Human delta-aminolevulinate synthase: assignment of the housekeeping gene to 3p21 and the erythroid-specific gene to the X chromosome

delta-Aminolevulinate synthase (ALAS) catalyzes the first committed step of heme biosynthesis. Previous studies suggested that there were erythroid and nonerythroid ALAS isozymes. We have isolated cDNAs encoding the ubiquitously expressed housekeeping ALAS isozyme and a related, but distinct, erythroid-specific isozyme. Using these different cDNAs, the human ALAS housekeeping gene (ALAS1) and the human erythroid-specific (ALAS2) gene have been localized to chromosomes 3p21 and X, respectively, by somatic cell hybrid and in situ hybridization techniques. The ALAS1 gene was concordant with chromosome 3 in all 26 human fibroblast/murine(RAG) somatic cell hybrid clones analyzed and was discordant with all other chromosomes in at least 6 of 26 clones. The regional localization of ALAS1 to 3p21 was accomplished by in situ hybridization using the 125I-labeled human ALAS1 cDNA. Of the 43 grains observed over chromosome 3, 63% were localized to the region 3p21. The gene encoding ALAS2 was assigned by examination of a DNA panel of 30 somatic cell hybrid lines hybridized with the ALAS2 cDNA. The ALAS2 gene segregated with the human X chromosome in all 30 hybrid cell lines analyzed and was discordant with all other chromosomes in at least 8 of the 30 hybrids. These results confirm the existence of two independent, but related, genes encoding human ALAS. Furthermore, the mapping of the ALAS2 gene to the X chromosome and the observed reduction in ALAS activity in X-linked sideroblastic anemia suggest that this disorder may be due to a mutation in the erythroid-specific gene.     

Selective Translational Control of the Alzheimer Amyloid Precursor Protein Transcript by Iron Regulatory Protein-1

Iron influx increases the translation of the Alzheimer amyloid precursor protein (APP) via an iron-responsive element (IRE) RNA stem loop in its 5′-untranslated region. Equal modulated interaction of the iron regulatory proteins (IRP1 and IRP2) with canonical IREs controls iron-dependent translation of the ferritin subunits. However, our immunoprecipitation RT-PCR and RNA binding experiments demonstrated that IRP1, but not IRP2, selectively bound the APP IRE in human neural cells. This selective IRP1 interaction pattern was evident in human brain and blood tissue from normal and Alzheimer disease patients. We computer-predicted an optimal novel RNA stem loop structure for the human, rhesus monkey, and mouse APP IREs with reference to the canonical ferritin IREs but also the IREs encoded by erythroid heme biosynthetic aminolevulinate synthase and Hif-2α mRNAs, which preferentially bind IRP1. Selective 2′-hydroxyl acylation analyzed by primer extension analysis was consistent with a 13-base single-stranded terminal loop and a conserved GC-rich stem. Biotinylated RNA probes deleted of the conserved CAGA motif in the terminal loop did not bind to IRP1 relative to wild type probes and could no longer base pair to form a predicted AGA triloop. An AGU pseudo-triloop is key for IRP1 binding to the canonical ferritin IREs. RNA probes encoding the APP IRE stem loop exhibited the same high affinity binding to rhIRP1 as occurs for the H-ferritin IRE (35 pm). Intracellular iron chelation increased binding of IRP1 to the APP IRE, decreasing intracellular APP expression in SH-SY5Y cells. Functionally, shRNA knockdown of IRP1 caused increased expression of neural APP consistent with IRP1-APP IRE-driven translation.     

Crystal structure of 5-aminolevulinate synthase, the first enzyme of heme biosynthesis, and its link to XLSA in humans

5-Aminolevulinate synthase (ALAS) is the first and rate-limiting enzyme of heme biosynthesis in humans, animals, other non-plant eukaryotes, and alpha-proteobacteria. It catalyzes the synthesis of 5-aminolevulinic acid, the first common precursor of all tetrapyrroles, from glycine and succinyl-coenzyme A (sCoA) in a pyridoxal 5'-phosphate (PLP)-dependent manner. X-linked sideroblastic anemias (XLSAs), a group of severe disorders in humans characterized by inadequate formation of heme in erythroblast mitochondria, are caused by mutations in the gene for erythroid eALAS, one of two human genes for ALAS. We present the first crystal structure of homodimeric ALAS from Rhodobacter capsulatus (ALAS(Rc)) binding its cofactor PLP. We, furthermore, present structures of ALAS(Rc) in complex with the substrates glycine or sCoA. The sequence identity of ALAS from R. capsulatus and human eALAS is 49%. XLSA-causing mutations may thus be mapped, revealing the molecular basis of XLSA in humans. Mutations are found to obstruct substrate binding, disrupt the dimer interface, or hamper the correct folding. The structure of ALAS completes the structural analysis of enzymes in heme biosynthesis.     

The fragile X mental retardation protein interacts with U-rich RNAs in a yeast three-hybrid system

We recently identified several ESTs that bind to the fragile X mental retardation protein (FMRP) in vitro. To determine whether they interacted in vivo we performed three-hybrid screens in a Saccharomyces cerevisiae histidine auxotroph. We demonstrate that two of the ESTs support growth on histidine and transduce beta-galactosidase activity when co-expressed with FMRP under selective growth conditions. In contrast, the iron response element (IRE) RNA does not. Likewise, the ESTs do not support growth or transduce beta-galactosidase activity when co-expressed with the iron response element binding protein (IRP). Each EST is relatively small and has 40% identity with a sequence in FMR1 mRNA harboring FMRP binding determinants. Interestingly, while neither the ESTs contain a G-quartet structural motif they do contain U-rich sequences that are found in mRNA with demonstrated in vitro binding and in vivo association with FMRP. This indicates that U-rich elements comprise another motif recognized by FMRP.     

cDNA cloning,expression pattern and RNA binding analysis of human selenocysteine insertion sequence (SECIS) binding protein 2

Selenocysteine and selenoprotein synthesis require a complex molecular machinery in mammals. Among the key players is the RNA-protein complex formed by the selenocysteine insertion sequence (SECIS) binding protein (SBP2) and the SECIS element, an RNA hairpin in the 3' untranslated regions of selenoprotein messenger RNAs (mRNAs). We have isolated the DNA complementary to mRNA of the human SBP2, enabling us to establish that it differs from a previously reported human SBP2-like protein. Examination of the expression pattern revealed that the human SBP2 protein is encoded by a 4 kb long mRNA that is over-expressed in testis. Compared to the rat SBP2 sequence, the human SBP2 protein displays two highly conserved domains with 92 and 95% amino acid identity, the latter one containing the RNA binding domain. The inter-domain section carries 55% sequence identity, the remainder of the SBP2 sequences showing about 65% identity, values lower than expected for two mammalian proteins. Interestingly, we could show that the binding of human SBP2 to the SECIS RNA is stimulated by the selenoprotein-specialized elongation translation factor mSelB/eEFsec.     

A novel early estrogen-regulated gene gec1 encodes a protein related to GABARAP

We have isolated, in guinea-pig endometrial cells, an estrogen-induced 1.8 kb RNA called gec1. Screening of a guinea-pig genomic library led to identification of gec1 gene consisting of 4 exons and 3 introns. Exon 1 contains the 5'UTR and the ATG initiation codon. A guinea-pig gec1 cDNA was obtained by 5'-RACE. The 351 bp coding sequence shares 76.8% identity with that of the human GABARAP 924 bp cDNA while UTRs of the two cDNAs differ. A gec1 probe from the 3'UTR revealed a 1.9 kb mRNA in human tissues and a human GEC1 cDNA was isolated from placenta. Its coding sequence shares 93 and 79% identity with that of guinea-pig gec1 and human GABARAP, respectively. The human and guinea-pig GEC1 proteins have 100% identity. GEC1 and GABARAP proteins have 87% identity and N terminus featuring a tubulin binding motif. Thus, estrogen-regulated gec1 is a new gene which could encode a microtubule-associated protein.     

Demonstration of a boar testicular protein band that is immunoreactive to proacrosin and proacrosin binding protein antibodies.

A testicular protein band has been identified and shown to be immunoreactive to both of the proacrosin (53-55 kd) and the proacrosin binding protein (28 kd) antibodies. pH 4.5 extracts of boar testis were prepared and subjected to Western blot analysis using polyclonal antibodies of the proacrosin and the proacrosin binding protein. In addition to their respective antigens, a distinct high molecular weight protein band of approximately 200 kd was detected by both of the antibodies. Gelatin SDS-PAGE analysis of the extracts showed that this protein band was proteinase active. These results suggest that the proacrosin molecule is present as a much higher molecular weight form in the boar testis than the currently known 53-55 kd forms that have been isolated from spermatozoa.     

Differential regulation of human ALAS1 mRNA and protein levels by heme and cobalt protoporphyrin

5-Aminolevulinic acid synthase 1 (ALAS1) is the first and rate-controlling enzyme of heme biosynthesis. This study was to determine the effects of heme and selected nonheme metalloporphyrins on human ALAS1 gene expression in hepatocytes. We found that, upon heme and cobalt protoporphyrin (CoPP) treatments, ALAS1 mRNA levels were down-regulated significantly by ca. 50% or more. Measurement of mRNA in the presence of actinomycin D showed that these down-regulations were due to the decreases in mRNA half-lives. Furthermore, the levels of mitochondrial mature ALAS1 protein were down-regulated by 60-70%, but those of the cytosolic precursor protein were up-regulated by 2-5-fold. Measurement of protein in the presence of cycloheximide (CHX) suggests that elevation of the precursor form is due to the increase in protein half-lives. These results provide novel insights into the mechanisms of heme repressional effects on ALAS1 and provide a rationale for further investigation of CoPP as a therapeutic agent for acute porphyric syndromes.