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Locus control regions (LCRs) comprise sets of DNA elements capable of establishing autonomous chromatin domains that support robust and physiologically appropriate expression of target genes, often working over extensive distances. Human growth hormone (hGH-N) expression in the pituitary is under the regulation of a well characterized LCR containing four DNase I hypersensitive sites (HSs). The two pituitary-specific HS, HSI and HSII, are located 14.5 and 15.5 kb 5' to the hGH-N promoter. HSI is essential for activation of hGH-N during pituitary development and for sustaining robust activity in the adult. To determine whether the closely linked HSII has a role in hGH-N expression, it was deleted from a previously validated hGH/P1 transgene. Analysis of three independent hGH/P1(ΔHSII) transgenic mouse lines revealed that this deletion had no adverse effect on the formation of HSI, yet resulted in a substantial loss (70%) in hGH-N mRNA expression. This loss of expression was accompanied by a corresponding reduction in recruitment of the pituitary-specific transcription factor Pit-1 to the hGH-N promoter and a selective decrease in promoter occupancy of the elongation-linked isoform of RNA polymerase II. Sufficiency of HSI and HSII in LCR activity was explored by establishing two additional sets of mouse transgenic lines in which DNA segments containing these HS were positioned within the λ phage genome. In this "neutral" DNA context, HSII was required for the recruitment of HAT activity. These data establish HSII as a nonredundant component of the hGH LCR essential for establishment of robust levels of hGH-N gene expression.

The alpha-globin poly(C)-binding proteins (alphaCPs) comprise an abundant and widely expressed set of K-homolog domain RNA-binding proteins. alphaCPs regulate the expression of a number of cellular and viral mRNAs at the levels of splicing, stability, and translation. Previous surveys have identified 160 mRNAs that are bound by alphaCP in the human hematopoietic cell line, K562. To explore the functions of these alphaCP/mRNA interactions, we identified mRNAs whose levels are altered in K562 cells acutely depleted of the two major alphaCP proteins, alphaCP1 and alphaCP2. Microarray analysis identified 27 mRNAs that are down-regulated and 14 mRNAs that are up-regulated in the alphaCP1/2-co-depleted cells. This alphaCP1/2 co-depletion was also noted to inhibit cell proliferation and trigger a G(1) cell cycle arrest. Targeted analysis of genes involved in cell cycle control revealed a marked increase in p21(WAF) mRNA and protein. Analysis of mRNP complexes in K562 cells demonstrates in vivo association of p21(WAF) mRNA with alphaCP1 and alphaCP2. In vitro binding assays indicate that a 127-nucleotide region of the 3'-untranslated region of p21(WAF) interacts with both alphaCP1 and alphaCP2, and co-depletion of alphaCP1/2 results in a marked increase in p21(WAF) mRNA half-life. p21(WAF) induction and G(1) arrest in the alphaCP1/2-co-depleted cells occur in the absence of p53 and are not observed in cells depleted of the individual alphaCP isoforms. The apparent redundancy in the actions of alphaCP1 and alphaCP2 upon p21(WAF) expression correlates with a parallel redundancy in their effects on cell cycle control. These data reveal a pivotal role for alphaCP1 and alphaCP2 in a p53-independent pathway of p21(WAF) control and cell cycle progression.

The KH domain mediates RNA binding in a wide range of proteins. Here we investigate the RNA-binding properties of two abundant RNA-binding proteins, alphaCP-2KL and heterogeneous nuclear ribonucleoprotein (hnRNP) K. These proteins constitute the major poly(C) binding activity in mammalian cells, are closely related on the basis of the structures and positioning of their respective triplicated KH domains, and have been implicated in a variety of post-transcriptional controls. By using SELEX, we have obtained sets of high affinity RNA targets for both proteins. The primary and secondary structures necessary for optimal protein binding were inferred in each case from SELEX RNA sequence comparisons and confirmed by mutagenesis and structural mapping. The target sites for alphaCP-2KL and hnRNP K were both enriched for cytosine bases and were presented in a single-stranded conformation. In contrast to these shared characteristics, the optimal target sequence for hnRNP K is composed of a single short "C-patch" compatible with recognition by a single KH domain whereas that for alphaCP-2KL encompassed three such C-patches suggesting more extensive interactions. The binding specificities of the respective SELEX RNAs were confirmed by testing their interactions with native proteins in cell extracts, and the importance of the secondary structure in establishing an optimized alphaCP-2KL-binding site was supported by comparison of SELEX target structure with that of the native human alpha-globin 3'-untranslated region. These data indicate that modes of macromolecular interactions of arrayed KH domains can differ even among closely related KH proteins and that binding affinities are substantially dependent on the presentation of the target site within the RNA secondary structure.

The extraordinary stability of globin mRNAs permits their accumulation to over 95% of total cellular mRNA during erythroid differentiation. The stability of human alpha-globin mRNA correlates with assembly of a sequence-specific ribonucleoprotein complex at its 3'-untranslated region. A naturally occurring anti-termination mutation, Constant Spring (CS), which permits ribosomes to enter the 3'-untranslated region of the alpha-globin mRNA, results in accelerated mRNA decay. To study the mechanism of this destabilization in vivo, we established transgenic mouse lines carrying the human alphaCS gene. Relative to wild-type human alpha-globin mRNA (alphawt), alphaCS mRNA is destabilized in marrow erythroid cells. The poly(A) tails of both the alphaCS and alphawt mRNAs show a periodicity of 20-25 nucleotides consistent with phased binding of poly(A) binding proteins. However, the mean size of poly(A) tails of the unstable alphaCS mRNA is significantly shorter than that of the alphawt mRNA. Unexpectedly, the alphawt and alphaCS mRNAs are of equal stability in peripheral reticulocytes, where their respective poly(A) tails shorten coordinately. These findings demonstrate a characteristic organization of the poly(A) tail on alpha-globin mRNA which is maintained during normal and accelerated decay, a correlation between poly(A) metabolism and anti-termination-mediated accelerated mRNA turnover, and a switch in the mechanism of mRNA decay during erythroid terminal differentiation.

The human growth hormone/human chorionic somatomammotropin (hGH/hCS) gene cluster contains five genes: hGH-N, hGH-V, hCS-A, hCS-B, and hCS-L. The expression of the first four genes has been well documented. In contrast, the hCS-L gene has been considered a pseudogene inactivated by loss of the normal intron 2 splice donor site. Previously our laboratory has shown that hCS-L transcripts are present in human placenta and that their levels are induced during the second trimester. More detailed studies of hCS-L transcript processing and mRNA structure are hindered by overwhelming levels of the structurally similar hCS-A and hCS-B transcripts in the placenta. To circumvent this problem, we have established stably transfected cell lines selectively expressing the hCS-L gene. Analysis of hCS-L mRNA from these cell lines demonstrates at least five major alternative splicing pathways, four of which could be confirmed qualitatively by parallel analysis of placental RNA. This analysis reveals an unexpectedly high frequency of exon 2 skipping (73%) as well as utilization of three competing exon 3 splice acceptor sites. Since exon 2 encodes the signal peptide, the majority of hCS-L transcripts are unable to express a secreted protein. Three of the defined hCS-L mRNAs contain an extended open reading frame similar to that present in the functional GH and CS genes. All three of these hCS-L transcripts are of minor abundance and only two, hCS-L(L) and hCS-L(L'), contain exon 2. In vitro translation and signal peptide processing of hCS-L(L) mRNA yields a 20-kDa hCS-L isoform in vitro. These data confirm the placental expression of the hCS-L gene, demonstrate surprising complexity in the splicing of its transcripts, predict that the majority of processed hCS-L mRNAs are nonfunctional, and identify specific, low abundance mRNAs that may encode novel gestational hormones.

The human growth hormone family of peptide hormones is encoded by five genes, pituitary growth hormone (hGH-N), and four placentally expressed genes, growth hormone variant (hGH-V), chorionic somatomammotropin A and B (hCS-A, hCS-B), and prolactin (hPrl). As part of an effort to define the local effects of the placentally expressed members of the GH/Prl family of hormones on the placenta, we have identified an isoform (hGHRd3) of the growth hormone receptor expressed in the placental villi. hGHRd3 mRNA differs from the liver GHR mRNA by the deletion of a 66-base pair segment encoding exon 3. In this study we show that hGHRd3 mRNA encodes a stable and functional receptor. hGHRd3 mRNA is efficiently translated and processed in a rabbit reticulocyte lysate translation system as well as in an in vivo Xenopus laevis oocyte expression system. In Xenopus oocytes hGHRd3 is stably integrated into the cell membrane and binds and internalizes ligand as efficiently as hGHR. hGHRd3 binds all three of the placentally expressed members of the GH/Prl gene family (hGH-V, hCS, and Prl) as well as both the 22 and 20 kDa isoforms of the pituitary hGH-N. The results of the present study strongly support the expression of a functional hGHRd3 isoreceptor in the placenta which may serve in autocrine, paracrine, and/or endocrine activation.

Consensus sequences at the splice donor, splice acceptor, and lariat branch point regions are necessary but insufficient determinants of splice-site selection in nuclear precursor mRNAs. Sequences outside of these regions can have a significant effect on the utilization of splice sites. Although the mode of action of such sequences is undefined in most cases, higher order RNA structures have been suggested as a potential contributor to splice-site selection. During a detailed analysis of the splicing patterns of the human growth hormone transcript, we located 2 bases in the vicinity of the exon 3 major splice-acceptor site (B) which facilitate the utilization of a competing downstream acceptor (B'). The effects of a series of site-specific mutations on the splicing pattern demonstrate that these 2 bases function by stabilizing a specific stem-loop structure in the native transcript. This defined secondary structure selectively encompasses the upstream B splice-acceptor site together with its lariat branch point region. Increasing the predicted stability of this stem by point mutations results in a corresponding shift in splicing towards the alternative B' splice-acceptor site. These results indicate that a specific secondary structure within the native human growth hormone transcript controls the relative utilization of two competing splice-acceptor sites with the consequent generation of two functionally distinct hormone isoforms.

Four of the five genes in the human growth hormone gene cluster are expressed in the villous layer of the placenta. We report that the expression of these genes, hCS-A, hCS-B, hCS-L, and hGH-V, are coordinately induced during fetal development, increasing between 12 and 20 weeks of gestation and then plateauing through term. Within the context of this coordinate activation, these genes are expressed at widely different levels and are alternatively spliced in different patterns. There is a developmentally regulated switch in the relative expression of the two chorionic somatomammotropin genes, hCS-A and hCS-B. Starting from approximately equal levels at 8 weeks of gestation, hCS-A is expressed 5-fold more abundantly than hCS-B by term. The proportion of alternatively spliced hGH-V transcripts that retain intron 4 is also developmentally regulated, increasing 3-fold during gestation to 15% at term. A small percentage of hCS transcripts stably retain intron 4 through gestation, the majority derived from the hCS-A gene. hCS-L transcripts undergo two distinct, developmentally stable, splicing pathways between exons 2 and 3. These result from the absence of the normal splice-donor site in intron 2 and the activation of two cryptic splice-acceptor sites. Despite high levels of sequence identity, the four placentally expressed genes in the growth hormone cluster generate a complex set of mRNAs based on alternative splicing and developmental regulation during gestation.

The human growth hormone (hGH-N) and the closely related human growth hormone-variant (hGH-V) genes differ in their patterns of splice-site selection. In 9% of hGH-N transcripts exon 2 is spliced to an alternative acceptor site located 45 bases within exon 3. mRNA spliced in this manner encodes a 20-kDa hGH-N isoform instead of the normal 22-kDa hGH-N. The hGH-V transcript fails to utilize this alternative splicing pathway. A region of the hGH-N and hGH-V genes critical to this difference in splice-site selection has been identified by reciprocal exchange of corresponding genomic fragments and has been defined in detail by a series of reciprocal site-specific exchanges. Three base differences located between the two potential splice-acceptor sites are both necessary and sufficient in defining the respective splicing patterns. One of these bases may serve as a lariat branch point critical for the alternative acceptor site activity while the remaining two bases appear to modulate the frequency with which this site is selected.

We have recently established that the human growth hormone-variant (hGH-V) gene is functional in vivo by documenting its expression in the placenta. We have subsequently generated transformed murine cell lines stably expressing the genes for normal pituitary growth hormone (hGH-N), hGH-V, and each of two chimeric genes generated by exon 3 exchanges, hGH-NV3 and hGH-VN3. In the present study, we utilize these cell lines as sources of hormone to characterize and compare the receptor binding profiles of hGH-N with hGH-V. hGH-V was found to displace 125I-ovine prolactin bound to rat liver microsomes (lactogen binding) and to displace 125I-hGH bound to rabbit liver microsomes (somatogen binding). Therefore, hGH-V would be predicted to display both somatogenic and lactogenic bioactivity, a dual specificity previously thought to be unique to hGH-N. The concentrations of hormone necessary to displace 50% (IC50) of the 125I-hGH from somatogen receptors and 125I-ovine prolactin from lactogen receptors was expressed as a ratio, IC50 somatogen: IC50 lactogen, for each hormone tested. A 7.4-fold difference in this ratio was observed for hGH-N compared to hGH-V, suggesting significantly greater selectivity by hGH-V in binding to the somatogen receptor. The intermediate binding ratios of the hGH-NV3 and hGH-VN3 chimeric proteins confirmed the distinct receptor binding profiles of the two parent hormones and served to identify three amino acids of potential importance in defining their respective receptor binding specificities.

We have sought direct evidence for the in vivo expression of the human growth hormone-variant (hGH-V) gene by screening a placental cDNA library with a hGH-V-specific oligonucleotide. Nine independent hGH-V cDNA clones were isolated and analyzed, and three distinct species were detected. Five of these hGH-V cDNAs represent mRNAs spliced and processed in a pattern analogous to that of the highly homologous human growth hormone and human chorionic somatomammotropin gene transcripts. Each of the remaining four hGH-V cDNAs contains an additional segment of 253 nucleotides corresponding in position and sequence to the fourth intron of the hGH-V gene. In addition, one of the mRNAs in this second group uses an alternative downstream polyadenylation site. The alternatively spliced hGH-V mRNA, which we refer to as hGH-V2 mRNA, constitutes approximately 30% of the hGH-V transcripts both in the human term placenta and in a stable mouse fibroblast line expressing the transfected hGH-V gene. The placental expression of the hGH-V gene is specific to villous tissue. The hGH-V2 mRNA is predicted to encode a protein which substitutes the 65 carboxyl-terminal amino acids of hGH-V with a new 104-residue carboxyl terminus resulting in significant divergence in their relative physical properties. The alternative splicing of the hGH-V transcripts to hGH-V and hGH-V2 mRNAs expands the potential complexity of the hGH-V gene's role in normal placental function.

In a previous study, we demonstrated that the ability of a cDNA fragment to hybrid-arrest the translation of its complementary mRNA in rabbit reticulocyte lysate depends on the position of the mRNA/cDNA duplex within the mRNA molecule. In the present report, we further characterize the mechanisms involved in the destabilization and subsequent translation of mRNA/cDNA hybrids by mapping in detail the positional dependence of hybrid-arrested translation of the human alpha- and beta-globin mRNAs and by directly assessing the stability of mRNA/cDNA duplexes in reticulocyte lysate under a variety of translational conditions. The mapping studies in this report demonstrate that the translation of a hybridized mRNA requires exposure of the 5' nontranslated region and the AUG initiation codon, as well as those bases 3' to the AUG which are typically protected by an initiating 80 S ribosome. The translation of these mRNA/cDNA hybrids is associated with the complete removal of cDNA from the mRNA coding region; this disruption of the mRNA/cDNA duplex is blocked by inhibitors of translational initiation and elongation. cDNAs which extend into the 3' nontranslated region remain associated with the mRNA during normal translation but are completely removed from the mRNA during translation if translational termination is suppressed. Taken together, these findings demonstrate that the disruption of mRNA/cDNA duplexes in rabbit reticulocyte lysate is tightly linked to the assembly and migration of 80 S ribosomes.

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.

Helix-destabilizing activity associated with mRNA translation was detected in rabbit reticulocyte lysate by assessing the ability of the ribosome to read through mRNA/cDNA duplexes. Hybridization of globin mRNA to cDNA fragments which extend into the 5' nontranslated region fully block the translation of the hybridized message. However, hybridization of the mRNA to cDNA fragments which cover regions 3' to the initiation codon has no adverse effect on its translation. The translation of mRNA hybridized to this second category of cDNA fragments occurs without the total removal of cDNA. These results imply that the ribosomal complex, once fully assembled at the AUG initiation codon, can locally destabilize secondary structures as it moves along the message. This activity may be critical for translational elongation by the ribosome.

To test the feasibility of synthesizing recombinant peptide hormones by exon deletion and exchange, we have constructed and expressed hybrid human growth hormone (hGH)-rat prolactin (rPrl) genes in which the third and fourth exons of the hGH gene are deleted and separately replaced by the corresponding exons of the rPrl gene. These exon deletion and exon exchange genes were inserted into an SV40 viral vector, packaged, and expressed following acute viral infection of monkey kidney cells. Expression of the hGH gene lacking the third exon (hGHd3) was not detectable at the level of protein production. However, replacement of the deleted third exon in the hGHd3 gene with exon 3 of the rPrl gene (hGHP3) resulted in the efficient synthesis of a secreted chimeric protein. When the fourth exon of the hGH gene was deleted (hGHd4), the encoded protein was found only in the cytosol despite signal sequence processing. Replacement of the deleted fourth exon in this hGHd4 gene with exon 4 of rPrl resulted in the synthesis and secretion of both a chimeric protein (hGHP4) as well as a larger protein corresponding in size to prehGHP4. These results suggest that exon exchange among distantly related genes in the GH family may be used to produce high levels of chimeric GH-related proteins, and regions internal to the hGH protein may be critical in establishing normal protein processing and secretion.

The relative translational efficiency of the two human alpha-globin mRNAs, alpha 1 and alpha 2, was measured in vitro in a rabbit reticulocyte lysate system. To differentiate the translational products of these two mRNAs which normally encode an identical alpha-globin protein product, we used reticulocyte mRNA from a recently described Chinese subject. In this subject, an electrophoretically distinct alpha-globin mutant is encoded at the alpha 2 locus and both the alpha 1- and alpha 2-globin genes are deleted from the homologous chromosome (--/alpha 125Pro alpha). As in normal controls, the concentration of alpha 2-globin mRNA exceeded alpha 1 by approximately 3-fold. However, alpha 1- and alpha 2-globin proteins were synthesized by this reticulocyte mRNA at equal rates. This data suggests that the equal expression of the two alpha-globin genes observed in human erythrocytes results from a balance between the 3-fold excess of alpha 2-globin mRNA and a 3-fold higher translational efficiency of alpha 1-globin mRNA. The disparate translational efficiencies of the two alpha-globin mRNAs may be determined by the divergent structure of their 3' noncoding regions.