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The 3'-untranslated region of c-myc mRNA contains a perinuclear localisation signal which is sufficient to target beta-globin coding sequences. The link between perinuclear mRNA localisation and translation has been investigated using cells transfected with chimeric gene constructs in which globin reporter sequences were linked to the c-myc 3'-untranslated region and the iron-responsive element from ferritin mRNA. Iron supplementation of the medium promoted translation of the chimeric mRNA as assessed by its presence in polysomes; in situ hybridisation showed that the mRNA was localised around the nucleus. Treatment with the iron chelator desferrioxamine for 16 h prevented both translation and mRNA localisation. In controls where the expressed mRNA lacked the iron-responsive element desferrioxamine had no effect upon localisation. In contrast, arrest of on-going global translation by puromycin treatment had no effect on mRNA localisation. The data suggest that if initiation of translation of a mRNA containing the c-myc localisation signal is prevented in some way then localisation does not occur, whereas once the mRNA has been localised further translation is not required to maintain mRNA localisation.

Ku protein is a relatively abundant DNA-binding nuclear protein complex composed of two polypeptide subunits, p70 and p80. Ku has been recently identified as the regulatory component of the DNA-dependent protein kinase that phosphorylates RNA polymerase II. To further characterize in vivo regulation of Ku protein, we studied the expression of the transcripts coding for the Ku p70 and p80 subunits in different human cell lines and normal tissues by Northern blot hybridization, using specific cDNA probes. The expression level of both genes was approximately 10-fold higher in established cell lines than in normal tissues. However, mRNA expression levels in permanent cell lines correlated more strongly with their proliferative state than with their level of malignant transformation. In purified T lymphocytes induced to proliferate by the combined action of monoclonal antibodies directed against the CD2 and CD28 adhesion molecules, Ku p70 and p80 mRNA steady-state levels increased as soon as 6 h after activation and lasted at least 72 h. The human genes coding for the Ku p70 and p80 subunits were localized by cytogenetic mapping, using fluorescence in situ hybridization, to 22q13 and 2q33→q35, respectively.

International audience; A strong block to the elongation of nascent RNA transcripts by RNA polymerase II occurs in the 5' part of the mammalian c-fos proto-oncogene. In addition to the control of initiation, this mechanism contributes to transcriptional regulation of the gene. In vitro transcription experiments using nuclear extracts and purified transcription templates allowed us to map a unique arrest site within the mouse first intron 385 nucleotides downstream from the promoter. This position is in keeping with that estimated from nuclear run-on assays performed with short DNA probes and thus suggests that it corresponds to the actual block in vivo. Moreover, we have shown that neither the c-fos promoter nor upstream sequences are absolute requirements for an efficient transcription arrest both in vivo and in vitro. Finally, we have characterized a 103-nucleotide-long intron 1 motif comprising the arrest site and sufficient for obtaining the block in a cell-free transcription assay.

International audience; We have isolated and sequenced a full-length cDNA clone encoding rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, E.C.1.2.1.12). The entire mRNA is 1269 nucleotides long exclusive of poly(A) and contains respectively 71 and 196 bases of 5' and 3' non-coding regions. Primer extension as well as S1 nuclease protection experiments clearly established that a single (or at least a highly prominent) GAPDH mRNA species is expressed in all rat tissues examined. This sequence allowed the determination of the hitherto unknown primary structure of rat GAPDH which is 333 aminoacids long. Comparison between GAPDH sequences from rat, man and chicken revealed a high degree of sequence conservation at both nucleotide and protein levels.

International audience; c-myc RNA rapidly decreases to barely detectable levels in Friend erythroleukemia cells induced to differentiate upon the addition of dimethylsulfoxide. We show here that c-myc gene is down-regulated both at the transcriptional level presumably by a block in the elongation of primary transcripts and at the post-transcriptional level by an increase in the degradation of its mRNA.

International audience; A synthetic 13-mer oligodeoxynucleotide designed to specifically initiate on lactic dehydrogenase (LDH) mRNA was shown to prime with high efficiency the synthesis of DNA complementary to the last 133 nucleotides of mitochondrial 16S ribosomal RNA on a mouse liver total poly(A)+ RNA template. One especially interesting feature of this reaction was that reverse transcriptase has been able to initiate at the 3' terminal C residue of the primer despite the fact that it was facing an A. This observation therefore establishes the ability of reverse transcriptase to initiate directly on a mismatched nucleotide.

Chromatography of different soluble extracts from HeLa cells on poly(A)-Sepharose columns has allowed the isolation of a protein fraction eluted by 0.2 M NaCl and localized predominantly in the cytoplasmic supernatant and in the 0.5 M KCl ribosomal wash. This fraction is present in large amounts (around 3% of total cytosolic proteins) and appears to contain a major protein species that is acidic on electrofocusing (pI around 4.5) and phosphorylated. It runs on glycerol gradients and Sephadex G-200 chromatography close to the aldolase marker (158,000 daltons) and dissociates into apparently identical subunits of 38,000 ± 2,000 daltons on sodium dodecyl sulfate-acrylamide gels, suggesting a tetrameric structure.

Although only one gene is known to be functional, numerous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) related sequences are scattered throughout Mus musculus and Rattus rattus genomes. In this report we show that: (1) GAPDH pseudogenes are repeated to comparable extents, at least 400 copies, in 12 other Muridae species; (2) the complete, or nearly so, sequence of GAPDH messenger RNA is amplified, and a high proportion, if not all of these copies, are intronless; (3) GAPDH pseudogenes are preferentially located in heavily methylated and DNAse I-insensitive regions of chromatin; and (4) the presence of atypical GAPDH-related mRNAs in different cellular contexts raises the possibility that more than one GAPDH gene is transcribed.

International audience; The recent observation made in our laboratory that cellular myc (c-myc) mRNA has a very short half-life in a variety of normal and transformed human cells emphasized the potential importance of post-transcriptional regulation of c-myc gene expression. Jonak and Knight [Jonak, G. J. & Knight, E., Jr. (1984) Proc. Natl. Acad. Sci. USA 81, 1747-1750] have reported a selective reduction of c-myc mRNA accumulation in lymphoblastoid Daudi cells treated with human beta interferon. This provided a suitable situation in which to examine a possible action of negative modulators of c-myc expression at the level of mRNA stability. Our results confirm the observation by Jonak and Knight that c-myc mRNA level is depressed in cells treated with beta interferon and extend it to alpha 2 interferon. Furthermore, we now demonstrate that interferon has no effect on c-myc transcription rate in isolated nuclei but rather reduces the half-life of its mRNA. Conversely, we show that it increases the level of HLA-A2 mRNA by stimulating its transcription.

To elucidate the mechanisms which achieve and regulate the transport from the nucleus to the cytoplasm of those sequences of heterogeneous nuclear RNA (HnRNA) destined to become cytoplasmic mRNA, we have started a systematic investigation of the proteins associated with these RNA species in the form of the so-called ‘RNP particles’. The general occurrence of such ribonucleoprotein complexes in eukaryotic cells is widely recognised although they have only recently been described in HeLa cells1,2. The occurrence of phosphorylated proteins in rat brain nuclear particles3, together with the observation that mRNA release from adenovirus-infected KB cell nuclei in vitro is an ATP-dependent process4, has prompted a search for protein kinase activity in HeLa nuclear RNP particles. The role of protein phosphorylation in the regulation of enzyme activity is now recognised5 and we have observed such an activity which does not require the addition of exogenous substrates like histones. The endogenous protein substrates present in HeLa nuclear RNP particles have also been characterised.

The complete nucleotide sequence for chicken glyceraldehyde-3-phosphate dehydrogenase mRNA has been determined, thereby extending the longest such sequence previously reported ( Dugaiczyk et al. Biochemistry, 1983, 22 , 1605–1613 ) by 27 nucleotides. The complete mRNA with the exclusion of poly(A) is 1284 nucleotides long and contains 56 nucleotides of 5′ non coding sequence and 229 nucleotides of 3′ non coding region. Knowledge of the complete sequence allows us to propose secondary structures models which may be of biological significance.

International audience; We have isolated and identified cDNA clones containing part of the coding sequence for rat glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, E.C. 1.2.1.12). By using one of these clones as a probe, we have shown that: i) the abundance of GAPDH mRNA is different in various tissues of the adult rat and in good correlation with the abundance of the enzyme; ii) the transcription rates are quite similar in all tissues tested. We therefore conclude that the tissue-specific differential GAPDH gene expression is regulated by adjusting the abundance of its mRNA at the post-transcriptional level.

Exon 1 deleted c-myc RNAs found in some mouse plasma cell tumors (Piechaczyk et al., 1985) and Burkitt’s lymphomas (Eick et al., 1985) are more stable than their normal counterparts in control cells. In this paper we extend our original observations to other B cell and we prove that the structural modification of c-myc RNA is responsible for its stabilization. However, we also show that exon 1 by itself does not account for the extreme instability of normal c-myc RNAs. Finally our results suggest that abnormal c-myc transcripts, while always stabilized in the cytoplasm, may sometimes be more actively degraded in the nucleus than normal ones in reference cell lines.