Fátima Gebauer

Molecular mechanisms of translational control

Translational control is widely used to regulate gene expression. This mode of regulation is especially relevant in situations where transcription is silent or when local control over protein accumulation is required. Although many examples of translational regulation have been described, only a few are beginning to be mechanistically understood. Instead of providing a comprehensive account of the examples that are known at present, we discuss instructive cases that serve as paradigms for different modes of translational control.

Translational control by cytoplasmic polyadenylation of c-mos mRNA is necessary for oocyte maturation in the mouse.

The c‐mos proto‐oncogene product is a key element in the cascade of events leading to meiotic maturation of vertebrate oocytes. We have investigated the role of cytoplasmic polyadenylation in the translational control of mouse c‐mos mRNA and its contribution to meiosis. Using an RNase protection assay we show that optimal cytoplasmic polyadenylation of c‐mos mRNA requires three cis elements in the 3′ UTR: the polyadenylation hexanucleotide AAUAAA and two U‐rich cytoplasmic polyadenylation elements (CPEs) located 4 and 51 nucleotides upstream of the hexanucleotide. When fused to CAT coding sequences, the wild‐type 3′ UTR of c‐mos mRNA, but not a 3′ UTR containing mutations in both CPEs, confers translational recruitment during maturation. This recruitment coincides with maximum polyadenylation. To assess whether c‐mos mRNA polyadenylation is necessary for maturation of mouse oocytes, we have ablated endogenous c‐mos mRNA by injecting an antisense oligonucleotide, which results in a failure to progress to meiosis II after emission of the first polar body. Such antisense oligonucleotide‐injected oocytes could be efficiently rescued by co‐injection of a c‐mos mRNA carrying a wild‐type 3′ UTR. However, co‐injection of a c‐mos mRNA lacking functional CPEs substantially lowered the rescue activity. These results demonstrate that translational control of c‐mos mRNA by cytoplasmic polyadenylation is necessary for normal development.

Antigenic homology among coronaviruses related to transmissible gastroenteritis virus.

Abstract The antigenic homology of 26 coronavirus isolates, of which 22 were antigenically related to transmissible gastroenteritis virus (TGEV), was determined with 42 monoclonal antibodies. Type, group, and interspecies specific epitopes were defined. Two group specific MAbs distinguished the enteric TGEV isolates from the respiratory variants. An antigenic subsite involved in neutralization was conserved in porcine, feline, and canine coronavirus. The classification of the human coronavirus 229E in a taxonomic cluster distinct from TGEV group is suggested.

Genetic evolution and tropism of transmissible gastroenteritis coronaviruses

Abstract Transmissible gastroenteritis virus (TGEV) is an enteropathogenic coronavirus isolated for the first time in 1946. Nonenteropathogenic porcine respiratory coronaviruses (PRCVs) have been derived from TGEV. The genetic relationship among six European PRCVs and five coronaviruses of the TGEV antigenic cluster has been determined based on their RNA sequences. The S protein of six PRCVs have an identical deletion of 224 amino acids starting at position 21. The deleted area includes the antigenic sites C and B of TGEV S glycoprotein. Interestingly, two viruses (NEB72 and TOY56) with respiratory tropism have S proteins with a size similar to the enteric viruses. NEB72 and TOY56 viruses have in the S protein 2 and 15 specific amino acid differences with the enteric viruses. Four of the residues changed (aa 219 of NEB72 isolate and as 92, 94, and 218 of TOY56) are located within the deletion present in the PRCVs and may be involved in the receptor binding site (RBS) conferring enteric tropism to TGEVs. A second RBS used by the virus to infect ST cells might be located in a conserved area between sites A and D of the S glycoprotein, since monoclonal antibodies specific for these sites inhibit the binding of the virus to ST cells. An evolutionary tree relating 13 enteric and respiratory isolates has been proposed. According to this tree, a main virus lineage evolved from a recent progenitor virus which was circulating around 1941. From this, secondary lineages originated PUR46, NEB72, TOY56, MIL65, BR170, and the PRCVs, in this order. Least squares estimation of the origin of TGEV-related coronaviruses showed a significant constancy in the fixation of mutations with time, that is, the existence of a well-defined molecular clock. A mutation fixation rate of 7 ± 2 × 10−4 nucleotide substitutions per site and per year was calculated for TGEV-related viruses. This rate falls in the range reported for other RNA viruses. Point mutations and probably recombination events have occurred during TGEV evolution.

Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 5' and 3' UTRs of msl-2 mRNA is independent of the poly(A) tail.

Translational repression of male‐specific‐lethal 2 (msl‐2) mRNA by Sex‐lethal (SXL) controls dosage compensation in Drosophila. In vivo regulation involves cooperativity between SXL‐binding sites in the 5′ and 3′ untranslated regions (UTRs). To investigate the mechanism of msl‐2 translational control, we have developed a novel cell‐free translation system from Drosophila embryos that recapitulates the critical features of mRNA translation in eukaryotes: cap and poly(A) tail dependence. Importantly, tight regulation of msl‐2 translation in this system requires cooperation between the SXL‐binding sites in both the 5′ and 3′ UTRs, as seen in vivo. However, in contrast to numerous other developmentally regulated mRNAs, the regulation of msl‐2 mRNA occurs by a poly(A) tail‐independent mechanism. The approach described here allows mechanistic analysis of translational control in early Drosophila development and has revealed insights into the regulation of dosage compensation by SXL.

Eukaryotic cold shock domain proteins: highly versatile regulators of gene expression

Cold shock domain (CSD)‐containing proteins have been found in all three domains of life and function in a variety of processes that are related, for the most part, to post‐transcriptional gene regulation. The CSD is an ancient β‐barrel fold that serves to bind nucleic acids. The CSD is structurally and functionally similar to the S1 domain, a fold with otherwise unrelated primary sequence. The flexibility of the CSD/S1 domain for RNA recognition confers an enormous functional versatility to the proteins that contain them. This review summarizes the current knowledge on eukaryotic CSD/S1 domain‐containing proteins with a special emphasis on UNR (upstream of N‐ras), a member of this family with multiple copies of the CSD.

Residues involved in the antigenic sites of transmissible gastroenteritis coronavirus S glycoprotein

Abstract The S glycoprotein of transmissible gastroenteritis virus (TGEV) has been shown to contain four major antigenic sites (A, B, C, and D). Site A is the main inducer of neutralizing antibodies and has been previously subdivided into the three subsites Aa, Ab, and Ac. The residues that contribute to these sites were localized by sequence analysis of 21 mutants that escaped neutralization or binding by TGEV-specific monoclonal antibodies (MAbs), and by epitope scanning (PEPSCAN). Site A contains the residues 538, 591, and 543, which are essential in the formation of subsites Aa, Ab, and Ac, respectively. In addition, mar mutant 1B.H6 with residue 586 changed had partially altered both subsite Aa and Ab, indicating that these subsites overlap in residue 586; i.e. this residue also is part of site A. The peptide 537-MKSGYGQPIA-547 represents, at least partially, subsite Ac which is highly conserved among coronaviruses. This site is relevant for diagnosis and could be of interest for protection. Other residues contribute to site B (residues 97 and 144), site C (residues 50 and 51), and site D (residue 385). The location of site D is in agreement with PEPSCAN results. Site C can be represented by the peptide 48-P-P/S-N-S-D/E-52 but is not exposed on the surface of native virus. Its accessibility can be modulated by treatment at pH >11 (at 4°) and temperatures >45°. Sites A and B are fully dependent on glycosylation for proper folding, while sites C and D are fully or partially independent of glycosylation, respectively. Once the S glycoprotein has been assembled into the virion, the carbohydrate moiety is not essential for the antigenic sites.

Translational control by 3′-UTR-binding proteins

The regulation of mRNA translation is a major checkpoint in the flux of information from the transcriptome to the proteome. Critical for translational control are the trans-acting factors, RNA-binding proteins (RBPs) and small RNAs that bind to the mRNA and modify its translatability. This review summarizes the mechanisms by which RBPs regulate mRNA translation, with special focus on those binding to the 3′-untranslated region. It also discusses how recent high-throughput technologies are revealing exquisite layers of complexity and are helping to untangle translational regulation at a genome-wide scale.

Molecular Characterization of Transmissible Gastroenteritis Coronavirus Defective Interfering Genomes: Packaging and Heterogeneity

Abstract Three transmissible gastroenteritis virus (TGEV) defective RNAs were selected by serial undiluted passage of the PUR46 strain in ST cells. These RNAs of 22, 10.6, and 9.7 kb (DI-A, DI-B, and DI-C, respectively) were detected at passage 30, remained stable upon further passage in cell culture, and significantly interfered with helper mRNA synthesis. RNA analysis from purified virions showed that the three defective RNAs were efficiently packaged. Virions of different densities containing either full-length or defective RNAs were sorted in sucrose gradients, indicating that defective and full-length genomes were independently encapsidated. DI-B and DI-C RNAs were amplified by the reverse transcription-polymerase chain reaction, cloned, and sequenced. DI-B and DI-C genomes are formed by three and four discontinuous regions of the wild-type genome, respectively. DI-C contains 2144 nucleotides (nt) from the 5′-end of the genome, two fragments of 4540 and 2531 nt mostly from gene 1b, and 493 nt from the 3′ end of the genome. DI-B and DI-C RNAs include sequences with the pseudoknot motif and encoding the polymerase, metal ion binding, and helicase motifs. DI-B RNA has a structure closely related to DI-C RNA with two main differences: it maintains the entire ORF 1b and shows heterogeneity in the size of the 3′ end deletion. This heterogeneity maps at the beginning of the S gene, where other natural TGEV recombination events have been observed, suggesting that either a process of template switching occurs with high frequency at this point or that the derived genomes have a selective advantage.

Cytoplasmic polyadenylation and translational control.

Cytoplasmic polyadenylation is the process by which dormant, translationally inactive mRNAs become activated via the elongation of their poly(A) tails in the cytoplasm. This process is regulated by the conserved cytoplasmic polyadenylation element binding (CPEB) protein family. Recent studies have advanced our understanding of the molecular code that dictates the timing of CPEB-mediated poly(A) tail elongation and the extent of translational activation. In addition, evidence for CPEB-independent mechanisms has accumulated, and the breath of biological circumstances in which cytoplasmic polyadenylation plays a role has expanded. These observations underscore the versatility of CPEB as a translational regulator, and highlight the diversity of cytoplasmic polyadenylation mechanisms.