Transcriptional and translational regulation of zebrafish connexin 55.5 (zf.Cx.55.5) and connexin 52.6 (zf.Cx52.6)

Zebrafish connexin 55.5 (zf.Cx55.5) and connexin 52.6 (zf.Cx52.6) show highly restricted expression patterns in the nervous system. Both connexins are confined to subsets of neurons in the fish retina. In order to get initial answers to the questions of pattern definition in neuronal subsets, we elucidated molecular mechanisms responsible for their expression. Different upstream DNA fragments were subcloned into a pGL3-basic vector and transiently transfected in HeLa and N2A cells. Luciferase activity showed the presence of two putative promoter elements in zfCx55.5 and a promoter element in zfCx52.6 that showed different promoter activities in HeLa and N2A cells. Moreover, fusion constructs of zfCx55.5 with EGFP revealed the presence of a new isoform with an additional short exon I.     

Re-programming of translation following cell stress allows IRES-mediated translation to predominate

There is now an overwhelming body of evidence to suggest that internal ribosome entry is required to maintain the expression of specific proteins during patho-physiological situations when cap-dependent translation is compromised, for example, following heat shock or during mitosis, hypoxia, differentiation and apoptosis. Translational profiling has been used by several groups to assess the extent to which alternative mechanisms of translation initiation selectively recruit mRNAs to polysomes during cell stress. The data from these studies have shown that under each condition 3-5% of coding mRNAs remain associated with the polysomes. Importantly, the genes identified in each of these studies do not show a significant amount of overlap, suggesting that 10-15% of all mRNAs have the capability for their initiation to occur via alternative mechanism(s).     

Re-programming of translation following cell stress allows IRES-mediated translation to predominate.

There is now an overwhelming body of evidence to suggest that internal ribosome entry is required to maintain the expression of specific proteins during patho-physiological situations when cap-dependent translation is compromised, for example, following heat shock or during mitosis, hypoxia, differentiation and apoptosis. Translational profiling has been used by several groups to assess the extent to which alternative mechanisms of translation initiation selectively recruit mRNAs to polysomes during cell stress. The data from these studies have shown that under each condition 3-5% of coding mRNAs remain associated with the polysomes. Importantly, the genes identified in each of these studies do not show a significant amount of overlap, suggesting that 10-15% of all mRNAs have the capability for their initiation to occur via alternative mechanism(s).     

Transcriptional and Translational Regulation of Zebrafish Connexin 55.5 (zf.Cx.55.5) and Connexin 52.6 (zf.Cx52.6)

Zebrafish connexin 55.5 (zf.Cx55.5) and connexin 52.6 (zf.Cx52.6) show highly restricted expression patterns in the nervous system. Both connexins are confined to subsets of neurons in the fish retina. In order to get initial answers to the questions of pattern definition in neuronal subsets, we elucidated molecular mechanisms responsible for their expression. Different upstream DNA fragments were subcloned into a pGL3-basic vector and transiently transfected in HeLa and N2A cells. Luciferase activity showed the presence of two putative promoter elements in zfCx55.5 and a promoter element in zfCx52.6 that showed different promoter activities in HeLa and N2A cells. Moreover, fusion constructs of zfCx55.5 with EGFP revealed the presence of a new isoform with an additional short exon I.     

Transcriptional and Translational Regulation of Zebrafish Connexin 55.5 (zf.Cx.55.5) and Connexin 52.6 (zf.Cx52.6)

Zebrafish connexin 55.5 (zf.Cx55.5) and connexin 52.6 (zf.Cx52.6) show highly restricted expression patterns in the nervous system. Both connexins are confined to subsets of neurons in the fish retina. In order to get initial answers to the questions of pattern definition in neuronal subsets, we elucidated molecular mechanisms responsible for their expression. Different upstream DNA fragments were subcloned into a pGL3-basic vector and transiently transfected in HeLa and N2A cells. Luciferase activity showed the presence of two putative promoter elements in zfCx55.5 and a promoter element in zfCx52.6 that showed different promoter activities in HeLa and N2A cells. Moreover, fusion constructs of zfCx55.5 with EGFP revealed the presence of a new isoform with an additional short exon I.     

Crystal structure of an RNA tertiary domain essential to HCV IRES-mediated translation initiation

The hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA drives internal initiation of viral protein synthesis during host cell infection. In the tertiary structure of the IRES RNA, two helical junctions create recognition sites for direct binding of the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). The 2.8 A resolution structure of the IIIabc four-way junction, which is critical for binding eIF3, reveals how junction nucleotides interact with an adjacent helix to position regions directly involved in eIF3 recognition. Two of the emergent helices stack to form a nearly continuous A-form duplex, while stacking of the other two helices is interrupted by the insertion of junction residues into the helix minor groove. This distorted stack probably serves as an important recognition surface for the translational machinery.     

The crystal structure of the carboxy-terminal domain of human translation initiation factor eIF5

The carboxy-terminal domain (CTD) of eukaryotic initiation factor 5 (eIF5) plays a central role in the formation of the multifactor complex (MFC), an important intermediate for the 43 S pre-initiation complex assembly. The IF5-CTD interacts directly with the translation initiation factors eIF1, eIF2-beta, and eIF3c, thus forming together with eIF2 bound Met-tRNA(i)(Met) the MFC. In this work we present the high resolution crystal structure of eIF5-CTD. This domain of the protein is exclusively composed out of alpha-helices and is homologous to the carboxy-terminal domain of eIF2B-epsilon (eIF2Bepsilon-CTD). The most striking difference in the two structures is an additional carboxy-terminal helix in eIF5. The binding sites of eIF2-beta, eIF3 and eIF1 were mapped onto the structure. eIF2-beta and eIF3 bind to non-overlapping patches of negative and positive electrostatic potential, respectively.     

Insights into the role of yeast eIF2A in IRES-mediated translation

Eukaryotic initiation factor 2A is a single polypeptide that acts to negatively regulate IRES-mediated translation during normal cellular conditions. We have found that eIF2A (encoded by YGR054w) abundance is reduced at both the mRNA and protein level during 6% ethanol stress (or 37°C heat shock) under conditions that mimic the diauxic shift in the yeast Saccharomyces cerevisiae. Furthermore, eIF2A protein is posttranslationally modified during ethanol stress. Unlike ethanol and heat shock stress, H(2)O(2) and sorbitol treatment induce the loss of eIF2A mRNA, but not protein and without protein modification. To investigate the mechanism of eIF2A function we employed immunoprecipitation-mass spectrometry and identified an interaction between eIF2A and eEF1A. The interaction between eIF2A and eEF1A increases during ethanol stress, which correlates with an increase in IRES-mediated translation from the URE2 IRES element. These data suggest that eIF2A acts as a switch to regulate IRES-mediated translation, and eEF1A may be an important mediator of translational activation during ethanol stress.     

Cellular IRES-mediated translation: The war of ITAFs in pathophysiological states

Translation of cellular mRNAs via initiation at internal ribosome entry sites (IRESs) has received increased attention during recent years due to its emerging significance for many physiological and pathological stress conditions in eukaryotic cells. Expression of genes bearing IRES elements in their mRNAs is controlled by multiple molecular mechanisms, with IRES-mediated translation favored under conditions when cap-dependent translation is compromised. In this review, we discuss recent advances in the field and future directions that may bring us closer to understanding the complex mechanisms that guide cellular IRES-mediated expression. We present examples in which the competitive action of IRES-transacting factors (ITAFs) plays a pivotal role in IRES-mediated translation and thereby controls cell-fate decisions leading to either pro-survival stress adaptation or cell death.Key words: translation initiation, IRES, canonical initiation factors, ITAFs, stress response, eIF2, angiogenesis, mitosis, nutrient-signaling, hyperosmolar stress     

Conservation of a cytoplasmic carboxy-terminal domain of connexin 43, a gap junctional protein,in mammal heart and brain

Summary According to the sequence of connexin 43, a cardiac gap junctional protein, the domain contained within residues 314–322 is located 60 amino acids away from the carboxy-terminus. Antibodies raised to a peptide corresponding to this domain label a unique 43-kD protein on immunoblots of both purified gap junctions and whole extracts from rat heart. Immunofluorescence investigations carried out on mammal heart sections reveal a pattern consistent with the known distribution of intercalated discs. Immunogold labeling performed with ultrathin frozen sections of rat heart or partially purified rat heart gap junctions demonstrate that antigenic determinants are associated exclusively with the cytoplasmic surfaces of gap junctions.The antibodies were shown to cross-react with a 43-kD protein on immunoblots of whole extracts from human, mouse and guinea pig heart. However, no labeling was seen when heart of lower vertebrates such as chicken, frog and trout, was investigated. These results, confirmed by immunofluorescence investigations, were interpreted as a loss of antigenic determinants due to sequence polymorphism of cardiac connexin 43.Proteins ofM _r 43 and 41 kD, immunologically related to cardiac connexin 43, were detected in immunoblots of mouse and rat brain whole extracts. mRNAs, homologous to those of cardiac connexin 43 and of the same size (3.0 kb), are also present in brain. Immunofluorescence investigations with primary cultures of unpermeabilized and permeabilized mouse neural cells showed that the antigenic determinants recognized by the antibodies specific for connexin 43 are cytoplasmic and that the labeling observed between clustered flat cells, is punctate, as expected for gap junctions. Double labeling experiments demonstrated that the immunoreactivity is associated with GFAP-positive cells, that is to say, astrocytes.     

Structural basis for the biological relevance of the invariant apical stem in IRES-mediated translation

RNA structure plays a fundamental role in internal initiation of translation. Picornavirus internal ribosome entry site (IRES) are long, efficient cis-acting elements that recruit the ribosome to internal mRNA sites. However, little is known about long-range constraints determining the IRES RNA structure. Here, we sought to investigate the functional and structural relevance of the invariant apical stem of a picornavirus IRES. Mutation of this apical stem revealed better performance of G:C compared with C:G base pairs, demonstrating that the secondary structure solely is not sufficient for IRES function. In turn, mutations designed to disrupt the stem abolished IRES activity. Lack of tolerance to accept genetic variability in the apical stem was supported by the presence of coupled covariations within the adjacent stem-loops. SHAPE structural analysis, gel mobility-shift and microarrays-based RNA accessibility revealed that the apical stem contributes to maintain IRES RNA structure through the generation of distant interactions between two adjacent stem-loops. Our results demonstrate that a highly interactive structure constrained by distant interactions involving invariant G:C base pairs plays a key role in maintaining the RNA conformation necessary for IRES-mediated translation.     

Diversity among beta-tubulins: a carboxy-terminal domain of yeast beta-tubulin is not essential in vivo.

Sequences of genes for beta-tubulins from many different organisms demonstrate that they encode highly conserved proteins but that these proteins diverge considerably at their carboxyl termini. The patterns of interspecies conservation of this diversity suggest that it may have functional significance. We have taken advantage of the properties of Saccharomyces cerevisiae to test this hypothesis in vivo. The sole beta-tubulin gene of this species is one of the most divergent of all beta-tubulins and encodes 12 amino acids which extend past the end of most other beta-tubulin molecules. We have constructed strains in which the only beta-tubulin gene is an allele lacking these 12 codons. We show here that this carboxy-terminal extension is not essential. The absence of these 12 amino acids had no effect on a number of microtubule-dependent functions, such as mitotic and meiotic division and mating. It did confer dominant supersensitivity to a microtubule-depolymerizing drug.     

IRES-mediated translation of utrophin A is enhanced by glucocorticoid treatment in skeletal muscle cells

Glucocorticoids are currently the only drug treatment recognized to benefit Duchenne muscular dystrophy (DMD) patients. The nature of the mechanisms underlying the beneficial effects remains incompletely understood but may involve an increase in the expression of utrophin. Here, we show that treatment of myotubes with 6alpha-methylprednisolone-21 sodium succinate (PDN) results in enhanced expression of utrophin A without concomitant increases in mRNA levels thereby suggesting that translational regulation contributes to the increase. In agreement with this, we show that PDN treatment of cells transfected with monocistronic reporter constructs harbouring the utrophin A 5'UTR, causes an increase in reporter protein expression while leaving levels of reporter mRNAs unchanged. Using bicistronic reporter assays, we further demonstrate that PDN enhances activity of an Internal Ribosome Entry Site (IRES) located within the utrophin A 5'UTR. Analysis of polysomes demonstrate that PDN causes an overall reduction in polysome-associated mRNAs indicating that global translation rates are depressed under these conditions. Importantly, PDN causes an increase in the polysome association of endogenous utrophin A mRNAs and reporter mRNAs harbouring the utrophin A 5'UTR. Additional experiments identified a distinct region within the utrophin A 5'UTR that contains the inducible IRES activity. Together, these studies demonstrate that a translational regulatory mechanism involving increased IRES activation mediates, at least partially, the enhanced expression of utrophin A in muscle cells treated with glucocorticoids. Targeting the utrophin A IRES may thus offer an important and novel therapeutic avenue for developing drugs appropriate for DMD patients.     

The carboxy-terminal peptides of in vitro precursor and in vivo mature kappa light chains are identical.

Protein structural analysis of several precursor light chains indicates that these in vitro products include a cysteine-containing tryptic peptide that is indistinguishable from the carboxy-terminal tryptic tripeptide present in in vivo mature kappa light chains. It is concluded that although in vitro precursor light chains contain an additional amino-terminal sequence, their carboxy-terminus is the same as that found in in vivo mature light chains.