A U-rich element in the 5' untranslated region is necessary for the translation of p27 mRNA

Increased translation of p27 mRNA correlates with withdrawal of cells from the cell cycle. This raised the possibility that antimitogenic signals might mediate their effects on p27 expression by altering complexes that formed on p27 mRNA, regulating its translation. In this report, we identify a U-rich sequence in the 5' untranslated region (5'UTR) of p27 mRNA that is necessary for efficient translation in proliferating and nonproliferating cells. We show that a number of factors bind to the 5'UTR in vitro in a manner dependent on the U-rich element, and their availability in the cytosol is controlled in a growth- and cell cycle-dependent fashion. One of these factors is HuR, a protein previously implicated in mRNA stability, transport, and translation. Another is hnRNP C1 and C2, proteins implicated in mRNA processing and the translation of a specific subset of mRNAs expressed in differentiated cells. In lovastatin-treated MDA468 cells, the mobility of the associated hnRNP C1 and C2 proteins changed, and this correlated with increased p27 expression. Together, these data suggest that the U-rich dependent RNP complex on the 5'UTR may regulate the translation of p27 mRNA and may be a target of antimitogenic signals.     

Cooperativity between the preproinsulin mRNA untranslated regions is necessary for glucose-stimulated translation

Glucose regulates proinsulin biosynthesis via stimulation of the translation of the preproinsulin mRNA in pancreatic beta-cells. However, the mechanism by which this occurs has remained unclear. Using recombinant adenoviruses that express the preproinsulin mRNA with defined alterations, the untranslated regions (UTRs) of the preproinsulin mRNA were examined for elements that specifically control translation of the mRNA in rat pancreatic islets. These studies revealed that the preproinsulin 5'-UTR was necessary for glucose stimulation of preproinsulin mRNA translation, whereas the 3'-UTR appeared to suppress translation. However, together the 5'- and 3'-UTRs acted cooperatively to markedly increase glucose-induced proinsulin biosynthesis. In primary hepatocytes the presence of the preproinsulin 3'-UTR led to reduced mRNA levels compared with the presence of the SV40 3'-UTR, consistent with the presence of mRNA stability determinants in the 3'-UTR that stabilize the preproinsulin mRNA in a pancreatic beta-cell-specific manner. Translation of these mRNAs in primary hepatocytes was not stimulated by glucose, indicating that regulated translation of the preproinsulin mRNA occurs in a pancreatic beta-cell-specific manner. Thus, the untranslated regions of the preproinsulin mRNA play crucial roles in regulating insulin production and therefore glucose homeostasis by regulating the translation and the stability of the preproinsulin mRNA.     

Complementarity between the mRNA 5' untranslated region and 18S ribosomal RNA can inhibit translation

In eubacteria, base pairing between the 3' end of 16S rRNA and the ribosome-binding site of mRNA is required for efficient initiation of translation. An interaction between the 18S rRNA and the mRNA was also proposed for translation initiation in eukaryotes. Here, we used an antisense RNA approach in vivo to identify the regions of 18S rRNA that might interact with the mRNA 5' untranslated region (5' UTR). Various fragments covering the entire mouse 18S rRNA gene were cloned 5' of a cat reporter gene in a eukaryotic vector, and translation products were analyzed after transient expression in human cells. For the largest part of 18S rRNA, we show that the insertion of complementary fragments in the mRNA 5' UTR do not impair translation of the downstream open reading frame (ORF). When translation inhibition is observed, reduction of the size of the complementary sequence to less than 200 nt alleviates the inhibitory effect. A single fragment complementary to the 18S rRNA 3' domain retains its inhibitory potential when reduced to 100 nt. Deletion analyses show that two distinct sequences of approximately 25 nt separated by a spacer sequence of 50 nt are required for the inhibitory effect. Sucrose gradient fractionation of polysomes reveals that mRNAs containing the inhibitory sequences accumulate in the fractions with 40S ribosomal subunits, suggesting that translation is blocked due to stalling of initiation complexes. Our results support an mRNA-rRNA base pairing to explain the translation inhibition observed and suggest that this region of 18S rRNA is properly located for interacting with mRNA.     

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.     

Aberrant regulation of mRNA 3' untranslated region in cancers and inflammation

Almost 10% of mammalian coding mRNAs contain in their 3' untranslated region a sequence rich in adenine and uridine residues known as AU-rich element (ARE). Many of them encode oncogenes (for instance c-Myc and c-Fos), cell cycle regulators (cyclin D1, A1, B1), cytokines (TNFalpha, IL2) and growth factors (GM-CSF) which are overexpressed in cancer or inflammatory diseases due to increased mRNA stability and/or translation. AREs are recognized by a group of proteins, collectively called AUBPs which display various functions. For instance, HuR/ELAV is mainly known to protect ARE-containing mRNAs from degradation, while AUF1, TTP and KSRP act to destabilize their bound target mRNAs and TIA/TIAR to inhibit their translation. Alterations in ARE sequences or AUBP abundance, cellular localization or activity due to post-translational modifications such as phosphorylation can promote or enhance malignancy or perturb immune homeostasis. Here, c-myc and TNFalpha are chosen as examples to illustrate how altered 3' UTR gene regulation impacts on pathologies.     

Identification of a translation inhibitory element (TIE) in the 3' untranslated region of the human interferon-beta mRNA

We have previously reported that the 3' untranslated region (UTR) of the human interferon-beta mRNA has an inhibitory effect on the mRNA translation both in vitro, in a rabbit reticulocyte lysate, and in vivo, in the Xenopus oocyte. In the present study, we identify the sequence in the 3' UTR which is responsible for this translation inhibition. We show that this sequence is located between the 100th and 161st nucleotides downstream from the translation stop codon. It contains several repeats of the A + U-rich consensus octanucleotide UUAUUUAU, which is also present in the 3' UTR of several mRNAs involved in the inflammatory response. We also demonstrate here that the inhibitory effect of the sequence on the mRNA translation does not depend on its position in relation to the termination codon. However, no inhibition of translation is observed when this sequence is inserted in the 5' UTR of the mRNA. The removal of the translation inhibitory sequence not only improves the mRNA translation in Xenopus oocytes but it also strongly decreases the IFN-beta mRNA stability in those cells. This suggests that, in this system at least, the mRNA degradation is linked to its translational efficiency.     

Binding of the La autoantigen to the 5' untranslated region of a chimeric human translation elongation factor 1A reporter mRNA inhibits translation in vitro.

Human translation elongation factor 1A (EF1A) is a member of a large class of mRNAs, including ribosomal proteins and other translation elongation factors, which are coordinately translationally regulated under various conditions. Each of these mRNAs contains a terminal oligopyrimidine tract (TOP) that is required for translational control. A human growth hormone (hGH) expression construct containing the promoter region and 5' untranslated region (UTR) of EF1A linked to the hGH coding region (EF1A/hGH) was translationally repressed following rapamycin treatment in similar fashion to endogenous EF1A in human B lymphocytes. Mutation of two nucleotides in the TOP motif abolished the translational regulation. Gel mobility shift assays showed that both La protein from human B lymphocyte cytoplasmic extracts as well as purified recombinant La protein specifically bind to an in vitro-synthesized RNA containing the 5' UTR of EF1A mRNA. Moreover, extracts prepared from rapamycin-treated cells showed increased binding activity to the EF1A 5' UTR RNA, which correlates with TOP mRNA translational repression. In an in vitro translation system, recombinant La dramatically decreased the expression of EF1A/hGH construct mRNA, but not mRNAs lacking an intact TOP element. These results indicate that TOP mRNA translation may be modulated through La binding to the TOP element.     

The 5' untranslated region of the maize alcohol dehydrogenase gene provides efficient translation of mRNA in plants under stress conditions

Reduced level of expression of most cell proteins under stress conditions is determined by low efficiency of cap-dependent translation of corresponding mRNAs. The maize gene encoding alcohol dehydrogenase, adh1, is an example of a gene which mRNA is efficiently translated under hypoxia. Using reporter gene assay we showed that the leader sequence of adh1 mRNA, provides efficient translation of reporter gene gfp in Nicotiana benthamiana cells under hypoxia and heat shock. The presence of this leader sequence in 5' UTR of mRNA does not change the level of expression in aerobic conditions, but under hypoxia and heat shock the levels of reporter gfp expression were reduced about 5-10 fold in the absence of leader and remained unaffected in its presence in 5'UTR. We found that this leader sequence does not change the level of mRNA stability and does not exhibit promoter activity. Consequently, leader sequence acts as translational enhancer providing efficient translation of mRNA in plant cells under stress conditions. Introduction of this sequence into standard expression cassettes may be used for development of new systems of expression of target proteins in plants, efficient under stress conditions.     

Characterization of protein-binding to the spinach chloroplast psbA mRNA 5' untranslated region.

RNA-binding proteins play a major role in regulating mRNA metabolism in chloroplasts. In this work we characterized two proteins, of 43 and 47 kDa, which bind to the spinach psbA mRNA 5' untranslated region (psbA encoding the D1 protein of photosystem II). The 43 kDa protein, which is present in the stroma and in membranes, co-sediments with a complex of 68S. It was purified, and the N-terminal sequence was determined. Upon homology search it was identified as the chloroplast homologue of the Escherichia coli ribosomal protein S1. The 47 kDa protein, which, in contrast with the 43 kDa protein, sediments with a small sedimentation coefficient, is only detected in the stromal fraction. It is soluble in an uncomplexed form. By deletion analysis, an element within the psbA mRNA 5' untranslated region was identified that is necessary but not sufficient for binding of stromal proteins. The 'central protein binding element' ranges from nucleotide -49 to -9 of the psbA mRNA 5' untranslated region. It comprises the Shine-Dalgarno-like GGAG motif and, 7 nucleotides upstream, an endonucleolytic cleavage site involved in psbA mRNA degradation in vitro . The mechanistic impacts of this region in relation to RNA-binding proteins are discussed.