The 3' UTR of the human CTLA4 mRNA can regulate mRNA stability and translational efficiency

T cell activation results from the integration of signals generated through the T cell antigen receptor-CD3 complex with those from additional positive and negative regulatory pathways mainly mediated by the engagement of costimulatory receptors on T cells. Disruption of this balance leads to a defective immune response or alternative over-activation of the immune system. CTLA-4 plays a critical role in downregulating T cell responses. Autoimmune diseases have shown genetic linkage to the CTLA4 locus. In this report we demonstrate that the 3' UTR of CTLA4 regulates firefly luciferase reporter gene expression, can confer instability to CTLA4 mRNA and can influence its translation efficiency in vitro.     

RNA complementary to the 5' UTR of mRNA triggers effective silencing in Saccharomyces cerevisiae

Conditional silencing of target genes in Saccharomyces cerevisiae by antisense RNAs expressed in vivo has been challenged. The MFalpha1::lacZ fusion present in S. cerevisiae SF51-3 was chosen as a model target, and fragments of this gene were cloned in reverse orientation into the expression vector pYES2, bearing the GAL1 promoter. Among the different antisense constructs tested, only the one complementary to the 5' UTR of target mRNA featured effective silencing. Nevertheless, the expression in vivo of this antisense RNA could not be properly tuned by the absence or presence of galactose in the culture medium. Accordingly, conditional silencing could not be attained by this antisense hosted into pYES2. On the contrary, cloning the same antisense construct into the expression vector pSAL4 yielded a fully conditional silencing linked to the control of antisense expression by the absence or presence of Cu(2+) into the culture medium.     

EJC-independent degradation of nonsense immunoglobulin-mu mRNA depends on 3' UTR length

Inconsistent with prevailing models for nonsense-mediated mRNA decay (NMD) in mammals, the mRNA levels of immunoglobulin-mu (Ig-mu) genes with premature termination codons (PTCs) in the penultimate exon are still reduced by NMD when the intron furthest downstream is deleted. As in yeast, this exon junction complex-independent NMD of Ig-mu mRNAs depends on the distance between the termination codon and the poly(A) tail and suggests an evolutionarily conserved mode of PTC recognition.     

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.     

Molecular architecture of a miRNA-regulated 3' UTR

Animal genomes contain hundreds of microRNAs (miRNAs), small regulatory RNAs that control gene expression by binding to complementary sites in target mRNAs. Some rules that govern miRNA/target interaction have been elucidated but their general applicability awaits further experimentation on a case-by-case basis. We use here an assay system in transgenic nematodes to analyze the interaction of the Caenorhabditis elegans lsy-6 miRNA with 3' UTR sequences. In contrast to many previously described assay systems used to analyze miRNA/target interactions, our assay system operates within the cellular context in which lsy-6 normally functions, a single neuron in the nervous system of C. elegans. Through extensive mutational analysis, we define features in the known and experimentally validated target of lsy-6, the 3' UTR of the cog-1 homeobox gene, that are required for a functional miRNA/target interaction. We describe that both in the context of the cog-1 3' UTR and in the context of heterologous 3' UTRs, one or more seed matches are not a reliable predictor for a functional miRNA/target interaction. We rather find that two nonsequence specific contextual features beyond miRNA target sites are critical determinants of miRNA-mediated 3' UTR regulation. The contextual features reside 3' of lsy-6 binding sites in the 3' UTR and act in a combinatorial manner; mutation of each results in limited defects in 3' UTR regulation, but a combinatorial deletion results in complete loss of 3' UTR regulation. Together with two lsy-6 sites, these two contextual features are capable of imparting regulation on a heterologous 3' UTR. Moreover, the contextual features need to be present in a specific configuration relative to miRNA binding sites and could either represent protein binding sites or provide an appropriate structural context. We conclude that a given target site resides in a 3' UTR context that evolved beyond target site complementarity to support regulation by a specific miRNA. The large number of 3' UTRs that we analyzed in this study will also be useful to computational biologists in designing the next generation of miRNA/target prediction algorithms.     

Isolation and characterization of a human U3 small nucleolar RNA gene

U3 RNA is an abundant, capped, small nucleolar RNA, implicated in the processing of preribosomal RNA. In this study, a DNA clone coding for U3 RNA (clone U3-1) was isolated from a human genomic library and characterized. The DNA sequence was identical to that of human U3 RNA isolated from HeLa cells. The flanking regions showed homology to the enhancer, promoter, and 3'-processing signal found in U1 and U2 snRNA genes. Further, the recently identified "U3 box" (GATTGGCTGCN10TATGTTAATTATGG) of rat U3 genes (Stroke and Weiner, (1985) J. Mol. Biol. 184, 183-193), was also found in the human U3 gene. This gene was transcribed in Xenopus oocytes; it is the first cloned true human U3 gene.     

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.     

hNUDT16: a universal decapping enzyme for small nucleolar RNA and cytoplasmic mRNA

Human NUDT16 (hNUDT16) is a decapping enzyme initially identified as the human homolog to the Xenopus laevis X29. As a metalloenzyme, hNUDT16 relies on divalent cations for its cap-hydrolysis activity to remove m⁷GDP and m²²⁷GDP from RNAs. Metal also determines substrate specificity of the enzyme. So far, only U8 small nucleolar RNA (snoRNA) has been identified as the substrate of hNUDT16 in the presence of Mg²⁺. Here we demonstrate that besides U8, hNUDT16 can also actively cleave the m⁷GDP cap from mRNAs in the presence of Mg²⁺ or Mn²⁺. We further show that hNUDT16 does not preferentially recognize U8 or mRNA substrates by our cross-inhibition and quantitative decapping assays. In addition, our mutagenesis analysis identifies several key residues involved in hydrolysis and confirms the key role of the REXXEE motif in catalysis. Finally an investigation into the subcellular localization of hNUDT16 revealed its abundance in both cytoplasm and nucleus. These findings extend the substrate spectrum of hNUDT16 beyond snoRNAs to also include mRNA, demonstrating the pleiotropic decapping activity of hNUDT16.     

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.     

Analysis of the 3' UTR of the ART3 and ART4 gene by 3' inverse RACE-PCR.

3' Rapid amplification of cDNA ends (3' RACE) is a polymerase chain reaction (PCR) based technique which has been developed to analyse 3' ends of partially known cDNA sequences. To improve the effectiveness of the technique, many investigators have modified the RACE protocol. Here, we describe an alternative procedure for analysing 3' mRNA ends which is based on DNA ligase-mediated self circularization and inverse PCR. This technique is simple and characterized by the exclusive use of gene-specific primers and the absence of unspecific adaptor sequences to obtain highly specific PCR products. We applied the method to analyze the 3' UTR of human mono-ADP-ribosyltransferase (ART) 3 mRNA in testis and heart muscle and of ART4 mRNA in HEL cells. The obtained sequences of ART3 and ART4 mRNA corresponded to data base entries of the respective mRNAs. No adenylate/uridylate-rich elements (AREs) were found in the 3' UTR of ART3 mRNA while one ARE class I motif was detected in the 3' UTR of ART4 mRNA.     

A variation in 3' UTR of hPTP1B increases specific gene expression and associates with insulin resistance

Protein tyrosine phosphatase 1B (PTP1B) inhibits insulin signaling and, when overexpressed, plays a role in insulin resistance (Ahmad et al. 1997). We identified, in the 3' untranslated region of the PTP1B gene, a 1484insG variation that, in two different populations, is associated with several features of insulin resistance: among male individuals, higher values of the insulin resistance HOMA(IR) index (P=.006), serum triglycerides (P=.0002), and total/HDL cholesterol ratio (P=.025) and, among female individuals, higher blood pressure (P=.01). Similar data were also obtained in a family-based association study by use of sib pairs discordant for genotype (Gu et al. 2000). Subjects carrying the 1484insG variant showed also PTP1B mRNA overexpression in skeletal muscle (6,166 plus minus 1,879 copies/40 ng RNA vs. 2,983 plus minus 1,620; P<.01). Finally, PTP1B mRNA stability was significantly higher (P<.01) in human embryo kidney 293 cells transfected with 1484insG PTP1B, as compared with those transfected with wild-type PTP1B. Our data indicate that the 1484insG allele causes PTP1B overexpression and plays a role in insulin resistance. Therefore, individuals carrying the 1484insG variant might particularly benefit from PTP1B inhibitors, a promising new tool for treatment of insulin resistance (Kennedy and Ramachandran 2000).