mRNA Translation Regulation by the Gly-Ala Repeat of Epstein-Barr Virus Nuclear Antigen 1

The glycine-alanine repeat (GAr) sequence of the Epstein-Barr virus-encoded EBNA-1 prevents presentation of antigenic peptides to major histocompatibility complex class I molecules. This has been attributed to its capacity to suppress mRNA translation in cis. However, the underlying mechanism of this function remains largely unknown. Here, we have further investigated the effect of the GAr as a regulator of mRNA translation. Introduction of silent mutations in each codon of a 30-amino-acid GAr sequence does not significantly affect the translation-inhibitory capacity, whereas minimal alterations in the amino acid composition have strong effects, which underscores the observation that the amino acid sequence and not the mRNA sequence mediates GAr-dependent translation suppression. The capacity of the GAr to repress translation is dose and position dependent and leads to a relative accumulation of preinitiation complexes on the mRNA. Taken together with the surprising observation that fusion of the 5′ untranslated region (UTR) of the c-myc mRNA to the 5′ UTR of GAr-carrying mRNAs specifically inactivates the effect of the GAr, these results indicate that the GAr targets components of the translation initiation process. We propose a model in which the nascent GAr peptide delays the assembly of the initiation complex on its own mRNA.Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA-1) and latency-associated nuclear antigen 1 (LANA-1), from Kaposi''s sarcoma-associated herpesvirus (KSHV), are major latency proteins of these two gammaherpesviruses that are essential for maintaining viral episomes in infected cells (21, 22). Independent studies suggest that both proteins have evolved mechanisms to remain largely invisible to the immune system, which could otherwise eliminate latently infected cells (8, 9, 19, 25). These mechanisms act in cis and are mediated via an internal repeat region. In the case of EBNA-1 this region consists of an N-terminal glycine-alanine repeat (GAr), and for LANA-1 the region consists of a glutamine-glutamate-aspartate central repeat (QED-CR). Although the two domains do not share amino acid homology, both retard their own synthesis to reduce the production of defective ribosomal products that can be processed for the major histocompatibility complex (MHC) class I-restricted antigen presentation pathway (23, 24), highlighting the importance of translation control in regulating MHC class I-restricted antigen presentation. To compensate for their low rates of synthesis, both proteins also have slow turnover rates (4, 8).Regulation of translation for most prokaryotic and eukaryotic mRNAs occurs at the level of initiation, but there are examples where regulation of protein synthesis depends on the elongation stage (17). The two main types of translation initiation are the classic cap-dependent and the less frequent cap-independent translation mechanisms (5, 7, 11, 14, 16). In the former, the preinitiation complex is formed around the cap structure in the 5′ untranslated region (UTR) of the message, whereas in the latter the 40S subunit is directed toward the mRNA via an internal ribosome entry site (IRES). The mechanism of GAr- and LANA-1-mediated control of translation seems different from other types of viral regulation in several aspects. The EBNA-1 GAr is 60 to 300 amino acids long, depending on virus isolate, and is positioned in the N-terminal part of the protein. The GAr message is GC rich but does not activate protein kinase R and eukaryotic initiation factor 2α phosphorylation (25). The fact that the GAr has to be encoded to suppress translation, coupled with the restricted use of GGG and GGA codons to express Gly and of GCA to express Ala in the GAr (GAT, GAG, and CAG for aspartic acid, glutamic acid, and glutamine, respectively, in the LANA sequence), could suggest that codon exhaustion might explain the effect of these repeats. However, manipulations of sequence order, orientation, and composition of the QED-CR and GAr domains and the observation that antibodies directed toward the GAr can stimulate translation in vitro instead favor a direct role for the amino acid sequence (8, 25).Here, we have studied GAr-mediated regulation of translation in vitro and in vivo. The results presented suggest that, once synthesized, the nascent GAr peptide sequence prevents the assembly of the following upstream ribosomes. This knowledge should further understanding of how amino acid repeat sequences can affect mRNA translation in cis and should shed light on a novel type of viral control of mRNA translation and its implications in regulating MHC class I-restricted antigen presentation.     

Dissection of the critical binding determinants of cellular retinoic acid binding protein II by mutagenesis and fluorescence binding assay

The binding of retinoic acid to mutants of Cellular Retinoic Acid Binding Protein II (CRABPII) was evaluated to better understand the importance of the direct protein/ligand interactions. The important role of Arg111 for the correct structure and function of the protein was verified and other residues that directly affect retinoic acid binding have been identified. Furthermore, retinoic acid binding to CRABPII mutants that lack all previously identified interacting amino acids was rescued by providing a carboxylic acid dimer partner in the form of a Glu residue. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Enhancement of exciton coupled circular dichroism with sterically encumbered bis-porphyrin tweezers

Porphyrin tweezers have been successfully used as hosts for the absolute stereochemical determination of a variety of chiral compounds. A set of new porphyrin tweezers with substituted aryl groups on the meso position of the porphyrin rings have been synthesized. The modified tweezers are used as hosts for the stereochemical determination of chiral diamines and carrier-derivatized alpha-chiral carboxylic acids in order to monitor the influence of the various substitutions of the aryl group on the amplitude and sign of the ECCD couplet. t-Butyl substitution at the meta positions of the porphyrin's meso phenyl substituents leads to enhanced ECCD amplitudes.     

Three new satellite sequences and a mobile element found inside HSP70 introns of the Mediterranean mussel (Mytilus galloprovincialis).

We report the characterization of 3 new repetitive sequences from the bivalve mollusc Mytilus galloprovincialis, designated Mg1, Mg2, and Mg3, with monomer lengths of 169, 260, and 70 bp, respectively. The 3 repeats together constitute approximately 7.8% of the M. galloprovincialis genome and were found, together with ApaI-type 2 repeats, inside the introns of 2 genes of the HSP70 family, hsc70 and hsc71. Both the monomer length and the genomic content of the repeats indicate satellite sequences. The Mg1 repetitive region and its flanking sequences exhibit significant homology to CvE, a member of the Pearl family of mobile elements found in the eastern oyster (Crassostrea virginica). Thus, the whole homologous region is designated MgE, the first putative transposable element characterized in M. galloprovincialis. The ApaI, Mg2, and Mg3 repeats are continuously arranged inside the introns of both the hsc70 and hsc71 genes. The presence of perfect inverted repeats flanking the ApaI-Mg2-Mg3 repetitive region, as well as a sequence analysis of the repeats, indicates a transposition-like insertion of this region. The genes of the HSP70 family are highly conserved, and the presence of repetitive DNA or of mobile elements inside their introns is reported here for the first time.     

Ovarian fluid in the three-spined stickleback Gasterosteus aculeatus: effects of egg overripening and sex steroid treatment

The ovarian fluid properties of the three-spined stickleback Gasterosteus aculeatus were studied in overripe and non-overripe ovulated female sticklebacks and in females that were implanted with Silastic capsules containing testosterone (T), oestradiol (E2), 17,20β-dihydroxypregn-4-en-3-one (17,20β-P) or progesterone (P4) into the abdominal cavity. Overripe females had less ovarian fluid than non-overripe females, but with higher dry mass, higher protein concentration and lower viscosity. T and 17,20β-P increased the amount of ovarian fluid and the fluid protein concentration was increased by 17,20β-P. 1-D sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) showed that ovarian fluid contains several proteins, with high individual variability but with no consistent differences between groups. Some of the ovarian fluid proteins appeared to correspond to proteins from the eggs. The results suggest that secretion of ovarian fluid may be influenced by steroid hormones and that changes in its properties are related to the overripening of ovulated eggs. In at least some respects it appears that the changes in the ovarian fluid is a result, rather than the cause of overripening.     

Bone mineral density and genetic markers involved in three connected pathways (focal adhesion, actin cytoskeleton regulation and cell cycle): the CUMAGAS-BMD information system

The focal adhesion, the actin cytoskeleton and cell-cycle are connected pathways and their genes are implicated in the pathogenesis of low BMD. Data from 211 studies that investigated the association between BMD and gene variants involved in these pathways were catalogued in a web-based information system and analyzed. In individual studies, significant association was found for 16 variants in lumbar spine, 11 in femoral neck and 5 in hip. In meta-analysis, significant results were shown for the variants COL1A1 rs1800012 (in lumbar spine and femoral neck), COL1A1 rs1107946 (in lumbar spine), TGFB1 rs1982073 (in femoral neck and hip) and TGFB1 rs1800469 (in lumbar spine).     

Linking Pre-Diabetes with Benign Prostate Hyperplasia. IGFBP-3: A Conductor of Benign Prostate Hyperplasia Development Orchestra?

Benign prostatic hyperplasia (BPH) represents a pattern of non-malignant growth of prostatic fibromuscular stroma. Metabolic disturbances such us pre-diabetes and metabolic syndrome may have a role in BPH pathophysiology. A potential explanation for the above relationship involves the insulin-like growth factor (IGF) axis as well as IGF binding proteins, (IGFBPs) of which the most abundant form is IGFBP-3. Therefore, the aim of the present study was to investigate the association between intra-prostatic levels of IGF-1, IGF-2 as well as to evaluate the role of locally expressed IGFBP-3 in BPH development in pre-diabetes. A total of 49 patients admitted to the Urology department of a tertiary urban Greek hospital, for transurethral prostate resection, or prostatectomy and with pre-diabetes [impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) or both] were finally included. The majority of the sample consisted of subjects with IGT (51.0%), followed by IFG and IGT (32.7%) and isolated IFG (16.3%). For all participants a clinical examination was performed and blood samples were collected. In addition, total prostate (TP) volume or transitional zone (TZ) volume were estimated by transrectal ultrasonography. The results of the multivariate analysis regarding TP volume showed that higher PSA (p<0.001), larger waist circumference (p=0.007) and higher IGFBP-3 expression levels (p<0.001) independently predicted higher TP volume. The results regarding the volume of the TZ showed that higher PSA (p<0.001), larger waist circumference (p<0.001) and higher IGFBP-3 expression levels (p=0.024) were independently associated with higher TZ volume. Our findings show that intra-prostatic levels of IGFBP-3, PSA and waist circumference, but not overall obesity, are positively associated with prostate volume. IGFBP-3 seems to be a multifunctional protein, which can potentiate or inhibit IGF activity.     

Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia

Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. We report a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. We find that Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. Our findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homeostatic contexts. They also suggest an epithelial contribution to Ret loss‐of‐function disorders such as Hirschsprung disease.