Chicken transferrin receptor gene: conservation 3'' noncoding sequences and expression in erythroid cells.

Recombinant clones of the chicken transferrin receptor gene and cDNA have been isolated and sequenced. Two highly conserved regions have been identified in the 3' noncoding sequence of the human and chicken TR gene. The conserved regions include sequences that have been shown to be involved in the iron-dependent regulation of human TR mRNA stability. These sequences can be modeled as two different types of RNA secondary structures, one containing stem-loop structures that are similar to the iron-responsive elements found in ferritin mRNA and the other being a stable, duplex/stem-loop structure. Both forms show considerable similarity between chicken and human mRNA. The expression of TR is developmentally regulated during erythroid maturation, and immature erythroid cells express exceptionally high levels of TR mRNA.     

Binding to the transferrin receptor is required for endocytosis of HFE and regulation of iron homeostasis

HFE, the protein that is mutated in hereditary haemochromatosis, binds to the transferrin receptor (TfR). Here we show that wild-type HFE and TfR localize in endosomes and at the basolateral membrane of a polarized duodenal epithelial cell line, whereas the primary haemochromatosis HFE mutant, and another mutant with impaired TfR-binding ability accumulate in the ER/Golgi and at the basolateral membrane, respectively. Levels of the iron-storage protein ferritin are greatly reduced and those of TfR are slightly increased in cells expressing wild-type HFE, but not in cells expressing either mutant. Addition of an endosomal-targeting sequence derived from the human low-density lipoprotein receptor (LDLR) to the TfR-binding-impaired mutant restores its endosomal localization but not ferritin reduction or TfR elevation. Thus, binding to TfR is required for transport of HFE to endosomes and regulation of intracellular iron homeostasis, but not for basolateral surface expression of HFE.     

Several distinct types of sequence elements are required for efficient mRNA 3'' end formation in a pea rbcS gene.

We have conducted an extensive linker substitution analysis of the polyadenylation signal from a pea rbcS gene. From these studies, we can identify at least two, and perhaps three, distinct classes of cis element involved in mRNA 3' end formation in this gene. One of these, termed the far-upstream element, is located between 60 and 120 nt upstream from its associated polyadenylation sites and appears to be largely composed of a series of UG motifs. A second, termed the near-upstream element, is more proximate to poly(A) sites and may be functionally analogous to the mammalian polyadenylation signal AAUAAA, even though the actual sequences involved may not be AAUAAA. The third possible class is the putative cleavage and polyadenylation site itself. We find that the rbcS-E9 far-upstream element can replace the analogous element in another plant polyadenylation signal, that from cauliflower mosaic virus, and that one near-upstream element can function with either of two poly(A) sites. Thus, these different cis elements are largely interchangeable. Our studies indicate that a cellular plant gene possesses upstream elements distinct from AAUAAA that are involved in mRNA 3' end formation and that plant genes probably have modular, multicomponent polyadenylation signals.     

Apolipoprotein B mRNA sequences 3'' of the editing site are necessary and sufficient for editing and editosome assembly.

Apolipoprotein B (apoB) mRNA is edited in rat liver and intestine through the direct conversion of cytidine to uridine at nucleotide 6666. Recently, we have proposed the 'Mooring Sequence' model, in which editing complexes (editosomes) assemble on specific apoB mRNA flanking sequences to direct this site-specific editing event. To test this model, apoB mRNA deletion and translocation mutants were constructed and analyzed. Specific sequences 3' of the editing site were absolutely required for editing, while specific sequences and bulk RNA 5' of the editing site were required for efficient editing. Translocation of apoB 3' flanking sequences induced editing of an upstream cytidine, demonstrating that 3' sequences are necessary and sufficient to direct editing in vitro. 3' flanking sequences were also shown to be necessary and sufficient for editosome complex assembly. These data provide strong support for a 'Mooring Sequence' model in which 3' apoB flanking sequences direct editosome assembly and subsequent editing in vitro, while 5' flanking sequences enhance these functions.     

5'' upstream sequences from the wun1 gene are responsible for gene activation by wounding in transgenic plants.

A 1.2-kilobase pair fragment of the 5' upstream region of a potato wound-inducible gene (wun1) was fused to different marker genes (wun1-CAT, wun1-NPTII). Stable integration of a wun1-CAT chimeric gene into the tobacco genome led to a high wound-inducible chloramphenicol acetyltransferase activity in leaves. Transient expression experiments in potato protoplasts showed that wun1 carries a strong promoter sequence similar in strength to the 35S promoter. The same intensity of expression was also observed using wun1 constructs in transient experiments with rice protoplasts. wun1 mRNA was shown to accumulate to high levels in potato leaves collapsing as a result of infection with the phytopathogen Phytophthora infestans. The wun1 product might, therefore, play a role in a general physiological reaction to stress correlated with cell death.     

Multiple cis-acting targeting sequences are required for orb mRNA localization during Drosophila oogenesis.

The targeting of positional information to specific regions of the oocyte or early embryo is one of the key processes in establishing anterior-posterior and dorsal-ventral polarity. In many developmental systems, this is accomplished by localization of mRNAs. The germ line-specific Drosophila orb gene plays a critical role in defining both axes of the developing oocyte, and its mRNA is localized in a complex pattern during oogenesis. We have identified a 280-bp sequence from the orb 3' untranslated region capable of reproducing this complex localization pattern. Furthermore, we have found that multiple cis-acting elements appear to be required for proper targeting of orb mRNA.     

RNA-RNA interaction is required for the formation of specific bicoid mRNA 3'' UTR-STAUFEN ribonucleoprotein particles.

The formation of the anterior pattern of the Drosophila embryo is dependent on the localization of the mRNA of the morphogen Bicoid (bcd) to the anterior pole of the egg cell. Staufen protein (STAU) is required in a late step of the localization to anchor the bcd mRNA in the anterior cytoplasm. We have shown previously that endogenous STAU associates specifically with injected bcd mRNA 3'-untranslated region (UTR), resulting in the formation of characteristic RNA-protein particles that are transported along microtubules of the mitotic spindles in a directed manner. The regions recognized by STAU in this in vivo assay are predicted to form three stem-loop structures involving large double-stranded stretches. Here, we show that the STAU interaction requires a double-stranded conformation of the stems within the RNA localization signal. In addition, base pairing between two single-stranded loops plays a major role in particle formation. This loop-loop interaction is intermolecular, not intramolecular; thus dimers or multimers of the RNA localization signal must be associated with STAU in these particles. The bcd mRNA 3' UTR can also dimerize in vitro in the absence of STAU. Thus, in addition to RNA-protein interactions, RNA-RNA interaction might be involved in the formation of ribonucleoprotein particles for transport and localization.     

Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels.

Classical glutathione peroxidase (GPX1) mRNA levels can decrease to less than 10% in selenium (Se)-deficient rat liver. The cis-acting nucleic acid sequence requirements for Se regulation of GPX1 mRNA levels were studied by transfecting Chinese hamster ovary (CHO) cells with GPX1 DNA constructs in which specific regions of the GPX1 gene were mutated, deleted, or replaced by comparable regions from unregulated genes such as phospholipid hydroperoxide glutathione peroxidase (GPX4). For each construct, stable transfectants were pooled two weeks after transfection, divided into Se-deficient (2 nM Se) or Se-adequate (200 nM Se) medium, and grown for an additional four days. On day of harvest, Se-deficient GPX1 and GPX4 activities averaged 13 +/- 2% and 15 +/- 2% of Se adequate levels, confirming that cellular Se status was dramatically altered by Se supplementation. RNA was isolated from replicate plates of cells and transfected mRNA levels were specifically determined by RNase protection assay. Analysis of chimeric GPX1/GPX4 constructs showed that the GPX4 3'-UTR can completely replace the GPX1 3'-UTR in Se regulation of GPX1 mRNA. We did not find any GPX1 coding regions that could be replaced by the corresponding GPX4 coding regions without diminishing or eliminating Se regulation of the transfected GPX1 mRNA. Further analysis of the GPX1 coding region demonstrated that the GPX1 Sec codon (UGA) and the GPX1 intron sequences are required for full Se regulation of transfected GPX1 mRNA levels. Mutations that moved the GPX1 Sec codon to three different positions within the GPX1 coding region suggest that the mechanism for Se regulation of GPX1 mRNA requires a Sec codon within exon 1. Lastly, we found that addition of the GPX1 3'-UTR to beta-globin mRNA can convey significant Se regulation to beta-globin mRNA levels when a UGA codon is placed within exon 1. We conclude that Se regulation of GPX1 mRNA requires a functional selenocysteine insertion sequence (SECIS) in the 3'-UTR and a Sec codon followed by an intron.