Competency for nonsense-mediated reduction in collagen X mRNA is specified by the 3' UTR and corresponds to the position of mutations in Schmid metaphyseal chondrodysplasia

Nonsense-mediated decay (NMD) is a eukaryotic cellular RNA surveillance and quality-control mechanism that degrades mRNA containing premature stop codons (nonsense mutations) that otherwise may exert a deleterious effect by the production of dysfunctional truncated proteins. Collagen X (COL10A1) nonsense mutations in Schmid-type metaphyseal chondrodysplasia are localized in a region toward the 3' end of the last exon (exon 3) and result in mRNA decay, in contrast to most other genes in which terminal-exon nonsense mutations are resistant to NMD. We introduce nonsense mutations into the mouse Col10a1 gene and express these in a hypertrophic-chondrocyte cell line to explore the mechanism of last-exon mRNA decay of Col10a1 and demonstrate that mRNA decay is spatially restricted to mutations occurring in a 3' region of the exon 3 coding sequence; this region corresponds to where human mutations have been described. This localization of mRNA-decay competency suggested that a downstream region, such as the 3' UTR, may play a role in specifying decay of mutant Col10a1 mRNA containing nonsense mutations. We found that deleting any of the three conserved sequence regions within the 3' UTR (region I, 23 bp; region II, 170 bp; and region III, 76 bp) prevented mutant mRNA decay, but a smaller 13 bp deletion within region III was permissive for decay. These data suggest that the 3' UTR participates in collagen X last-exon mRNA decay and that overall 3' UTR configuration, rather than specific linear-sequence motifs, may be important in specifying decay of Col10a1 mRNA containing nonsense mutations.     

Recruitment of the Puf3 protein to its mRNA target for regulation of mRNA decay in yeast

The Puf family of RNA-binding proteins regulates mRNA translation and decay via interactions with 3' untranslated regions (3' UTRs) of target mRNAs. In yeast, Puf3p binds the 3' UTR of COX17 mRNA and promotes rapid deadenylation and decay. We have investigated the sequences required for Puf3p recruitment to this 3' UTR and have identified two separate binding sites. These sites are specific for Puf3p, as they cannot bind another Puf protein, Puf5p. Both sites use a conserved UGUANAUA sequence, whereas one site contains additional sequences that enhance binding affinity. In vivo, presence of either site partially stimulates COX17 mRNA decay, but full decay regulation requires the presence of both sites. No other sequences outside the 3' UTR are required to mediate this decay regulation. The Puf repeat domain of Puf3p is sufficient not only for in vitro binding to the 3' UTR, but also in vivo stimulation of COX17 mRNA decay. These experiments indicate that the essential residues involved in mRNA decay regulation are wholly contained within this RNA-binding domain.     

Cell and molecular regulation of endothelin-1 production during hepatic wound healing

During hepatic wound healing, activation of key effectors of the wounding response known as stellate cells leads to a multitude of pathological processes, including increased production of endothelin-1 (ET-1). This latter process has been linked to enhanced expression of endothelin-converting enzyme-1 (ECE-1, the enzyme that converts precursor ET-1 to the mature peptide) in activated stellate cells. Herein, we demonstrate up-regulation of 56- and 62-kDa ECE-1 3'-untranslated region (UTR) mRNA binding proteins in stellate cells after liver injury and stellate cell activation. Binding of these proteins was localized to a CC-rich region in the proximal ECE-1 3' UTR base pairs (the 56-kDa protein) and to a region between 60 and 193 base pairs in the ECE-1 3' UTR mRNA (62 kDa). A functional role for the 3' UTR mRNA/protein interaction was established in a series of reporter assays. Additionally, transforming growth factor-beta1, a cytokine integral to wound healing, stimulated ET-1 production. This effect was due to ECE-1 mRNA stabilization and increased ECE-1 expression in stellate cells, which in turn was a result of de novo synthesis of the identified 56- and 62-kDa ECE-1 3' UTR mRNA binding proteins. These data indicate that liver injury and the hepatic wound healing response lead to ECE-1 mRNA stabilization in stellate cells via binding of 56- and 62-kDa proteins, which in turn are regulated by transforming growth factor-beta. The possibility that the same or similar regulatory events are present in other forms of wound healing is raised.     

The yeast EDC1 mRNA undergoes deadenylation-independent decapping stimulated by Not2p, Not4p, and Not5p

A major mechanism of eukaryotic mRNA degradation initiates with deadenylation followed by decapping and 5' to 3' degradation. We demonstrate that the yeast EDC1 mRNA, which encodes a protein that enhances decapping, has unique properties and is both protected from deadenylation and undergoes deadenylation-independent decapping. The 3' UTR of the EDC1 mRNA is sufficient for both protection from deadenylation and deadenylation-independent decapping and an extended poly(U) tract within the 3' UTR is required. These observations highlight the diverse forms of decapping regulation and identify a feedback loop that can compensate for decreases in activity of the decapping enzyme. Surprisingly, the decapping of the EDC1 mRNA is slowed by the loss of Not2p, Not4p, and Not5p, which interact with the Ccr4p/Pop2p deadenylase complex. This indicates that the Not proteins can affect decapping, which suggests a possible link between the mRNA deadenylation and decapping machinery.     

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.     

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.     

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.