Immunohistochemical localisation of pterin-4α-carbinolamine dehydratase in rat peripheral organs

 The bifunctional protein PCD/DCoH is both a pterin-4α-carbinolamine dehydratase (PCD) involved in the recycling of tetrahydrobiopterin (BH₄) and a dimerisation cofactor (DCoH) of the hepatic nuclear factor 1α (HNF-1α). An antiserum raised against rat PCD/DCoH was used to localise the protein in peripheral organs. In liver, all the hepatocytes but not the other cell types are immunoreactive. In kidney, the protein is prevalent in the proximal and distal convoluted tubules. In adrenals, all the cells of the medulla are labelled. Positive nerve cells occur in myenteric ganglia of the whole gastrointestinal tract and in the intestinal submucous ganglia. Many positive endocrine cells are present in the epithelium. The immunoreactivity is either cytoplasmic (hepatocytes, convoluted tubules of the kidney and part of the gastrointestinal endocrine cells) or prominently nuclear (kidney collecting tubules, adrenals, intestinal neural plexuses and part of the gastrointestinal endocrine cells). Our results show that PCD/DCoH is present in cells expressing enzymes that use BH₄ as a cofactor and/or HNF-1α. In addition, PCD/DCoH is present in other cells, for example, neurons in the submucosal plexus. This fact and the prominent nuclear immunoreactivity found in all the positive cells derived from the neural crests argue in favour of a new, still unknown function for the protein. Accepted: 18 January 1999     

Role of the 5' TAR stem--loop and the U5-AUG duplex in dimerization of HIV-1 genomic RNA

The HIV-1 genome consists of two identical RNAs that are linked together through noncovalent interactions involving nucleotides from the 5' untranslated region (5' UTR) of each RNA strand. The 5' UTR is the most conserved part of the HIV-1 RNA genome, and its 335 nucleotide residues form regulatory motifs that mediate multiple essential steps in the viral replication cycle. Here, studying the effect of selected mutations both singly and together with mutations disabling SL1 (SL1 is a 5' UTR stem-loop containing a palindrome called the dimerization initiation site), we have done a rather systematic survey of the 5' UTR requirements for full genomic RNA dimerization in grown-up (i.e., predominantly >/=10 h old) HIV-1 viruses produced by transfected human and simian cells. We have identified a role for the 5' transactivation response element (5' TAR) and a contribution of a long-distance base pairing between a sequence located at the beginning of the U5 region and nucleotides surrounding the AUG Gag initiation codon. The resulting intra- or intermolecular duplex is called the U5-AUG duplex. The other regions of the 5' UTR have been shown to play no systematic role in genomic RNA dimerization, except for a sequence located around the 3' end of a large stem-loop enclosing the primer binding site, and the well-documented SL1. Our data are consistent with a direct role for the 5' TAR in genomic RNA dimerization (possibly via a palindrome encompassing the apical loop of the 5' TAR).     

The 5' UTR of protein kinase C epsilon confers translational regulation in vitro and in vivo

We have examined translational regulation conferred by the 5' untranslated region (UTR) of PKCepsilon on expression of the luciferase reporter gene in vitro, using rabbit reticulocyte lysates and in vivo, in contact-inhibiting mouse Swiss 3T3 fibroblasts and non-contact-inhibiting Swiss 3T6 fibroblasts. In rabbit reticulocyte lysates, the 5' UTR of PKCepsilon significantly represses translation. In 3T3 and 3T6 cells, the 5' UTR of PKCepsilon reduces luciferase activity, but not to the same extent as it does in vitro. In rabbit reticulocyte lysate, the degree of repression mediated by different PKCepsilon 5' UTR-deletion constructs correlates with the free energy (DeltaG) of their predicted secondary structures. However, in cells, secondary structure is not the only determinant of repression; an internal region of the 5' UTR is both necessary and sufficient for repression. Mutation of an upstream AUG (uAUG) motif in this region partially relieves repression. We conclude that the 5' UTR of PKCepsilon can mediate translational regulation and that translation inhibition in vivo involves the uAUG motif. Our findings also suggest that there are factors present in fibroblasts, but not in rabbit reticulocyte lysates that substantially overcome the repressive qualities of the long, structured 5' UTR. Thus, we have identified a potential new level of regulation of PKC in mammalian cells.     

Regulatory Role of the Conserved Stem-Loop Structure at the 5′ End of Collagen α1(I) mRNA

Three fibrillar collagen mRNAs, alpha1(I), alpha2(I), and alpha1(III), are coordinately upregulated in the activated hepatic stellate cell (hsc) in liver fibrosis. These three mRNAs contain sequences surrounding the start codon that can be folded into a stem-loop structure. We investigated the role of this stem-loop structure in expression of collagen alpha1(I) reporter mRNAs in hsc's and fibroblasts. The stem-loop dramatically decreases accumulation of mRNAs in quiescent hsc's and to a lesser extent in activated hsc's and fibroblasts. The stem-loop decreases mRNA stability in fibroblasts. In activated hsc's and fibroblasts, a protein complex binds to the stem-loop, and this binding requires the presence of a 7mG cap on the RNA. Placing the 3' untranslated region (UTR) of collagen alpha1(I) mRNA in a reporter mRNA containing this stem-loop further increases the steady-state level in activated hsc's. This 3' UTR binds alphaCP, a protein implicated in increasing stability of collagen alpha1(I) mRNA in activated hsc's (B. Stefanovic, C. Hellerbrand, M. Holcik, M. Briendl, S. A. Liebhaber, and D. A. Brenner, Mol. Cell. Biol. 17:5201-5209, 1997). A set of protein complexes assembles on the 7mG capped stem-loop RNA, and a 120-kDa protein is specifically cross-linked to this structure. Thus, collagen alpha1(I) mRNA is regulated by a complex interaction between the 5' stem-loop and the 3' UTR, which may optimize collagen production in activated hsc's.