Identification in the rat brain of a set of nuclear proteins interacting with H1° mRNA.

Synthesis of H1° histone, in the developing rat brain, is also regulated at post-transcriptional level. Regulation of RNA metabolism depends on a series of RNA-binding proteins (RBPs); therefore, we searched for H1° mRNA-interacting proteins. With this aim, we used in vitro transcribed, biotinylated H1° RNA as bait to isolate, by a chromatographic approach, proteins which interact with this mRNA, in the nuclei of brain cells. Abundant RBPs, such as heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP A1, and molecular chaperones (heat shock cognate 70, Hsc70) were identified by mass spectrometry. Western blot analysis also revealed the presence of cold shock domain-containing protein 2 (CSD-C2, also known as PIPPin), a brain-enriched RBP previously described in our laboratory. Co-immunoprecipitation assays were performed to investigate the possibility that identified proteins interact with each other and with other nuclear proteins. We found that hnRNP K interacts with both hnRNP A1 and Hsc70 whereas there is no interaction between hnRNP A1 and Hsc70. Moreover, CSD-C2 interacts with hnRNP A1, Y box-binding protein 1 (YB-1), and hnRNP K. We also have indications that CSD-C2 interacts with Hsc70. Overall, we have contributed to the molecular characterization of a ribonucleoprotein particle possibly controlling H1° histone expression in the brain.

Nuclear-mitochondrial interaction.

The biogenesis of mitochondria depends on the coordinated expression of nuclear and mitochondrial genomes. Consequently, the control of mitochondrial biogenesis and function depends on extremely complex processes requiring a variety of well orchestrated regulatory mechanisms. It is clear that the interplay of transcription factors and coactivators contributes to the expression of both nuclear and mitochondrial respiratory genes. In addition, the regulation of mitochondria biogenesis depends on proteins that, interacting with messenger RNAs for mitochondrial proteins, influence their metabolism and expression. Moreover, a tight regulation of the import and final assembly of mitochondrial protein is essential to endow mitochondria with functional complexes. These studies represent the basis for understanding the mechanisms involved in the nucleus-mitochondrion communication, a cross-talk essential for the cell.

Analysis of cytochrome C oxidase subunits III and IV expression in developing rat brain.

Cytochrome c oxidase (COX) complex is built up with both nucleus- and mitochondrion-encoded subunits. Biogenesis and assembly of the complex thus requires fine cross-talk between the two compartments. In order to shed light on the regulation of nuclear-mitochondrial interactions, we studied the expression of COXIII (mitochondrion-encoded) and COXIV (nucleus-encoded) in adult rat tissues and rat developing brain. We found that the levels of COXIV protein and mRNA are not linearly related, thus suggesting a post-transcriptional mode of regulation. In agreement with this observation, we report the presence of a protein that specifically binds to the 3'-untranslated region of COXIV mRNA. This factor, that forms with RNA a complex of about 60 kDa, is present both in the cytoplasm and mitochondria, where its concentration decreases throughout development with inverse correlation with COXIV accumulation. Interestingly, using an antibody raised in our laboratory, we found that, in developing rat brain, COXIII does not localize exclusively to mitochondria, but is also present in the cytosol, where it could exert a yet unknown regulatory role.

3'-untranslated regions of oxidative phosphorylation mRNAs function in vivo as enhancers of translation.

Recent findings have indicated that the 3'-untranslated region (3'-UTR) of the mRNA encoding the beta-catalytic subunit of the mitochondrial H(+)-ATP synthase has an in vitro translation-enhancing activity (TEA) [Izquierdo and Cuezva, Mol. Cell. Biol. (1997) 17, 5255-5268; Izquierdo and Cuezva, Biochem. J. (2000) 346, 849-855]. In the present work, we have expressed chimaeric plasmids that encode mRNA variants of green fluorescent protein in normal rat kidney and liver clone 9 cells to determine whether the 3'-UTRs of nuclear-encoded mRNAs involved in the biogenesis of mitochondria have an intrinsic TEA. TEA is found in the 3'-UTR of the mRNAs encoding the alpha- and beta-subunits of the rat H(+)-ATP synthase complex, as well as in subunit IV of cytochrome c oxidase. No TEA is present in the 3'-UTR of the somatic mRNA encoding rat mitochondrial transcription factor A. Interestingly, the TEA of the 3'-UTR of mRNAs of oxidative phosphorylation is different, depending upon the cell type analysed. These data provide the first in vivo evidence of a novel cell-specific mechanism for the control of the translation of mRNAs required in mitochondrial function.

Mitochondrial biogenesis in the liver during development and oncogenesis.

The analysis of the expression of oxidative phosphorylation genes in the liver during development reveals the existence of two biological programs involved in the biogenesis of mitochondria. Differentiation is a short-term program of biogenesis that is controlled at post-transcriptional levels of gene expression and is responsible for the rapid changes in the bioenergetic phenotype of mitochondria. In contrast, proliferation is a long-term program controlled both at the transcriptional and post-transcriptional levels of gene expression and is responsible for the increase in mitochondrial mass in the hepatocyte. Recently, a specific subcellular structure involved in the localization and control of the translation of the mRNA encoding the beta-catalytic subunit of the H(+)-ATP synthase (beta-mRNA) has been identified. It is suggested that this structure plays a prominent role in the control of mitochondrial biogenesis at post-transcriptional levels. The fetal liver has many phenotypic manifestations in common with highly glycolytic tumor cells. In addition, both have a low mitochondrial content despite a paradoxical increase in the cellular representation of oxidative phosphorylation transcripts. Based on the paradigm provided by the fetal liver we hypothesize that the aberrant mitochondrial phenotype of fast-growing hepatomas represents a reversion to a fetal program of expression of oxidative phosphorylation genes by the activation, or increased expression, of an inhibitor of beta-mRNA translation.

The amplicons in HL60 cells contain novel cellular sequences linked to MYC locus DNA.

We have previously determined that the amplified DNA present in the HL60 promyelocytic leukaemia cell line contained 70 kb of continuous DNA sequences around the c-myc gene. In the work presented here we have further defined the HL60 amplicon and find it to be of the order of 160 kb and to contain a large region of DNA from chromosome 8q24 that is located at least 260 kb telomeric to the c-myc gene, joined to the 70 kb of DNA from the c-myc gene region. The novel chromosome 8 DNA coamplified sequences are not lost during multiple passage of HL60 cells since the composition of the chimeric amplicon is the same in both early passage HL60 cells containing only double minutes (DMs) and late passage isolates containing homogeneous staining regions (HSRs) at different chromosomal locations. This shows that the HL60 HSRs found in late passage cells are not generated anew but are directly derived from the precursor DMs. The HL60 cells contain two copies of chromosome 8, each with different polymorphic markers. Both these chromosome 8 homologues retain a c-myc gene as well as the region containing the coamplified DNA sequences indicating that the HL60 amplicons were not generated by simple DNA deletion. The constraint to maintain the novel DNA sequences coamplified with c-myc gene DNA suggests that these sequences may play some role in maintaining the growth potential and/or differentiation capacity of the HL60 cells.

The DNA region around the c-myc gene and its amplification in human tumour cell lines.

In order to study amplicons containing the c-myc locus in human tumour cells we have analysed the region around the human c-myc gene. Using pulsed field gel electrophoresis we have constructed a 1.8 Mb map of the c-myc locus using six rare-cutting endonucleases. In addition we have cloned 130 kb of continuous DNA around the c-myc gene which includes 65 kb of 5' sequence and 65 kb of 3' sequence. The first exon of the pvt-1 gene was located 59 kb 3' of the c-myc gene. Using nine neighbouring probes, covering a region of about 395 kb around the c-myc gene, we were able to determine the extent and composition of c-myc amplicons in the HL60, NCI-N417, Colo320HSR, SkBr3 and the Sk-N-MC human tumour cell lines. Furthermore we analysed the chromosomal location of amplified c-myc DNA in NCI-N417 and SkBr3 cells. Similarities and differences between the amplified DNA in the different cell lines are discussed.

The dynamic properties of neuronal chromatin are modulated by triiodothyronine.

The effect of triiodothyronine (T3) on the rate of synthesis of nuclear proteins was studied during terminal differentiation of rat cortical neurons cultured in a serum-free medium. To this aim total and acid soluble nuclear proteins were analyzed by different electrophoretic techniques. Our results show that: 1) during maturation in vitro, neuronal nuclei undergo a dramatic change in the rate at which different classes of histones and high mobility group (HMG) proteins are synthesized; the synthetic activity, measured as incorporation of radioactive precursors into nuclear proteins, slows indeed down with age: especially evident is the decrease in core histones synthesis; at day 15, on the other hand, HMG 14 and 17 and ubiquitinated H2A (A24) are synthesized at a high rate, especially in T3-treated neurons; 2) neurons treated with T3 show, at any age tested, a higher level of lysine incorporation into nuclear proteins; 3) even if during the first days of culture neurons synthesize core histones more actively in the presence of T3, there is no accumulation of these proteins at later stages, as compared with untreated cells. Possible implications of these data and relationship with the chromatin rearrangement which accompanies neuronal terminal differentiation are discussed.

Accumulation of different c-erbA transcripts during rat brain development and in cortical neurons cultured in a synthetic medium.

1. Accumulation of different c-erbA transcripts was studied, during rat brain maturation and in cortical neurons differentiating in a serum-free medium, by quantitative Northern blot hybridization. 2. The alpha and beta forms of c-erbA mRNAs exhibit different patterns of accumulation, with a precocious increase in the alpha forms compared with the beta forms both in vivo and in culture. 3. erbA alpha 2 mRNA (2.6 kb) is by far the predominant form, with a maximum at birth (PO). 4. The accumulation patterns of both alpha and beta forms show discrete differences in isolated neurons compared to brain cortices; in particular the pattern of alpha 2 mRNA accumulation in culture suggests its predominant localization to neurons. 5. The presence of T3 in the culture medium does not have significant effects on the level of any of erbA mRNAs. 6. Possible implications and relationships with neuronal terminal differentiation are discussed.

Qualitative differences in nuclear proteins correlate with neuronal terminal differentiation.

1. Protein composition of neuronal nuclei was studied at two stages of brain maturation, i.e., before (embryonic day 16; E16) and after (postnatal day 10; P10) shortening of the nucleosomal repeat length. Glial nuclei were analyzed in parallel as a control. 2. Total nuclear or HCl- and 5% perchloric acid (PCA)-soluble proteins were analyzed by different electrophoretic techniques. 3. Our results show an increase in the concentration of histone H1 zero with differentiation, although the H1 class undergoes an overall decrease. 4. The chromatin of mature neurons is also enriched in the ubiquinated form of histone H2A (A24), while the high-mobility group (HMG) proteins 1 and 2 seem to decrease slightly relative to core histones. 5. Both quantitative and qualitative differences in the abundance of nonhistone proteins relative to histones accompany neuronal terminal differentiation.

Effects of triiodothyronine (T3) on differentiation of rat cortical neurons in primary cultures.

Some of the events which characterize neuronal terminal differentiation have been studied in rat cortical neurons cultured in a selective synthetic medium for a period which corresponds to terminal brain maturation in vivo. In particular, we have studied the effect of T3 on the synthesis of nuclear proteins and the expression of the mRNAs which encode different variants of T3 nuclear receptors (c erb A proteins). We have shown that: a) T3 stimulates the turnover of nuclear proteins, with a more evident effect on the non-histone component; b) for the whole lifespan of cultures the predominant form of c erb Aα mRNA is the α2 variant (which encodes a protein unable to bind T3); whatever the function of α2 protein this finding suggests that its predominance on α1 is settled very early during mammalian brain maturation.

Use of ZetaPrep cartridge for the purification of human recombinant interleukin 1 beta.

Zetaprep mass ion-exchange media represent a rapid and efficient chromatographic tool in the separation of proteins, in place of the conventional agarose or cellulose-based gels. We adopted this method, combined with classical steps, to purify to homogeneity human recombinant interleukin 1 beta (IL-1 beta) produced from E. coli and from S. cerevisiae. An anion exchanger QAE-ZetaPrep was used to achieve a rapid partial purification of both proteins. The IL-1 beta purification was completed by gel permeation chromatography on Sephadex G-50. When the protein was produced from yeast, an intermediate chromatographic step on a hydroxylapatite column was also necessary. The isolated proteins proved to be homogeneous by electrophoresis and amino acid analysis. The biological activity of IL-1 beta produced by E. coli is comparable to that of the natural protein, while the protein produced by yeast showed very low specific activity.

The in vitro transcription of the 7SK RNA gene by RNA polymerase III is dependent only on the presence of an upstream promoter.

Deletion analysis was carried out on the human 7SK RNA gene to map regions essential for in vitro transcription by RNA polymerase III. The sequence promoting transcription is located between 37 and 3 bp upstream of the 7SK RNA coding region. RNA polymerase III transcription of adjacent plasmid sequences can be directed by this promoter in the complete absence of the 7SK RNA coding region, indicating that no internal promoter sequences are required. Transcription is terminated by a stretch of T residues, typical of RNA polymerase III transcription. The promoter contains a TATA box at position -25, mutations within which dramatically reduce the efficiency of transcription. Upstream sequences from position -37 to -243 increase the promoter's efficiency. The promoter recognized by RNA polymerase III is structurally and functionally similar to the promoter of genes transcribed by RNA polymerase II.