) localized between -9 and -18 kb 5' of DES and reproducibly drives full physiological levels of expression in all muscle cell types. The DES LCR DNase I HS regions are highly conserved between humans and other mammals and can potentially bind a broad range of muscle-specific and ubiquitous transcription factors. Bioinformatics and direct molecular analysis show that the DES locus consists of three muscle-specific (DES) or muscle preferentially expressed genes (APEG1 and SPEG, the human orthologue of murine striated-muscle-specific serine/threonine protein kinase, Speg). The DES LCR may therefore regulate expression of SPEG and APEG1 as well as DES.

Amino Acid Sequence↗

PubMed2005

CpG-island fragments from the HNRPA2B1/CBX3 genomic locus reduce silencing and enhance transgene expression from the hCMV promoter/enhancer in mammalian cells.

BACKGROUND: The hCMV promoter is very commonly used for high level expression of transgenes in mammalian cells, but its utility is hindered by transcriptional silencing. Large genomic fragments incorporating the CpG island region of the HNRPA2B1 locus are resistant to transcriptional silencing. RESULTS: In this report we describe studies on the use of a novel series of vectors combining the HNRPA2B1 CpG island with the hCMV promoter for expression of transgenes in CHO-K1 cells. We show that the CpG island gives at least twenty-fold increases in the levels of EGFP and EPO observed in pools of transfectants, and that transgene expression levels remain high in such pools for more than 100 generations. These novel vectors also allow facile isolation of clonal CHO-K1 cell lines showing stable, high-level transgene expression. CONCLUSION: Vectors incorporating the hnRPA2B1 CpG island give major benefits in transgene expression from the hCMV promoter, including substantial improvements in the level and stability of expression. The utility of these vectors for the improved production of recombinant proteins in CHO cells has been demonstrated.

Animals↗

PubMed2004

Analysis of intergenic transcription in the human IL-4/IL-13 gene cluster.

During the differentiation of naïve CD4+ precursors to T helper 1 (Th1) or Th2 effector cells, several epigenetic changes occur in a lineage-specific manner at the IFN-gamma or IL-4/IL-13 loci. These changes result in alterations in the chromatin structure of these loci and, hence, lineage-restricted expression of the corresponding cytokines. Intergenic transcripts have recently been shown to regulate the expression of genes in the beta-globin locus; therefore, we have examined the Th2 cytokine gene cluster during human Th1/Th2 differentiation and in a transgenic mouse line containing the human IL-4/IL-13 genes for intergenic transcripts. We show for the first time that intergenic transcription of this locus is restricted to tissues and lineages in which IL-4 and IL-13 are expressed. We also show that intergenic transcription in the IL-4/IL-13 locus is up-regulated after Th2 differentiation. Furthermore, we demonstrate that the Th2 cytokines and intergenic transcripts are detectable in the thymus. We propose that intergenic transcription is tightly associated with transcriptional competence for the Th2 cytokines and may play a role in their regulation. These results support a progressive differentiation model of T cell lineage commitment.

Animals↗

PubMed2004

Drugs in crops.

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Biotechnology↗

PubMed2004

Intraarterial delivery of naked plasmid DNA expressing full-length mouse dystrophin in the mdx mouse model of duchenne muscular dystrophy.

Our previous studies have demonstrated that the intraarterial delivery of naked plasmid DNA leads to high levels of foreign gene expression throughout the muscles of the targeted limb. Although the procedure was first developed in rats and then extended to nonhuman primates, the present study has successfully implemented the procedure in normal mice and the mdx mouse model for Duchenne muscular dystrophy. After intraarterial delivery of plasmid DNA expressing the normal, full-length mouse dystrophin from either the cytomegalovirus promoter or a muscle-specific human desmin gene control region, mdx mouse muscle stably expressed dystrophin in 1-5% of the myofibers of the injected hind limb for at least 6 months. This expression generated an antibody response but no apparent cellular response.

Animals↗

PubMed2004

Thalassaemia mutations within the 5'UTR of the human beta-globin gene disrupt transcription.

The mechanisms by which mutations within the 5' untranslated region (UTR) of the human beta-globin gene (HBB) cause thalassaemia are currently not well understood. We present here the first comprehensive comparative functional analysis of four 'silent' mutations in the human beta-globin 5'UTR, namely: +10(-T), +22(G --> A), +33(C --> G) and +(40-43)(-AAAC), which are present in patients with beta-thalassaemia intermedia. Expression of these genes under the control of the beta-globin locus control region in stable transfected murine erythroleukaemia cells showed that all four mutations decreased steady state levels of mRNA to 61.6%, 68%, 85.2% and 70.6%, respectively, compared with the wildtype gene. These mutations did not interfere with either mRNA transport from the nucleus to the cytoplasm, 3' end processing or mRNA stability. Nuclear run-on experiments demonstrated that mutations +10(-T) and +33(C --> G) reduced the rate of transcription to a degree that fully accounted for the observed lower level of mRNA accumulation, suggesting a disruption of downstream promoter sequences. Interestingly, mutation +22(G --> A) decreased the rate of transcription to a low degree, indicating the existence of a mechanism that acts post-transcriptionally. Generally, our data demonstrated the significance of functionally analysing mutants of this type in the presence of a full complement of transcriptional regulatory elements within a stably integrated chromatin context in an erythroid cell environment.

5' Untranslated Regions↗

PubMed2004

In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nuclei.

Studies over the past years indicate that there is extensive coupling between nuclear export of mRNA and pre-mRNA processing. Here, we visualized the distribution of exon junction complex (EJC) proteins and RNA export factors relative to sites of abundant pre-mRNA synthesis in the nucleus. We analyzed both HeLa cells infected with adenovirus and murine erythroleukemia (MEL) cells stably transfected with the human beta-globin gene. Using in situ hybridization and confocal microscopy, we observe accumulation of EJC proteins (REF/Aly, Y14, SRm160, UAP56, RNPS1, and Magoh) and core spliceosome components (U snRNPs) at sites of transcription. This suggests that EJC proteins bind stably to pre-mRNA cotranscriptionally. No concentration of the export factors NXF1/TAP, p15, and Dbp5 was detected on nascent transcripts, arguing that in mammalian cells these proteins bind the mRNA shortly before or after release from the sites of transcription. These results also suggest that binding of EJC proteins to the mRNA is not sufficient to recruit TAP-p15, consistent with recent findings showing that the EJC does not play a crucial role in mRNA export. Contrasting to the results obtained in MEL cells expressing normal human beta-globin transcripts, mutant pre-mRNAs defective in splicing and 3'end processing do not colocalize with SRm160, REF, UAP56, or Sm proteins. This shows that the accumulation of EJC proteins at transcription sites requires efficient processing of the nascent pre-mRNAs, arguing that transcription per se is not sufficient for the stable assembly of the EJC.

Adenoviridae↗

PubMed2003

Transgenes encompassing dual-promoter CpG islands from the human TBP and HNRPA2B1 loci are resistant to heterochromatin-mediated silencing.

The genetic elements that are responsible for establishing a transcriptionally competent, open chromatin structure at a region of the genome that consists only of ubiquitously expressed, housekeeping genes are currently unknown. We demonstrate for the first time through functional analysis in stably transfected tissue culture cells that transgenes containing methylation-free CpG islands spanning the dual divergently transcribed promoters from the human TATA binding protein (TBP)-proteasome component-B1 (PSMB1) and heterogeneous nuclear ribonucleoprotein A2/B1 (HNRPA2B1)-heterochromatin protein 1Hs-gamma (chromobox homolog 3, CBX3) gene loci are sufficient to prevent transcriptional silencing and a variegated expression pattern when integrated within centromeric heterochromatin. In addition, only transgene constructs extending over both the HNRPA2B1 and the CBX3 promoters, and not the HNRPA2B1 promoter alone, were able to confer high and stable long-term EGFP reporter gene expression. These observations suggest that methylation-free CpG islands associated with dual, divergently transcribed promoters possess an independent dominant chromatin opening function and may therefore be major determinants in establishing and maintaining a region of open chromatin at housekeeping gene loci.

Base Sequence↗

PubMed2002

Beta -Globin mRNA decay in erythroid cells: UG site-preferred endonucleolytic cleavage that is augmented by a premature termination codon.

Previous work showed that human beta-globin mRNAs harboring a premature termination codon are degraded in the erythroid tissues of mice to products that lack sequences from the mRNA 5' end but contain a 5' cap-like structure. Whether these decay products are the consequence of endonucleolytic or 5'-to-3' exonucleolytic activity is unclear. We report that this beta-globin mRNA decay pathway is recapitulated in cultured mouse erythroleukemia (MEL) cells and targets nonsense-free mRNA to a lesser extent than nonsense-containing mRNA. S1 nuclease mapping and primer extension demonstrated that 70-80% of decay product 5' ends contain a UG dinucleotide. Detection of upstream counterparts of these decay products indicates that they are generated by endonucleolytic activity. Both crude and partially purified polysome extracts prepared from MEL cells contain an endonucleolytic activity that generates decay products comparable to those observed in vivo. These data suggest that an endonuclease with preference for UG dinucleotides is involved in the degradation of nonsense-containing and, to a lesser extent, nonsense-free human beta-globin mRNAs in mouse erythroid cells.

Animals↗

PubMed2002

A novel function for the U2AF 65 splicing factor in promoting pre-mRNA 3'-end processing.

Splicing and 3'-end processing (including cleavage and polyadenylation) of vertebrate pre-mRNAs are tightly coupled events that contribute to the extensive molecular network that coordinates gene expression. Sequences within the terminal intron of genes are essential to stimulate pre-mRNA 3'-end processing, although the factors mediating this effect are unknown. Here, we show that the pyrimidine tract of the last splice acceptor site of the human beta-globin gene is necessary to stimulate mRNA 3'-end formation in vivo and binds the U2AF 65 splicing factor. Naturally occurring beta-thalassaemia-causing mutations within the pyrimidine tract reduces both U2AF 65 binding and 3'-end cleavage efficiency. Significantly, a fusion protein containing U2AF 65, when tethered upstream of a cleavage/polyadenylation site, increases 3'-end cleavage efficiency in vitro and in vivo. Therefore, we propose that U2AF 65 promotes 3'-end processing, which contributes to 3'-terminal exon definition.

Cell Nucleus↗

PubMed2002

Transcriptional regulation of the human TATA binding protein gene.

The human TATA binding protein (TBP) locus consists of a functional domain of three closely linkedhousekeeping genes (TBP, PSMB1 (proteasomal C5 subunit), and PDCD2 (programmed cell death-2)) within a 50-kb interval at chromosome position 6q27. Here we demonstrate that a genomic clone spanning the 20-kb TBP gene, with 12 kb 5' and 3' flanking sequences, was fully functional in stable, transfected L-cells harboring a single copy of this transgene, including after long-term (60 day) culture in the absence of drug selective pressure. Furthermore, we were only able to detect DNaseI hypersensitive sites at the TBP and PSMB1 promoters present within this 44-kb fragment. Our data suggest that this 44-kb genomic region possesses genetic regulatory elements that not only drive ubiquitous expression of TBP but also negate chromatin and DNA methylation induced silencing, which is normally associated with transgenes stably integrated into tissue culture cells.

Chromosomes, Human, Pair 6↗

PubMed2002

Genomic characterization of the human prion protein (PrP) gene locus.

Prion protein (PrP) is intimately linked with a class of neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). Employing bioinformatics and direct molecular analysis, we demonstrated that the human PrP gene (PRNP) locus, which is situated at Chromosome (Chr) position 20p12-ter, contains three genes within a 55-kb interval: PRNP; DOPPEL or PRND, located 20 kb 3' of PRNP; and a novel gene, designated PRNT, that maps 3 kb 3' to PRND and is transcribed to generate at least three alternatively spliced mRNAs. All three genes of this locus demonstrate low sequence homology, implying that, although they may be evolutionarily related, they are functionally distinct. Analysis of both adult and fetal human tissues confirmed the ubiquitous but variable expression profile of PRNP, with the highest levels observed in the CNS and testis. Contrastingly, although PRND shows a wide tissue expression pattern in fetal tissues, it is expressed exclusively in adult testis, whereas all three PRNT isoforms were detected only in adult testis, implying that PRND is developmentally regulated. An investigation of the regulatory mechanisms underlying this complex gene expression pattern from the PRNP locus should provide insight into the function of these genes and the possible involvement of the non-PrP proteins in the development of TSEs.

Adult↗

PubMed2002

The beta-globin C-->G mutation at 6 bp 3' to the termination codon causes beta-thalassaemia by decreasing the mRNA level.

We have studied the expression of the silent beta-thalassaemia term+6 (C-->G) mutation, at nucleotide 6 after the stop codon within the human beta-globin 3' untranslated regions (3'UTR), by stable transfection in murine erythroleukaemia (MEL) cells. Steady state mRNA levels from transfected MEL cells containing the term+6 mutant allele were reduced by 52-60%, compared with those obtained from the normal beta-globin gene, in both total and cytoplasmic RNA fractions, showing that the mutation itself is responsible for the similar data obtained from patients. Upon analysis of nuclear RNA, the term+6 mutation was found to also lower the ratio of cleaved/uncleaved transcripts by 22-30%, thus revealing that it interferes with correct 3'-end formation of beta-globin mRNA. The term+6 mutation lies within a polypyrimidine track, similar to that in the beta-intervening sequence II (beta-IVSII), which is known to be an important contributor to the promotion of premRNA 3'-end formation. We propose that the two polypyrimidine tracks flanking the translated region of exon III of the human beta-globin gene may co-operate during beta-globin mRNA biogenesis.

Child↗