Analysis of 5' flanking regions of the gamma globin genes from major African haplotype backgrounds associated with sickle cell disease.

There are at least three major African haplotype backgrounds on which the beta s mutation arises. Sequence changes in the immediate 5' flanking area of the gamma-globin genes may account for differences in fetal hemoglobin expression among the three haplotypes. We determined the sequence from -350 to 10 bp 5' of the G gamma and A gamma fetal globin genes from one beta s-containing chromosome on each of the three major haplotype backgrounds. The Senegal chromosome had a T at -158 5' to the G gamma gene; the Benin (BEN) chromosome had an A to G change at -309 5' to the G gamma gene; and the Central African Republic (CAR) chromosome had a C to T change at -271 5' to the A gamma gene. Genomic DNA from patients with sickle cell disease was analyzed using the polymerase chain reaction and radiolabeled allele-specific oligonucleotide probes. The -309 G variant 5' to the G gamma gene is associated with BEN chromosomes, and the -271 T variant 5' to A gamma with CAR. The -309 change was also found on beta A-containing chromosomes, while the -271 change was not. The -309 change may have predated the beta s mutation on the BEN chromosome.

An mRNA stability complex functions with poly(A)-binding protein to stabilize mRNA in vitro.

The stable globin mRNAs provide an ideal system for studying the mechanism governing mammalian mRNA turnover. alpha-Globin mRNA stability is dictated by sequences in the 3' untranslated region (3'UTR) which form a specific ribonucleoprotein complex (alpha-complex) whose presence correlates with mRNA stability. One of the major protein components within this complex is a family of two polycytidylate-binding proteins, alphaCP1 and alphaCP2. Using an in vitro-transcribed and polyadenylated alpha-globin 3'UTR, we have devised an in vitro mRNA decay assay which reproduces the alpha-complex-dependent mRNA stability observed in cells. Incubation of the RNA with erythroleukemia K562 cytosolic extract results in deadenylation with distinct intermediates containing a periodicity of approximately 30 nucleotides, which is consistent with the binding of poly(A)-binding protein (PABP) monomers. Disruption of the alpha-complex by sequestration of alphaCP1 and alphaCP2 enhances deadenylation and decay of the mRNA, while reconstitution of the alpha-complex stabilizes the mRNA. Similarly, PABP is also essential for the stability of mRNA in vitro, since rapid deadenylation resulted upon its depletion. An RNA-dependent interaction between alphaCP1 and alphaCP2 with PABP suggests that the alpha-complex can directly interact with PABP. Therefore, the alpha-complex is an mRNA stability complex in vitro which could function at least in part by interacting with PABP.

Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins.

Defects in RNA-binding proteins have been implicated in human genetic disorders. However, efforts in understanding the functions of these proteins have been hampered by the inability to obtain their mRNA substrates. To identify cognate cellular mRNAs associated with an RNA-binding protein, we devised a strategy termed isolation of specific nucleic acids associated with proteins (SNAAP). The SNAAP technique allows isolation and subsequent identification of these mRNAs. To assess the validity of this approach, we utilized cellular mRNA and protein from K562 cells and alphaCP1, a protein implicated in a-globin mRNA stability, as a model system. Immobilization of an RNA-binding protein with the glutathione-S-transferase (GST) domain enables isolation of mRNA within an mRNP context and the identity of the bound mRNAs is determined by the differential display assay. The specificity of protein-RNA interactions was considerably enhanced when the interactions were carried out in the presence of cellular extract rather than purified components. Two of the mRNAs specifically bound by alphaCP1 were mRNAs encoding the transmembrane receptor protein, TAPA-1, and the mitochondrial cytochrome c oxidase subunit II enzyme, coxII. A specific poly(C)-sensitive complex formed on the TAPA-1 and coxII 3' UTRs consistent with the binding of aCP1. Furthermore, direct binding of purified alphaCP proteins to these 3' UTRs was demonstrated and the binding sites determined. These results support the feasibility of the SNAAP technique and suggest a broad applicability for the approach in identifying mRNA targets for clinically relevant RNA-binding proteins that will provide insights into their possible functions.

Purification and RNA binding properties of the polycytidylate-binding proteins alphaCP1 and alphaCP2.

Regulation of mRNA turnover is a critical control mechanism of gene expression and is influenced by ribonucleoprotein (RNP) complexes that form on cis elements. All mRNAs have an intrinsic half-life and in many cases these half-lives can be altered by a variety of stimuli that are manifested through the formation or disruption of an RNP structure. The stability of alpha-globin mRNA is determined by elements in the 3' untranslated region that are bound by an RNP complex (alpha-complex) which appears to control the erythroid-specific accumulation of alpha-globin mRNA. The alpha-complex could consist of up to six distinct proteins or protein families. One of these families is a prominent polycytidylate binding activity which consists of two highly homologous proteins, alpha-complex proteins 1 and 2 (alphaCP1 and alphaCP2). This article focuses on various methodologies for the detection and manipulation of alphaCP1 and alphaCP2 binding to RNA and details means of isolating and characterizing mRNA bound by these proteins to study mRNA turnover and its regulation.

Simian virus 40 T antigen can transcriptionally activate and mediate viral DNA replication in cells which lack the retinoblastoma susceptibility gene product.

Simian virus 40 T antigen is a multifunctional protein which has recently been shown to form a complex with the retinoblastoma susceptibility gene product (Rb protein) (J.A. DeCaprio, J.W. Ludlow, J. Figge, J.-Y. Shaw, C.-M. Huang, W.-H. Lee, E. Marsilio, E. Paucha, and D.M. Livingston, Cell 54:275-283, 1988; P. Whyte, K.J. Buchkovich, J.M. Horowitz, S.H. Friend, M. Raybuck, R.A. Weinberg, and E. Harlow, Nature (London) 334:124-129, 1988). This interaction may facilitate some of the functions of T antigen. The ability of simian virus 40 T antigen to mediate transcriptional activation and viral DNA replication was tested in human osteosarcoma cell lines U-2OS and Saos-2, which are Rb positive and Rb negative, respectively. Both functions of T antigen were efficient in both cell lines. Hence, these functions can occur in the absence of Rb protein.

Identification of four novel delta-globin gene mutations in Greek Cypriots using polymerase chain reaction and automated fluorescence-based DNA sequence analysis.

The molecular basis of most beta-thalassemia syndromes has been defined, while the spectrum of mutations causing delta-thalassemia is not well characterized. In an attempt to identify such mutations, the region encompassing the delta-globin gene from three Greek Cypriot families suspected of having delta-thalassemia was amplified by polymerase chain reaction (PCR), and DNA sequence determined using an automated fluorescence-based sequencer. Four novel mutations were identified: a G----T change at codon 27 that results in an alanine to serine change; a C----T change at codon 116 converting arginine to cysteine; a T----C change at codon 141 converting leucine to proline; and an AG----GG change at the consensus 3'-acceptor site in IVS-2. While the latter is clearly a thalassemic mutation, the low hemoglobin A2 in the first three may be due to either decreased production or instability of the altered delta-globin chain. All four mutations may be detected by PCR amplification of genomic DNA followed by restriction enzyme digestion. Two mutations abolish restriction sites while two create new cleavage sites. Screening for molecular defects that cause delta-thalassemia or unstable delta-globin by PCR amplification and restriction enzyme digestion will lead to correct diagnosis of beta/delta-thalassemia compound heterozygotes and improved genetic counseling.

Expression studies of delta-globin gene alleles associated with reduced hemoglobin A2 levels in Greek Cypriots.

We previously identified five delta-globin gene alleles associated with reduced hemoglobin (Hb) A2 (Trifillis, P., Ioannou, P., Schwartz, E., and Surrey, S. (1991) Blood 78, 3298-3305). We have now evaluated functional consequences of the changes after expression in COS-1 cells to monitor effects on RNA splicing. In addition, variant Hb A2 tetramers were expressed in yeast to assess effects of amino acid changes on oxygen binding and stability to heat and mechanical agitation. The G --> T change at codon 27 and the A --> G change in IVS-2 both affect RNA splicing, whereas the C --> T change at codon 97 and the AT deletion in IVS-2 have no effect. Oxygen equilibrium curves of the Hb A2 variants expressed in yeast were similar to that of wild type Hb A2. None of the three variant Hb A2 tetramers (Thr --> Ile at codon 4 (Hb deltaT4I), Ala --> Ser at codon 27 (Hb deltaA27S), and Arg --> Cys at codon 116 (Hb deltaR116C)) showed decreased heat stability compared with Hb A2, whereas the Hb deltaT4I variant showed highest instability to mechanical agitation. Co-expression in yeast of alpha-globin chain and the delta-chain variant containing a Leu --> Pro change at codon 141 yielded no identifiable tetramers, suggesting lack of assembly or severe tetramer instability. These studies show the probable cause for decreased Hb A2 for two alleles is due to defective splicing, whereas decreased protein stability, increased tetramer association with red cell membranes, increased interdisulfide bond formation of delta-chains, which inhibits assembly with alpha-chains, and/or reduced assembly is suggested for the other three alleles.

Characterization of the biochemical properties of the human Upf1 gene product that is involved in nonsense-mediated mRNA decay.

The Upf1 protein in yeast has been implicated in the modulation of efficient translation termination as well as in the accelerated turnover of mRNAs containing premature stop codons, a phenomenon called nonsense-mediated mRNA decay (NMD). A human homolog of the yeast UPF1, termed HUpf1/RENT1, has also been identified. The HUpf1 has also been shown to play a role in NMD in mammalian cells. Comparison of the yeast and human UPF1 proteins demonstrated that the amino terminal cysteine/histidine-rich region and the region comprising the domains that define this protein as a superfamily group I helicase have been conserved. The yeast Upf1p demonstrates RNA-dependent ATPase and 5' --> 3' helicase activities. In this paper, we report the expression, purification, and characterization of the activities of the human Upf1 protein. We demonstrate that human Upf1 protein displays a nucleic-acid-dependent ATPase activity and a 5'--> 3' helicase activity. Furthermore, human Upf1 is an RNA-binding protein whose RNA-binding activity is modulated by ATP. Taken together, these results indicate that the activities of the Upf1 protein are conserved across species, reflecting the conservation of function of this protein throughout evolution.

Analysis of delta-globin gene mutations in Greek cypriots.

We recently described four delta-globin gene mutations in Greek Cypriots studied by polymerase chain reaction (PCR) amplification and automated fluorescence-based DNA sequence analysis (Blood 78:3298, 1991). Selective restriction enzyme digestion of PCR products facilitated direct mutation detection. Twenty-eight additional samples from unrelated Cypriots with Hb A2 levels ranging from 0.6% to 3.6% were studied by PCR and showed the following: twelve had the delta 27 (ala-->ser) mutation, one was heterozygous for the delta IVS-2 AG-->GG change, and none had either the delta 116 (arg-->cys) or delta 141 (leu-->pro) mutations. The remaining samples were divided into two groups: 11 with borderline normal Hb A2 values that were not pursued; and four with abnormal Hb A2 values. The delta-globin genes from these four samples were sequenced and the same four changes identified in each: a C-->T at -199, a C-->T at codon 4 (thr-->ile), a silent C-->T at codon 97, and an AT deletion at position 722 in IVS-2. The codon 4 change abolishes a Ple I site whereas the codon 97 creates an Nla III site, thus facilitating rapid identification. All four changes are in cis position, suggesting that the -199 C-->T, the C-->T at codon 97, and the AT deletion in IVS-2 are neutral polymorphisms present on the codon 4 (thr-->ile) chromosome. DNA haplotype analysis suggests all five delta-globin gene mutant alleles arose independently on different chromosomal backgrounds.

Polymerization of three hemoglobin A2 variants containing Val6 and inhibition of hemoglobin S polymerization by hemoglobin A2.

To understand determinants for hemoglobin (Hb) stability and Hb A2 inhibition of Hb S polymerization, three Valdelta6 Hb A2 variants (Hb A2 deltaE6V, Hb A2 deltaE6V,deltaQ87T, and Hb A2 deltaE6V, deltaA22E,deltaQ87T) were expressed in yeast, and stability to mechanical agitation and polymerization properties were assessed. Oxy forms of Hb A2 deltaE6V and Hb A2 deltaE6V,deltaQ87T were 2- and 1.6-fold, respectively, less stable than oxy-Hb S, while the stability of Hb A2 deltaE6V,deltaA22E,deltaQ87T was similar to that of Hb S, suggesting that Aladelta22 and Glndelta87 contribute to the surface hydrophobicity of Hb A2. Deoxy Hb A2 deltaE6V polymerized without a delay time, like deoxy Hb F gammaE6V, while deoxy Hb A2 deltaE6V,deltaQ87T and deoxy Hb A2 deltaE6V,deltaA22E,deltaQ87T polymerized after a delay time, like deoxy Hb S, suggesting that beta87 Thr is required for the formation of nuclei. Deoxy Hb F gammaE6V,gammaQ87T showed no delay time and required a 3.5-fold higher concentration than deoxy Hb S for polymerization, suggesting that Thr effects on Valdelta6 Hb A2 and Valgamma6 Hb F variants are different. Mixtures of deoxy Hb S/Hb A2 deltaE6V,deltaQ87T polymerized, like deoxy Hb S, while polymerization of Hb S/Hb A2 deltaE6V mixtures was inhibited, like Hb S/Hb F gammaE6V mixtures. These results suggest alpha2betaSdelta6 Val, 87 Thr hybrids and Hb A2 deltaE6V,deltaQ87T participate in Hb S nucleation, while only 50% of alpha2betaSdelta6 Val hybrids and none of the Hb A2 deltaE6V participate. These findings are in contrast to those of mixtures of Hb S with Hb F gammaE6V or Hb F gammaE6V,Q87T, which both inhibit Hb S polymerization. Our results also suggest participation in nucleation of some alpha2betaSdelta hybrids in A2S mixtures but not alpha2betaSgamma hybrids in FS mixtures.

Different hematological phenotypes caused by the interaction of triplicated alpha-globin genes and heterozygous beta-thalassemia.

The pathophysiology and clinical severity of beta-thalassemia are related to the degree of alpha/non-alpha-chain imbalance. A triplicated alpha-globin gene locus can exacerbate effects of excess alpha-chains caused by a defective beta-globin gene, although this is not observed in all cases. Extensive studies on this condition are lacking. We report a group of 17 patients who are heterozygous for both the alpha alpha alpha(anti-3.7) allele and a mutation in the beta-globin gene cluster. Their clinical phenotypes varied: six had mild anemia with microcytosis and hypochromia, while 11 had more severe anemia with splenomegaly requiring splenectomy (three cases) and blood transfusions (four cases). Different phenotypes were also evident in the presence of the same beta-thalassemia mutation: in one family, two individuals had the same alpha- and beta-globin genotypes but presented with different hematologic phenotypes. In addition, the complex interaction involving a triplicated alpha-globin gene, beta39- and delta+27-thalassemia mutations is studied in a family with two siblings presenting with hemolytic anemia, normal Hb A2 and increased Hb F. Analysis of this series of patients suggests that additional genetic determinants play a role in modulating phenotypic expression in individuals with identical alpha- and beta-globin genotypes. Interaction with a triplicated alpha-gene can play a role in the clinical presentation of patients with defective beta-globin gene expression and should be considered in the diagnosis of atypical cases.