Mos 3' UTR regulatory differences underlie species-specific temporal patterns of Mos mRNA cytoplasmic polyadenylation and translational recruitment during oocyte maturation

The Mos proto-oncogene is a critical regulator of vertebrate oocyte maturation. The maturation-dependent translation of Mos protein correlates with the cytoplasmic polyadenylation of the maternal Mos mRNA. However, the precise temporal requirements for Mos protein function differ between oocytes of model mammalian species and oocytes of the frog Xenopus laevis. Despite the advances in model organisms, it is not known if the translation of the human Mos mRNA is also regulated by cytoplasmic polyadenylation or what regulatory elements may be involved. We report that the human Mos 3' untranslated region (3' UTR) contains a functional cytoplasmic polyadenylation element (CPE) and demonstrate that the endogenous Mos mRNA undergoes maturation-dependent cytoplasmic polyadenylation in human oocytes. The human Mos 3' UTR interacts with the human CPE-binding protein and exerts translational control on a reporter mRNA in the heterologous Xenopus oocyte system. Unlike the Xenopus Mos mRNA, which is translationally activated by an early acting Musashi/polyadenylation response element (PRE)-directed control mechanism, the translational activation of the human Mos 3' UTR is dependent on a late acting CPE-dependent process. Taken together, our findings suggest a fundamental difference in the 3' UTR regulatory mechanisms controlling the temporal induction of maternal Mos mRNA polyadenylation and translational activation during Xenopus and mammalian oocyte maturation.     

A 250-nucleotide UA-rich element in the 3' untranslated region of Xenopus laevis Vg1 mRNA represses translation both in vivo and in vitro.

Xenopus laevis Vgl mRNA undergoes both localization and translational control during oogenesis. Vg1 protein does not appear until late stage IV, after localization is complete. To determine whether Vg1 translation is regulated by cytoplasmic polyadenylation, the RACE-PAT method was used. Vg1 mRNA has a constant poly(A) tail throughout oogenesis, precluding a role for cytoplasmic polyadenylation. To identify cis-acting elements involved in Vg1 translational control, the Vg1 3' UTR was inserted downstream of the luciferase ORF and in vitro transcribed, adenylated mRNA injected into stage III or stage VI oocytes. The Vg1 3' UTR repressed luciferase translation in both stages. Deletion analysis of the Vg1 3' UTR revealed that a 250-nt UA-rich fragment, the Vg1 translational element or VTE, which lies 118 nt downstream of the Vg1 localization element, could repress translation as well as the full-length Vg1 3' UTR. Poly(A)-dependent translation is not necessary for repression as nonadenylated mRNAs are also repressed, but cap-dependent translation is required as introduction of the classical swine fever virus IRES upstream of the luciferase coding region prevents repression by the VTE. Repression by the Vg1 3' UTR has been reproduced in Xenopus oocyte in vitro translation extracts, which show a 10-25-fold synergy between the cap and poly(A) tail. A number of proteins UV crosslink to the VTE including FRGY2 and proteins of 36, 42, 45, and 60 kDa. The abundance of p42, p45, and p60 is strikingly higher in stages I-III than in later stages, consistent with a possible role for these proteins in Vg1 translational control.     

Deletion of a conserved regulatory element in the Drosophila Adh gene leads to increased alcohol dehydrogenase activity but also delays development

In vivo levels of enzymatic activity may be increased through either structural or regulatory changes. Here we use Drosophila melanogaster alcohol dehydrogenase (ADH) in an experimental test for selective differences between these two mechanisms. The well-known ADH-Slow (S)/Fast (F) amino acid replacement leads to a twofold increase in activity by increasing the catalytic efficiency of the enzyme. Disruption of a highly conserved, negative regulatory element in the Adh 3' UTR also leads to a twofold increase in activity, although this is achieved by increasing in vivo Adh mRNA and protein concentrations. These two changes appear to be under different types of selection, with positive selection favoring the amino acid replacement and purifying selection maintaining the 3' UTR sequence. Using transgenic experiments we show that deletion of the conserved 3' UTR element increases adult and larval Adh expression in both the ADH-F and ADH-S genetic backgrounds. However, the 3' UTR deletion also leads to a significant increase in developmental time in both backgrounds. ADH allozyme type has no detectable effect on development. These results demonstrate a negative fitness effect associated with Adh overexpression. This provides a mechanism whereby natural selection can discriminate between alternative pathways of increasing enzymatic activity.     

Characterization of the 5' and 3' untranslated regions in murine mdm2 mRNAs

The murine double minute 2 (mdm2) gene is essential for embryogenesis in mice that express the p53 tumor suppressor protein. Mdm2 levels must be regulated tightly because overexpression of mdm2 contributes to tumorigenesis. We investigated whether the 5' and 3' untranslated regions (UTRs) of murine mdm2 affect the expression of MDM2 proteins. Induction of mdm2 expression by p53 results in synthesis of an mdm2 mRNA with a short 5' UTR. The long 5' UTR increases internal initiation of translation of a minor MDM2 protein, p76(MDM2), without affecting the efficiency of translation of the full-length p90(MDM2). We discovered two alternative 3' untranslated regions in murine mdm2 mRNA expressed in the testis. The longer 3' UTR contains a consensus instability element, but mdm2 mRNAs containing the long and short 3' UTRs have comparable half-lives. The 3' UTRs do not affect either initiation codon use or translation efficiency. Thus, the murine 5' UTR, but not the 3'UTR, influences the ratio of the two MDM2 proteins but neither UTR affects MDM2 abundance significantly.     

The nanos translational control element represses translation in somatic cells by a Bearded box-like motif

Developmental control of translation is frequently mediated by regulatory elements that reside within 3' untranslated regions (3' UTRs). Two stem-loops within the nanos 3' UTR translational control element (TCE) act independently to direct translational repression of maternal nanos mRNA in the ovary or embryo. We have previously shown that the nanos TCE can also function in select somatic sites. Using an ectopic expression screen, we now identify a new site of TCE function, the dorsal pouch epithelium. Analysis of TCE mutants reveals that TCE activity in the dorsal pouch does not depend on either of the stem-loops required for maternal TCE function, but instead requires a third feature-a sequence that closely matches the Bearded box, a regulatory motif found in the 3' UTRs of several Notch pathway genes. In addition, we identify pleiohomeotic mRNA as an endogenous candidate for regulation by Bearded box-like motifs in the dorsal pouch. Together, these results suggest that the TCE has appropriated a conserved regulatory motif to expand its function to somatic tissues.     

A novel mRNA 3' untranslated region translational control sequence regulates Xenopus Wee1 mRNA translation

Cell cycle progression during oocyte maturation requires the strict temporal regulation of maternal mRNA translation. The intrinsic basis of this temporal control has not been fully elucidated but appears to involve distinct mRNA 3′ UTR regulatory elements. In this study, we identify a novel translational control sequence (TCS) that exerts repression of target mRNAs in immature oocytes of the frog, Xenopus laevis, and can direct early cytoplasmic polyadenylation and translational activation during oocyte maturation. The TCS is functionally distinct from the previously characterized Musashi/polyadenylation response element (PRE) and the cytoplasmic polyadenylation element (CPE). We report that TCS elements exert translational repression in both the Wee1 mRNA 3′ UTR and the pericentriolar material-1 (Pcm-1) mRNA 3′ UTR in immature oocytes. During oocyte maturation, TCS function directs the early translational activation of the Pcm-1 mRNA. By contrast, we demonstrate that CPE sequences flanking the TCS elements in the Wee1 3′ UTR suppress the ability of the TCS to direct early translational activation. Our results indicate that a functional hierarchy exists between these distinct 3′ UTR regulatory elements to control the timing of maternal mRNA translational activation during oocyte maturation.     

Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5' untranslated region optimization

Microalgae have the potential to be a valuable biotechnological platform for the production of recombinant proteins. However, because of the complex regulatory network that tightly controls chloroplast gene expression, heterologous protein accumulation in a wild-type, photosynthetic-competent algal chloroplast remains low. High levels of heterologous protein accumulation have been achieved using the psbA promoter/5' untranslated region (UTR), but only in a psbA-deficient genetic background, because of psbA/D1-dependent auto-attenuation. Here, we examine the effect of fusing the strong 16S rRNA promoter to the 5' UTR of the psbA and atpA genes on transgene expression in the chloroplast of Chlamydomonas reinhardtii. We show that fusion of the 16S promoter had little impact on protein accumulation from the psbA 5' UTR in a psbA-deficient genetic background. Furthermore, the 16S/psbA promoter/UTR fusion was silenced in the presence of wild-type levels of D1 protein, confirming that the psbA 5' UTR is the primary target for D1-dependent auto-repression. However, fusion of the 16S promoter to the atpA 5' UTR significantly boosts mRNA levels and supports high levels of heterologous protein accumulation in photosynthetic-competent cells. The 16S/atpA promoter/UTR drove LUXCT protein accumulation to levels close to that of psbA in a psbA- background, and drove expression of a human therapeutic protein to levels only twofold lower than the psbA 5' UTR. The 16S/atpA promoter/UTR combination should have utility for heterologous protein production when expression from a photosynthetic-competent microalgal strain is required.     

Translation of the chloroplast psbC mRNA is controlled by interactions between its 5' leader and the nuclear loci TBC1 and TBC3 in Chlamydomonas reinhardtii.

Translation of the chloroplast psbC mRNA in Chlamydomonas reinhardtii has been shown previously to require interactions between its 5' untranslated region (5' UTR) and the functions encoded by two nuclear loci, which we name here TBC1 and TBC2. We show that a 97-nucleotide (nt) region located in the middle of the psbC 5' UTR is required for translation initiation. Unlike most procaryotic cis-acting translational control elements, this region has a translational activation function and is located 236 nt upstream from the GUG translation initiation codon. In vivo pulse-labeling of chloroplast-encoded proteins and analyses of the expression of chimeric reporter genes in vivo reveal that a mutation of a newly described locus, TBC3, restores translation from the psbC 5' UTR in the absence of either this cis-acting element or the wild-type trans-acting TBC1 function. These data demonstrate that sequences located in the middle of the psbC 5' UTR, TBC1, and TBC3 functionally interact to control the translation of the psbC mRNA.     

Identification of a highly conserved 3' UTR in the translationally regulated mRNA for prostaglandin synthase

Prostaglandin H synthase (E.C. 1.14.99.1) is induced by growth factors and lymphokines such as EGF and IL-1, and is suppressed by anti-inflammatory glucocorticoids. Inhibition of enzyme synthesis by glucocorticoids is mediated by a novel translational control that appears to involve conversion of the PG synthase mRNA into a cryptic non-hybridizable form. In order to understand expression of the enzyme in more detail, a full length 2.8 Kb cDNA was cloned from a human embryonic lung cell cDNA library and the complete mRNA including the 3' untranslated region (3' UTR), was sequenced. The coding sequence for the human PG synthase shows greater than 90% homology with the sheep and mouse enzymes. A high degree of conservation (70%), however, was also observed in the approximately 750 nucleotide sequence that comprises the 3' non-coding domain of both sheep and human PG synthase mRNA's and with the approximately 900 nucleotide 3' UTR of the mouse RNA (68% sheep vs mouse; 47% human vs mouse). Extensive microregions of 10-30 nucleotides are distributed throughout the 3' UTR where homology between species is 95-100%. This high degree of conservation in a non-coding region and recent evidence from other genes suggests that these 3' UTR sequences have important regulatory functions possibly related to translational control of this mRNA by growth factors and glucocorticoids.     

Interplay between hnRNP A1 and a cis-acting element in the 3' UTR of CYP2A5 mRNA is central for high expression of the gene

Our previous evidence suggests that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 plays a part in the regulation of the Cyp2a5 gene by interacting with the 3' untranslated region (UTR) of the CYP2A5 mRNA. However, the exact role of this interaction is not clear. The aim of the present work was to gain further insight into the regulation process of Cyp2a5. For this purpose the 3' UTR of CYP2A5 was fused to the coding region of luciferase mRNA. Luciferase recombinants containing either the full length 3' UTR, or the 3' UTR lacking a previously described 71 nucleotide (nt) region (the hnRNP A1 primary binding site), were transiently expressed in cells expressing or lacking hnRNP A1. The expression of the luciferase recombinants was examined both at mRNA and enzyme activity levels. The results disclosed that the presence of hnRNP A1 was required for the high expression of the recombinant carrying the full length 3' UTR of CYP2A5. Deletion of the hnRNP A1 primary binding site dramatically modified the expression pattern: the mRNA levels and luciferase activities of the deletion mutant were independent from hnRNP A1. These results conclusively demonstrate that the 71 nt region in the 3' UTR of CYP2A5 mRNA can confer hnRNP A1-dependent regulation to a gene. In addition, comparison of RNA levels and luciferase activities suggested that regions flanking the hnRNP A1 binding site could regulate translation of the CYP2A5 mRNA. These results are consistent with a model in which the binding of hnRNP A1 to the 71 nt putative hairpin-loop region in the CYP2A5 mRNA 3' UTR upregulates mRNA levels possibly by protecting the mRNA from degradation.