3' non-translated region of human alpha-globin messenger RNA does not affect translation efficiency

The relative translation efficiency of three synthetic alpha-globin mRNAs differing by their 3' non-translated end was measured in vitro in a rabbit reticulocyte lysate. Results showed that substituting the 3' non-translated end of human alpha 2 globin mRNA by the 3' non-translated end of chimpanzee alpha 1 or alpha 2 mRNAs has no effect on translation efficiency. In contrast, the introduction of the alpha-Quong-Sze mutation (alpha 125, Leu----Pro) in human alpha 2 mRNA led to a 50% apparent reduction in globin synthesis due to the instability of the alpha-Quong-Sze globin chain. We conclude that human alpha 1 and alpha 2 globin mRNAs have the same translation efficiency, and that the reduction, previously reported, in the kinetics of alpha-globin synthesis by alpha 2 mRNA carrying the alpha-Quong-Sze mutation is due to the instability of the alpha-Quong-Sze globin chain only.     

Primate evolution of the alpha-globin gene cluster and its Alu-like repeats

The arrangement of alpha-globin genes in Old World and New World monkeys and a prosimian, galago, has been determined by restriction mapping. Recombinant DNAs containing galago and Old World monkey alpha-globin genes have been isolated and subjected to a partial sequence determination for comparison to alpha-globin genes in human, chimpanzee and non-primate mammals. The results of this extensive structural analysis are relevant to several topics concerning the evolution of primate alpha-globin genes and Alu family repeats. All orders of higher primates (i.e. Old and New World monkeys, chimpanzee and human) have the same arrangement of alpha-globin genes. In contrast, the arrangement and correction of galago alpha-globin genes differ from those of higher primates, but are similar to those of non-primate mammals. The 5' and 3'-flanking regions of the human alpha 1 gene are orthologous to the corresponding region in galago, identifying the human alpha 2 gene as the more recently duplicated gene. The human psi alpha 1 gene is found to be inactivated after divergence of the human and galago lineages but prior to the divergence of human and monkey. Orthologous Alu family members in human and monkey DNAs indicate that the dispersion of some Alu repeats occurred prior to the divergence of these lineages. However, the Alu-like repeats of prosimian and higher primates result from entirely independent events giving rise to different repeat elements inserted at distinct genomic positions.     

Human alpha-globin gene expression. The dominant role of the alpha 2-locus in mRNA and protein synthesis

The two human alpha-globin genes, alpha 1 and alpha 2, are coexpressed in normal erythroid cells and encode identical alpha-globin protein products. Based upon genetic studies, it has been assumed that these two adjacent and highly homologous genes are equally expressed. In previous studies we have, however, demonstrated that the alpha 2 gene encodes a 2-3-fold higher steady state level of mRNA than the alpha 1 gene. In the present study, we monitor the relative levels of protein production from these two loci by quantitating the synthesis of specific alpha-globin structural mutants encoded by each alpha-globin gene. These values are then used to infer the relative contributions of the normal alpha 1 and alpha 2 loci to total alpha-globin production. The results of eight separate studies, each based upon a different alpha-globin structural mutant mapped to either the alpha 1 or the alpha 2 locus, are internally consistent. The data demonstrate that the alpha 2 gene encodes 2-3-fold more protein than the alpha 1 gene. These results suggest that the human alpha-globin gene cluster contains a major and a minor locus. The dominant expression of the alpha 2 gene predicts a greater impact of mutations at this locus, in comparison to mutations at the alpha 1 locus, in the generation of the alpha-thalassemia phenotype.     

Identification of two KH domain proteins in the alpha-globin mRNP stability complex.

Accumulation of globin mRNAs during erythroid differentiation is dependent on their extraordinary stability. The longevity of human alpha-globin mRNA is associated with a ribonucleoprotein complex (alpha-complex) formed on the 3' untranslated region (3'UTR). One or more of the proteins within this alpha-complex contain strong polycytosine [poly(C)] binding (alpha PCB) activity. In the present report we purify alpha PCB activity from human erythroid K562 cells. Although not able to bind the alpha-globin 3'UTR directly, alpha PCB activity is sufficient to complement alpha-complex formation in a cytosolic extract depleted of poly(C) binding activity. Peptide microsequencing demonstrates that alpha PCB activity contains two structurally related poly(C) binding proteins. These two proteins, alpha-complex protein (alpha CP)-1 and -2, have an overall structural identity of 80% and contain three repeats of the K homology (KH) domain which is found in a subset of RNA binding proteins. Epitope-tagged recombinant alpha CP-1 and alpha CP-2 expressed in cells are each incorporated into the alpha-complex. We conclude that alpha CP-1 and alpha CP-2, members of the KH domain RNA binding protein family, are involved in formation of a sequence-specific alpha-globin mRNP complex associated with alpha-globin mRNA stability. As such this represents the first example of a specific function for this class of proteins and suggests potential roles for other members of this protein family.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

The KH-domain protein alpha CP has a direct role in mRNA stabilization independent of its cognate binding site

Previous studies suggest that high-level stability of a subset of mammalian mRNAs is linked to a C-rich motif in the 3' untranslated region (3'UTR). High-level expression of human alpha-globin mRNA (h alpha-globin mRNA) in erythroid cells has been specifically attributed to formation of an RNA-protein complex comprised of a 3'UTR C-rich motif and an associated 39-kDa poly(C) binding protein, alpha CP. Documentation of this RNA-protein alpha-complex has been limited to in vitro binding studies, and its impact has been monitored by alterations in steady-state mRNA. Here we demonstrate that alpha CP is stably bound to h alpha-globin mRNA in vivo, that alpha-complex assembly on the h alpha-globin mRNA is restricted to the 3'UTR C-rich motif, and that alpha-complex assembly extends the physical half-life of h alpha-globin mRNA selectively in erythroid cells. Significantly, these studies also reveal that an artificially tethered alpha CP has the same mRNA-stabilizing activity as the native alpha-complex. These data demonstrate a unique contribution of the alpha-complex to h alpha-globin mRNA stability and support a model in which the sole function of the C-rich motif is to selectively tether alpha CP to a subset of mRNAs. Once bound, alpha CP appears to be fully sufficient to trigger downstream events in the stabilization pathway.     

Intergenic DNA sequences flanking the pseudo alpha globin genes of human and chimpanzee.

We have determined the sequence of 2400 base pairs upstream from the human pseudo alpha globin (psi alpha) gene, and for comparison, 1100 base pairs of DNA within and upstream from the chimpanzee psi alpha gene. The region upstream from the promoter of the psi alpha gene shows no significant homology to the intergenic regions of the adult alpha 2 and alpha 1 globin genes. The chimpanzee gene has a coding defect in common with the human psi alpha gene, showing that the product of this gene, if any, was inactivated before the divergence of human and chimpanzee. However the chimpanzee gene contains a normal ATG initiation codon in contrast to the human gene which has GTG as the initiation codon. The psi alpha genes of both human and chimpanzee are flanked by the same Alu family member. The structure and position of this repeat have not been altered since the divergence of human and chimpanzee, and it is at least as well conserved as its immediate flanking sequence. Comparing human and chimpanzee, the 300 bp Alu repeat has accumulated only two base substitutions and one length mutation; the adjacent 300 bp flanking region has accumulated five base substitutions and twelve length mutations.     

Analysis of a 70 kb segment of DNA containing the human zeta and alpha-globin genes linked to their regulatory element (HS-40) in transgenic mice.

We have ligated two cosmids through an oligonucleotide linker to produce a single fragment spanning 70 kb of the human alpha-globin cluster, in which the alpha-like globin genes (zeta 2, alpha 2 and alpha 1), their regulatory element (HS-40) and erythroid-specific DNase I hypersensitive sites accurately retain their normal genomic organization. The zeta (embryonic) and alpha (embryonic, fetal and adult) globin genes were expressed in all 17 transgenic embryos. Similarly, all fetal and adult mice from seven transgenic lines that contained one or more copies of the fragment, produced up to 66% of the level of endogenous mouse alpha-globin mRNA. However, as for smaller constructs containing these elements, human alpha-globin expression was not copy number dependent and decreased by 1.5-9.0 fold during development. These findings suggest that either it is not possible to obtain full regulation of human alpha-globin expression in transgenic mice or, more likely, that additional alpha-globin regulatory elements lie beyond the 70 kb segment of DNA analysed.     

Complementary change in cis determinants and trans factors in the evolution of an mRNP stability complex.

RNA-protein (RNP) complexes play significant roles in the fate and expression of mRNAs. The prolonged half-life of human alpha-globin mRNA, a major determinant of normal erythroid differentiation, is dependent on the assembly of a sequence-specific 3'-untranslated region (3'UTR) RNP (alpha-complex). We demonstrate that the stability of murine alpha-globin mRNA is controlled by a parallel mechanism. Unexpectedly, however, the respective 3'UTR RNP complexes that stabilize the h(alpha)- and m(alpha)-globin mRNAs differ in structure. While the cis determinants in both species are encoded in polypyrimidine tracks, the human determinant is C-rich (CCUCC motif) while the mouse alpha-3'UTR consists of an equal distribution of Cs and Us (CCUUCU motif). The protein components of the corresponding human and murine alpha-complexes differ in a complementary manner: the previously described 39 kDa poly(C) binding protein (PCBP) present in the human alpha-complex is replaced in the mouse alpha-complex by a 48 kDa cytoplasmic poly(CU) binding protein (CUBP). These results reveal that drift in the primary sequences of the alpha-globin mRNA 3'UTR polypyrimidine tracks in a comparison between mouse and human is paralleled by an alteration in the composition of the corresponding trans-acting components. Surprisingly, these structurally distinct complexes appear to perform the identical function of stabilizing the corresponding alpha-globin mRNAs.     

Opposite responses of rabbit and human globin mRNAs to translational inhibition by cap analogues

The translational efficiency of an mRNA may be determined at the step of translational initiation by the efficiency of its interaction with the cap binding protein complex. To further investigate the role of these interactions in translational control, we compare in vitro the relative sensitivities of rabbit and human alpha- and beta-globin mRNAs to translational inhibition by cap analogues. We find that rabbit beta-globin mRNA is more resistant to translational inhibition by cap analogues than rabbit alpha-globin mRNA, while in contrast, human beta-globin mRNA is more sensitive to cap analogue inhibition than human alpha-globin mRNA. This opposite pattern of translational inhibition by cap analogues of the rabbit and human alpha- and beta-globin mRNAs is unexpected as direct in vivo and in vitro comparisons of polysome profiles reveal parallel translational handling of the alpha- and beta-globin mRNAs from these two species. This discordance between the relative translational sensitivities of these mRNAs to cap analogues and their relative ribosome loading activities suggests that cap-dependent events may not be rate limiting in steady-state globin translation.     

Concerted evolution of the primate immunoglobulin alpha-gene through gene conversion.

We determined four nucleotide sequences of the hominoid immunoglobulin alpha (C alpha) genes (chimpanzee C alpha 2, gorilla C alpha 2, and gibbon C alpha 1 and C alpha 2 genes), which made possible the examination of gene conversions in all hominoid C alpha genes. The following three methods were used to detect gene conversions: 1) phenetic tree construction; 2) detection of a DNA segment with extremely low variability between duplicated C alpha genes; and 3) a site by site search of shared nucleotide changes between duplicated C alpha genes. Results obtained from method 1 indicated a concerted evolution of the duplicated C alpha genes in the human, chimpanzee, gorilla, and gibbon lineages, while results obtained from method 2 suggested gene conversions in the human, gorilla, and gibbon C alpha genes. With method 3 we identified clusters of shared nucleotide changes between duplicated C alpha genes in human, chimpanzee, gorilla, and gibbon lineages, and in their hypothetical ancestors. In the present study converted regions were identified over the entire C alpha gene region excluding a few sites in the coding region which have escaped from gene conversion. This indicates that gene conversion is a general phenomenon in evolution, that can be clearly observed in non-functional regions.     

Complementation of α-thalassaemia in α-globin Knockout Mice with a 191 kb Transgene Containing the Human α-Globin Locus

alpha-thalassaemia is an inherited blood disorder caused by a decrease in the synthesis of alpha-globin due to mutations in one or both of the alpha-globin genes located on human chromosome 16. A 191 kb transgene derived from a sequenced bacterial artificial chromosome (BAC) clone carrying the human alpha-globin gene cluster, together with about 100 kb of sequence upstream of DNase1 hypersensitive site HS-40 and 30 kb downstream of the alpha1-globin gene, was introduced into fertilised mouse oocytes by pronuclear microinjection. Three transgenic founder mice were obtained. Analysis of one transmitting line by fluorescent in situ hybridisation and quantitative PCR demonstrated a single copy integration of the human alpha-globin transgene on chromosome 1. Analysis of haemoglobins from the peripheral blood by cellulose acetate electrophoresis and high performance liquid chromatography (HPLC) demonstrated synthesis of human alpha-globin to about 36% of the level of each mouse alpha-globin locus. Breeding of transgenic mice with mice heterozygous for a knockout (KO) deletion of both murine alpha-globin genes showed that the human alpha-globin locus restored haemoglobin levels and red cell distribution width to normal in double heterozygous mice and significantly normalised other haematological parameters. Interestingly the human transgene also induced a significant increase in red cell production and haematocrit above wild type values. This is the first report demonstrating complementation of a murine alpha-globin KO mutation by human alpha-globin gene expression from an intact human alpha-globin locus. The transgenic mouse model described in this report should be very useful for the study of human alpha-globin gene regulation and for the development of strategies to down regulate alpha-globin production as a means of ameliorating the severity of beta-thalassaemia.     

High-level production of human alpha- and beta-globins in insect cells.

High-level production of human alpha- and beta-globins in cultured Spodoptera frugiperda (Sf-9) cells infected with recombinant baculoviruses is described. The expressed globins are produced to 70-140 mg protein/liter of cell culture or 5-10% of the total cellular protein. Two recombinant baculoviruses for alpha-globin, H alpha and H beta alpha, differ in their construction in that the 5'-untranslated region of the beta-globin gene is inserted 5' to the alpha-globin mRNA coding region in H beta alpha. This insertion results in a 40% increase in yield of alpha-globin over that of H alpha. Consistent with previous observations of the processing of recombinant proteins in Sf-9 cells, both alpha- and beta-globins expressed in Sf-9 cells are correctly processed to remove the initiating methionine from the amino termini of the globins. Sequencing of the expressed globins in Sf-9 cells confirms their identity with globins purified from human normal adult hemoglobin.     

Codon usage in the vertebrate hemoglobins and its implications

A study of codon usage in vertebrate hemoglobins revealed an evolutionary trend toward elevated numbers of CpG codon boundary pairs in mammalian hemoglobin alpha genes. Selection for CpG codon boundaries countering the generally observed CpG suppression is strongly suggested by these data. These observations parallel recently published experimental results that indicate that constitutive expression of the human alpha-globin gene appears to be determined by regulatory information encoded within the structural gene. The possibility is raised that, in the absence of selection, CpG decay can be used to date the evolutionary origin of a mammalian alpha pseudogene from its active alpha gene.      

Rhesus macaque class I duplicon structures, organization, and evolution within the alpha block of the major histocompatibility complex

The alpha block of the human and chimpanzee major histocompatibility complex (MHC) class I genomic region contains 10 to 11 duplicated MHC class I genes, including the HLA/Patr-A, -G, and -F genes. In comparison, the alpha block of the rhesus macaque (Macaca mulatta, Mamu) has an additional 20 MHC class I genes within this orthologous region. The present study describes the identification and analysis of the duplicated segmental genomic structures (duplicons) and genomic markers within the alpha block of the rhesus macaque and their use to reconstruct the duplication history of the genes within this region. A variety of MHC class I genes, pseudogenes, transposons, and retrotransposons, such as Alu and ERV16, were used to categorize the 28 duplicons into four distinct structural categories. The phylogenetic relationship of MHC class I genes, Alu, and LTR16B sequences within the duplicons was examined by use of the Neighbor-Joining (NJ) method. Two single-duplicon tandem duplications, two polyduplicon tandem duplications with an accompanying inversion product per duplication, eight polyduplicon tandem duplications steps, 12 deletions, and at least two recombinations were reconstructed to explain the highly complex organization and evolution of the 28 duplicons (nine inversions) within the Mamu alpha block. On the basis of the phylogenetic evidence and the reconstructed tandem duplication history of the 28 duplicons, the Mamu/Patr/HLA-F ortholog was the first MHC class I gene to have been fixed without further duplication within the alpha block of primates. Assuming that the rhesus macaque and the chimpanzee/human lineages had started with the same number of MHC class I duplicons at the time of their divergence approximately 24 to 31 MYA, then the number of genes within the alpha block have been duplicated at an approximately three times greater rate in the rhesus macaque than in either the human or chimpanzee.     

The mammalian alphaD-globin gene lineage and a new model for the molecular evolution of alpha-globin gene clusters at the stem of the mammalian radiation

We have explored the evolution of the alpha-globin gene family by comparative sequence and phylogenetic analyses of mammalian alpha-globin genes. Our analyses reveal the existence of a new alpha-globin gene lineage in mammals that is related to the alpha(D)-globin genes of birds, squamates and turtles. The gene is located in the middle of the alpha-globin gene cluster of a marsupial, Sminthopsis macroura and of humans. It exists in a wide variety of additional mammals, including pigs, cows, cats, and dogs, but is a pseudogene in American marsupials. Evolutionary analyses suggest that the gene has generally evolved under purifying selection, indicative of a functional gene. The presence of mRNA products in humans, pigs, and cows also suggest that the gene is expressed and likely to be functional. The analyses support the hypothesis that the alpha(D)-globin gene lineage has an ancient evolutionary origin that predates the divergence of amniotes. The structural similarity of alpha-globin gene clusters of marsupials and humans suggest that an eight gene cluster (5'-zeta2-zeta1-alpha(D)-alpha3-alpha2-alpha1-theta-omega-3'), including seven alpha-like genes and one beta-like globin gene (omega-globin) existed in the common ancestor of all marsupial and eutherian mammals. This basic structure has remained relatively stable in marsupials and in the lineage leading to humans, although omega-globin has been lost from the alpha-globin gene cluster of humans.     

CACCC and GATA-1 sequences make the constitutively expressed alpha-globin gene erythroid-responsive in mouse erythroleukemia cells.

Although the human alpha-globin and beta-globin genes are co-regulated in adult life, they achieve the same end by very different mechanisms. For example, a transfected beta-globin gene is expressed in an inducible manner in mouse erythroleukemia (MEL) cells while a transfected alpha-globin gene is constitutively expressed at a high level in induced and uninduced MEL cells. Interestingly, when the alpha-globin gene is transferred into MEL cells as part of human chromosome 16, it is appropriately expressed in an inducible manner. We explored the basis for the lack of erythroid-responsiveness of the proximal regulatory elements of the human alpha-globin gene. Since the alpha-globin gene is the only functional human globin gene that lacks CACCC and GATA-1 motifs, we asked whether their addition to the alpha-globin promoter would make the gene erythroid-responsive in MEL cells. The addition of each of these binding sites to the alpha-globin promoter separately did not result in inducibility in MEL cells. However, when both sites were added together, the alpha-globin gene became inducible in MEL cells. This suggests that erythroid non-responsiveness of the alpha-globin gene results from the lack of erythroid binding sites and is not necessarily a function of the constitutively active, GC rich promoter.