The 5' end of the 18S rRNA can be positioned from within the mature rRNA.
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In yeast, the 5' end of the mature 18S rRNA is generated by endonucleolytic cleavage at site A1, the position of which is specified by two distinct signals. An evolutionarily conserved sequence immediately upstream of the cleavage site has previously been shown to constitute one of these signals. We report here that a conserved stem-loop structure within the 5' region of the 18S rRNA is recognized as a second positioning signal. Mutations predicted to either extend or destabilize the stem inhibited the normal positioning of site A1 from within the 18S rRNA sequence, as did substitution of the loop nucleotides. In addition, these mutations destabilized the mature 18S rRNA, indicating that recognition of the stem-loop structure is also required for 18S rRNA stability. Several mutations tested reduced the efficiency of pre-rRNA cleavage at site A1. There was, however, a poor correlation between the effects of the different mutations on the efficiency of cleavage and on the choice of cleavage site, indicating that these involve recognition of the stem-loop region by distinct factors. In contrast, the cleavages at sites A1 and A2 are coupled and the positioning signals appear to be similar, suggesting that both cleavages may be carried out by the same endonuclease. (+info)
The TATA-binding protein and its associated factors are differentially expressed in adult mouse tissues.
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We have investigated the expression levels of the TATA-binding protein (TBP) and several TBP-associated factors (TAFIIs) in differentiated adult mouse tissues. Immunoblots performed using monoclonal antibodies show that there are considerable variations in the levels of TBP and many TAFII proteins present in various tissues. Consequently, the relative levels of TAFIIs and TBP vary significantly from one tissue to another. TBP and several TAFIIs are overexpressed in both testis and small intestine, while in marked contrast, many of these proteins, including TBP itself, were substantially down-regulated in nervous tissues and in the heart. These tissues do, however, show a high expression level of the TBP-like factor, which thus may represent an alternative factor for the specialized transcription program in some differentiated tissues. While there are significant variations in the levels of TAFII28 protein, reverse transcription-coupled polymerase chain reaction shows similar expression of the TAFII28 mRNA in different tissues. The variations in TAFII28 protein levels therefore result from post-transcriptional regulatory events. (+info)
Role of protein kinase C isoforms in phorbol ester-induced vascular endothelial growth factor expression in human glioblastoma cells.
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Aberrant expression of the potent angiogenic cytokine, vascular endothelial growth factor (VEGF), has been demonstrated to be associated with most human solid tumors. Both transcriptional and post-transcriptional mechanisms have been shown to modulate VEGF expression in a multitude of cell types. Here we report that when protein kinase C (PKC) pathways were activated in human glioblastoma U373 cells by phorbol 12-myristate 13-acetate (PMA), VEGF mRNA expression was up-regulated via a post-transcriptional mRNA stabilization mechanism. PMA treatment exhibited no increase in VEGF-specific transcriptional activation as determined by run-off transcription assays and VEGF promoter-luciferase reporter assays. However, PMA increased VEGF mRNA half-life from 0.8 to 3.6 h which was blocked by PKC inhibitors but not by protein kinase A or cyclic nucleotide-dependent protein kinase inhibitors. When U373 cells were transfected with antisense oligonucleotide sequences to the translation start sites of PKC-alpha, -beta, -gamma, -delta, -epsilon, or -zeta isoforms, both PKC-alpha and -zeta antisense oligonucleotides showed substantial inhibition of PMA-induced VEGF mRNA. In addition, overexpression of PKC-zeta resulted in a strong constitutive up-regulation of VEGF mRNA expression. This study demonstrates for the first time that specific PKC isoforms regulate VEGF mRNA expression through post-transcriptional mechanisms. (+info)
Differentiation lineage-specific expression of human heat shock transcription factor 2.
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Differentiation of multipotential hematopoietic cells into lineage-committed precursors involves the selection and maintenance of appropriate programs of gene expression, regulated by specific transcription factors. Using human K562 erythroleukemia cells capable of differentiating along erythroid and megakaryocytic lineages, we explore the differentiation-related role of heat shock transcription factor 2 (HSF2), which belongs to a family of transcription factors generally known to regulate heat shock gene expression. We demonstrate that enhanced HSF2 expression and the acquisition of HSF2 DNA binding activity are strictly specific for erythroid characteristics of K562 cells. Our results reveal a multistep regulatory process of HSF2 gene expression. In K562 cells undergoing hemin-mediated erythroid differentiation, the increase in HSF2 protein levels is preceded by transcriptional induction of the HSF2 gene, accompanied by increased HSF2 mRNA stability. In contrast, during megakaryocytic differentiation induced by the phorbol ester TPA, expression of HSF2 is rapidly down-regulated, leading to a complete loss of the HSF2 protein. These results indicate that the determination of HSF2 expression occurs at the early stages of lineage commitment. Taken together, our data suggest that HSF2 could function as a lineage-restricted transcription factor during differentiation of K562 cells along either the erythroid or the megakaryocytic pathway. (+info)
The nucleolar antigen Nop52, the human homologue of the yeast ribosomal RNA processing RRP1, is recruited at late stages of nucleologenesis.
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We report the molecular characterization of a novel nucleolar protein, Nop52, and its subcellular distribution during the cell cycle and nucleologenesis. This protein was originally identified with human autoantibodies which were subsequently used to clone its corresponding cDNA. Transfection experiments in mammalian cells have confirmed that this cDNA encodes a nucleolar protein that accumulates in the nucleoli and at the periphery of the chromosomes. Nop52 is the putative human homologue of the yeast ribosomal RNA processing protein RRP1 which is involved in pre-rRNA processing from 27S to 25S and 5.8S. In nucleoli, Nop52 is excluded from the ribosomal RNA transcription sites, accumulates in the granular external domain and mainly colocalizes with nucleolar proteins involved in the late processing step such as hPop1 and protein B23. During the building process of the nucleolus at the end of mitosis, a sequential order was observed in the assembly of nucleolar proteins of early and late processing mainly via the prenucleolar body pathway. The order is the following: fibrillarin, nucleolin, Nop52 together with protein B23 in the prenucleolar bodies, and simultaneously with hPop1, and finally Ki-67. The evolutionary conservation of Nop52 and the lethal effects observed in gene disruption experiments, predict a critical role for Nop52 in the generation of 28S rRNA. (+info)
HuD, a neuronal-specific RNA-binding protein, is a putative regulator of N-myc pre-mRNA processing/stability in malignant human neuroblasts.
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N-myc gene copy numbers and transcription rates are similar in N (neuroblastic, tumorigenic) and S (non-neuronal, non-tumorigenic) neuroblastoma cells with chromosomally integrated amplified N-myc genes. However, N cells show significantly higher N-myc mRNA levels than S cells. Therefore, post-transcriptional control of N-myc gene expression must differ between these cell types. Since no differences in N-myc mRNA half-life were found between N and S cells from two cell lines, steady-state levels of N-myc pre-mRNA processing intermediates were analysed. Results suggest that the differences in N-myc expression arise primarily at the nuclear post-transcriptional level. The neuronal-specific RNA-binding Hu proteins are present in cytoplasmic and nuclear fractions of N cells and one of them, HuD, binds specifically to both exonic and intronic N-myc RNA sequences. In sense and antisense HuD-transfected N cells, there are coordinate changes in HuD and N-myc expression levels. Thus, we propose that HuD plays a role in the nuclear processing/stability of N-myc pre-mRNA in N-type neuroblastoma cells. (+info)
The suppressor of forked gene of Drosophila, which encodes a homologue of human CstF-77K involved in mRNA 3'-end processing, is required for progression through mitosis.
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The Suppressor of forked (Su(f)) protein of Drosophila melanogaster is a homologue of the 77K subunit of human cleavage stimulation factor required for cleavage of pre-mRNAs before addition of poly(A). We have previously shown that the Su(f) protein is not ubiquitously distributed: it accumulates in dividing cells at various stages of Drosophila development. In this paper, we show that phenotypes of su(f) temperature-sensitive mutants result from a defect in cell proliferation. Analysis of the mitotic phenotype of su(f) temperature-sensitive alleles in larval brain and in imaginal discs reveals an increase in the number of metaphases with overcondensed chromosomes and asymmetric or reduced mitotic spindles. In contrast, neural differentiation in eye imaginal discs of the same mutant flies does not appear to be affected. These results indicate that su(f) is required during cell division for progression through metaphase. Taken together, these data suggest that a decrease in su(f) activity preferentially affects 3'-end formation of particular mRNAs, some of which are involved in mitosis, and are in agreement with a role of su(f) in the regulation of poly(A) site utilization. (+info)
Mitochondrial transcript processing and restoration of male fertility in T-cytoplasm maize.
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Cytoplasmic male sterility (CMS) systems have been useful in the production of hybrid seed in a number of crops. The Texas or T-cytoplasmic male-sterile (cms-T) system was used extensively in the 1960s to eliminate the need for hand detasseling in hybrid maize production. As a consequence of the 1970 epidemic of southern corn leaf blight, cms-T is no longer widely used commercially. However, it has been developed as a model system to study the genetic and molecular mechanisms underlying male sterility and fertility restoration. Male sterility in T-cytoplasm maize results from the action of a T-cytoplasm-specific mitochondrial gene, T-urf13. Full (or partial) fertility restoration of T-cytoplasm maize is mediated by the Rf2 nuclear restorer in combination with one of three other restorers: Rf1, Rf8, or Rf*. Rf2 encodes a protein highly similar to mitochondrial aldehyde dehydrogenases; Rf1, Rf8, and Rf* each mediate discrete T-urf13 mitochondrial transcript processing events. To test the functionality of Rf1, Rf8, or Rf*, a T-cytoplasm transformation system is under development. AFLP bulk-segregant analysis has been used to identify DNA markers closely linked to the Rf8 locus. These tools will provide a foundation for determining mechanisms of nuclear-directed mitochondrial RNA processing and fertility restoration. (+info)