Long-term expression of γ-globin mRNA in mouse erythrocytes from retrovirus vectors containing the human γ-globin gene fused to the ankyrin-1 promoter

Gene therapy for patients with hemoglobin disorders has been hampered by the inability of retrovirus vectors to transfer globin genes and their cis-acting regulatory sequences into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells due to position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Recently, we have shown that the human ankyrin (Ank) gene promoter directs position-independent, copy number-dependent expression of a linked γ-globin gene in transgenic mice. We inserted the Ank/Aγ-globin gene into retrovirus vectors that could transfer one or two copies of the Ank/Aγ-globin gene to target cells. Both vectors were stable, transferring only intact proviral sequences into primary mouse hematopoietic stem cells. Expression of Ank/Aγ-globin mRNA in mature red blood cells was 3% (single copy) and 8% (double copy) of the level of mouse α-globin mRNA. We conclude that these novel retrovirus vectors may be valuable for treating a variety of red cell disorders by gene replacement therapy including severe β-thalassemia if the level of expression can be further increased.

Phytochrome B binds with greater apparent affinity than phytochrome A to the basic helix–loop–helix factor PIF3 in a reaction requiring the PAS domain of PIF3

The signaling pathways by which the phytochrome (phy) family of photoreceptors transmits sensory information to light-regulated genes remain to be fully defined. Evidence for a relatively direct pathway has been provided by the binding of one member of the family, phyB, to a promoter-element-bound, basic helix–loop–helix protein, PIF3, specifically upon light-induced conversion of the photoreceptor molecule to its biologically active conformer (Pfr). Here, we show that phyA also binds selectively and reversibly to PIF3 upon photoconversion to Pfr, but that the apparent affinity of PIF3 for phyA is 10-fold lower than for phyB. This result is consistent with previous in vivo data from PIF3-deficient Arabidopsis, indicating that PIF3 has a major role in phyB signaling, but a more minor role in phyA signaling. We also show that phyB binds stoichiometrically to PIF3 at an equimolar ratio, suggesting that the resultant complex is the unit active in transcriptional regulation at target promoters. Deletion mapping suggests that a 37-aa segment present at the N terminus of phyB, but absent from phyA, contributes strongly to the high binding affinity of phyB for PIF3. Conversely, deletion mapping and point mutation analysis of PIF3 for determinants involved in recognition of phyB indicates that the PAS domain of PIF3 is a major contributor to this interaction, but that a second determinant in the C-terminal domain is also necessary.

The 3′ untranslated region of a rice α-amylase gene functions as a sugar-dependent mRNA stability determinant

In plants, sugar feedback regulation provides a mechanism for control of carbohydrate allocation and utilization among tissues and organs. The sugar repression of α-amylase gene expression in rice provides an ideal model for studying the mechanism of sugar feedback regulation. We have shown previously that sugar repression of α-amylase gene expression in rice suspension cells involves control of both transcription rate and mRNA stability. The α-amylase mRNA is significantly more stable in sucrose-starved cells than in sucrose-provided cells. To elucidate the mechanism of sugar-dependent mRNA turnover, we have examined the effect of αAmy3 3′ untranslated region (UTR) on mRNA stability by functional analyses in transformed rice suspension cells. We found that the entire αAmy3 3′ UTR and two of its subdomains can independently mediate sugar-dependent repression of reporter mRNA accumulation. Analysis of reporter mRNA half-lives demonstrated that the entire αAmy3 3′ UTR and the two subdomains each functioned as a sugar-dependent destabilizing determinant in the turnover of mRNA. Nuclear run-on transcription analysis further confirmed that the αAmy3 3′ UTR and the two subdomains did not affect the transcription rate of promoter. The identification of sequence elements in the α-amylase mRNA that dictate the differential stability has very important implications for the study of sugar-dependent mRNA decay mechanisms.

A phytochrome from the fern Adiantum with features of the putative photoreceptor NPH1

In plant photomorphogenesis, it is well accepted that the perception of red/far-red and blue light is mediated by distinct photoreceptor families, i.e., the phytochromes and blue-light photoreceptors, respectively. Here we describe the discovery of a photoreceptor gene from the fern Adiantum that encodes a protein with features of both phytochrome and NPH1, the putative blue-light receptor for second-positive phototropism in seed plants. The fusion of a functional photosensory domain of phytochrome with a nearly full-length NPH1 homolog suggests that this polypeptide could mediate both red/far-red and blue-light responses in Adiantum normally ascribed to distinct photoreceptors.

Expression of sterol regulatory element-binding protein 1c (SREBP-1c) mRNA in rat hepatoma cells requires endogenous LXR ligands

The current paper describes a line of cultured rat hepatoma cells (McA-RH7777 cells) that mimics the behavior of rat liver by producing an excess of mRNA for sterol regulatory element-binding protein 1c (SREBP-1c) as opposed to SREBP-1a. These two transcripts are derived from a single gene by use of alternative promoters that are separated by many kilobases in the genome. The high level of SREBP-1c mRNA is abolished when cholesterol synthesis is blocked by compactin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase that inhibits cholesterol synthesis. Levels of SREBP-1c mRNA are restored by mevalonate, the product of the HMG CoA reductase reaction, and by ligands for the nuclear hormone receptor LXR, including 22(R)-hydroxycholesterol and T0901317. These data suggest that transcription of the SREBP-1c gene in hepatocytes requires tonic activation of LXR by an oxysterol intermediate in the cholesterol biosynthetic pathway. Reduction of this intermediate lowers SREBP-1c levels, and this in turn is predicted to lower the rates of fatty acid biosynthesis in liver.

Luteinizing hormone-releasing hormone (LH-RH) antagonist Cetrorelix down-regulates the mRNA expression of pituitary receptors for LH-RH by counteracting the stimulatory effect of endogenous LH-RH

The mechanisms through which LH-RH antagonists suppress gonadotroph functions and LH-RH receptor (LH-RH-R) production are incompletely understood. To elucidate these mechanisms, we investigated the effects of Cetrorelix on the mRNA expression of pituitary LH-RH-R and luteinizing hormone (LH) secretion in three experimental systems with different pituitary LH-RH environments. Ovariectomy induced 3.61-fold and 6.34-fold increases in the mRNA expression of pituitary LH-RH-R in rats after 11 and 21 days, respectively. After (5 h) a single injection of 100 μg Cetrorelix, no significant decrease occurred in the mRNA levels of pituitary LH-RH-R in ovariectomized (OVX) rats with high pituitary exposure to LH-RH, but there was a significant 23.2% reduction in cycling rats with normal hypophysial LH-RH environment. Prolonged treatment for 10 days with a Cetrorelix depot formulation releasing 100 μg/day decreased the concentration of mRNA for pituitary LH-RH-R by 72.6% in OVX rats, but only by 32.9% in normal rats. The decline in serum LH was 98.7% in OVX rats and 63.2% in normal rats, resulting in a minimal 0.1–0.2 ng/ml LH concentration in both groups. A continuous exposure of pituitary cells to 100 nM Cetrorelix in the superfusion system, which is devoid of LH-RH, did not cause any significant changes in LH-RH-R mRNA level. These studies demonstrate that prolonged exposure to Cetrorelix in vivo, but not in vitro, down-regulates the mRNA expression of the pituitary receptors for LH-RH. Our findings indicate that LH-RH antagonists exert their inhibitory effects on the gene expression of pituitary LH-RH-R by counteracting the stimulatory effect of endogenous LH-RH.

A zinc finger-containing papain-like protease couples subgenomic mRNA synthesis to genome translation in a positive-stranded RNA virus

The genome expression of positive-stranded RNA viruses starts with translation rather than transcription. For some viruses, the genome is the only viral mRNA and expression is regulated primarily at the translational level and by limited proteolysis of polyproteins. Other virus groups also generate subgenomic mRNAs later in the reproductive cycle. For nidoviruses, subgenomic mRNA synthesis (transcription) is discontinuous and yields a 5′ and 3′ coterminal nested set of mRNAs. Nidovirus transcription is not essential for genome replication, which relies on the autoprocessing products of two replicase polyproteins that are translated from the genome. We now show that the N-terminal replicase subunit, nonstructural protein 1 (nsp1), of the nidovirus equine arteritis virus is in fact dispensable for replication but crucial for transcription, thereby coupling replicase expression and subgenomic mRNA synthesis in an unprecedented manner. Nsp1 is composed of two papain-like protease domains and a predicted N-terminal zinc finger, which was implicated in transcription by site-directed mutagenesis. The structural integrity of nsp1 is essential, suggesting that the protease domains form a platform for the zinc finger to operate in transcription.

Polymorphism in the 3′-untranslated region of TNFα mRNA impairs binding of the post-transcriptional regulatory protein HuR to TNFα mRNA

Tumor necrosis factor α (TNFα) acts as a beneficial mediator in the process of host defence. In recent years major interest has focused on the AU-rich elements (AREs) present in the 3′-untranslated region (3′-UTR) of TNFα mRNA as this region plays a pivotal role in post-transcriptional control of TNFα production. Certain stimuli, such as lipopolysaccharides, a component of the Gram-negative bacterial cell wall, have the ability to relinquish the translational suppression of TNFα mRNA imposed by these AREs in macrophages, thereby enabling the efficient production of the TNFα. In this study we show that the polymorphism (GAU trinucleotide insertional mutation) present in the regulatory 3′-UTR of TNFα mRNA of NZW mice results in the hindered binding of RNA-binding proteins, thereby leading to a significantly reduced production of TNFα protein. We also show that the binding of macrophage proteins to the main ARE is also decreased by another trinucleotide (CAU) insertion in the TNFα 3′-UTR. One of the proteins affected by the GAU trinucleotide insertional mutation was identified as HuR, a nucleo-cytoplasmic shuttling protein previously shown to play a prominent role in the stability and translatability of mRNA containing AREs. Since binding of this protein most likely modulates the stability, translational efficiency and transport of TNFα mRNA, these results suggest that mutations in the ARE of TNFα mRNA decrease the production of TNFα protein in macrophages by hindering the binding of HuR to the ARE.

Polysomal ribonuclease 1 exists in a latent form on polysomes prior to estrogen activation of mRNA decay

Estrogen induces a global change in the translation profile of Xenopus hepatocytes, replacing serum protein synthesis with production of the yolk protein precursor vitellogenin. This is accomplished by the coordinate destabilization of serum protein mRNAs and the transcriptional induction and subsequent stabilization of vitellogenin mRNA. Previous work identified an endonuclease activity whose appearance on polysomes correlated with the disappearance of serum protein mRNAs. This enzyme, polysomal ribonuclease 1 (PMR1), is a novel member of the peroxidase gene family. The current study examined the association of PMR1 with its mRNA targets on polysomes and mRNPs. The highest amount of polysome-bound PMR1 was observed prior to estrogen induction of mRNA decay. Its distribution on sucrose density gradients matched the absorbance profile of polysome-bound mRNA, suggesting that PMR1 forms a latent complex with mRNA. Following dissociation with EDTA the 62 kDa PMR1 sedimented with a larger complex of >670 kDa. Estrogen induces a 22-fold increase in unit enzymatic activity of polysome-bound PMR1, and a time-dependent loss of PMR1 from polysomes in a manner that mirrors the disappearance of albumin mRNA. These data suggest that the key step in the extensive estrogen-induced change in mRNA decay in Xenopus liver is activation of a latent mRNA endonuclease associated with its target mRNA.

Quantitative analysis of mRNA amplification by in vitro transcription

Effective transcript profiling in animal systems requires isolation of homogenous tissue or cells followed by faithful mRNA amplification. Linear amplification based on cDNA synthesis and in vitro transcription is reported to maintain representation of mRNA levels, however, quantitative data demonstrating this as well as a description of inherent limitations is lacking. We show that published protocols produce a template-independent product in addition to amplifying real target mRNA thus reducing the specific activity of the final product. We describe a modified amplification protocol that minimizes the generation of template-independent product and can therefore generate the desired microgram quantities of message-derived material from 100 ng of total RNA. Application of a second, nested round of cDNA synthesis and in vitro transcription reduces the required starting material to 2 ng of total RNA. Quantitative analysis of these products on Caenorhabditis elegans Affymetrix GeneChips shows that this amplification does not reduce overall sensitivity and has only minor effects on fidelity.

PromEC: An updated database of Escherichia coli mRNA promoters with experimentally identified transcriptional start sites

PromEC is an updated compilation of Escherichia coli mRNA promoter sequences. It includes documentation on the location of experimentally identified mRNA transcriptional start sites on the E.coli chromosome, as well as the actual sequences in the promoter region. The database was updated as of July 2000 and includes 472 entries. PromEC is accessible at http://bioinfo.md.huji.ac.il/marg/promec

ARED: human AU-rich element-containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins

The adenylate uridylate-rich elements (AREs) mediate the rapid turnover of mRNAs encoding proteins that regulate cellular growth and body response to exogenous agents such as microbes, inflammatory and environmental stimuli. However, the full repertoire of ARE-containing mRNAs is unknown. Here, we explore the distribution of AREs in human mRNA sequences. Computational derivation of a 13-bp ARE pattern was performed using multiple expectation maximization for motif elicitations (MEME) and consensus analyses. This pattern was statistically validated for the specificity towards the 3′-untranslated region and not coding region. The computationally derived ARE pattern is the basis of a database which contains non-redundant full-length ARE-mRNAs. The ARE-mRNA database (ARED; http://rc.kfshrc.edu.sa/ared) reveals that ARE-mRNAs encode a wide repertoire of functionally diverse proteins that belong to different biological processes and are important in several disease states. Cluster analysis was performed using the ARE sequences to demonstrate potential relationships between the type and number of ARE motifs, and the functional characteristics of the proteins.

RNA editing in Trypanosoma brucei: characterization of gRNA U-tail interactions with partially edited mRNA substrates

Guide RNAs (gRNAs), key components of the RNA editing reaction in Trypanosoma brucei, direct the insertion and deletion of uridylate (U) residues. Analyses of gRNAs reveal three functional elements. The 5′-end of the gRNA contains the anchor, which is responsible for selection and binding to the pre-edited mRNA. The second element (the guiding region) provides the information required for editing. At the 3′-end of the gRNA is a non-encoded U-tail, whose function remains unclear. However, the cleavage–ligation model for editing proposes that the U-tail binds to purine-rich regions upstream of editing sites, thereby strengthening the interaction and holding onto the 5′ cleavage product. Our previous studies demonstrated that the U-tail interacts with upstream sequences and may play roles in both stabilization and tethering. These studies also indicated that the U-tail interactions involved mRNA regions that were to be subsequently edited. This raised the question of what happens to the mRNA–U-tail interaction as editing proceeds in the 3′→5′ direction. We examined gCYb-558 and its U-tail interaction with 5′CYbUT and two partially edited 5′CYb substrates. Our results indicate that the 3′-end of the U-tail interacts with the same sequence in all three mRNAs. Predicted secondary structures using crosslinking data suggest that a similar structure is maintained as editing proceeds. These results indicate that the role of the U-tail may also involve maintenance of important secondary structure motifs.

A yeast-like mRNA capping apparatus in Plasmodium falciparum

Analysis of the mRNA capping apparatus of the malaria parasite Plasmodium falciparum illuminates an evolutionary connection to fungi rather than metazoans. We show that P. falciparum encodes separate RNA guanylyltransferase (Pgt1) and RNA triphosphatase (Prt1) enzymes and that the triphosphatase component is a member of the fungal/viral family of metal-dependent phosphohydrolases, which are structurally and mechanistically unrelated to the cysteine-phosphatase-type RNA triphosphatases found in metazoans and plants. These results highlight the potential for discovery of mechanism-based antimalarial drugs designed to specifically block the capping of Plasmodium mRNAs. A simple heuristic scheme of eukaryotic phylogeny is suggested based on the structure and physical linkage of the triphosphatase and guanylyltransferase enzymes that catalyze cap formation.