La protein has a positive effect on the translation of TOP mRNAs in vivo

In vertebrates, the mRNAs encoding ribosomal proteins, as well as other proteins implicated in translation, are characterized by a 5' -untranslated region (5'-UTR), including a stretch of pyrimidines at the 5'-end. The 5'-terminal oligopyrimidine (5'-TOP) sequence, which is involved in the growth-dependent translational regulation characteristic of this class of genes (so-called TOP genes), has been shown to specifically bind the La protein in vitro, suggesting that La might be implicated in translational regulation in vivo. In order to substantiate this hypothesis, we have examined the effect of La on TOP mRNA translational control in both stable and transient transfection experiments. In particular we have constructed and analyzed three stably transfected Xenopus cell lines inducible for overexpression of wild-type La or of putative dominant negative mutated forms. Moreover, La-expressing plasmids have been transiently co-transfected together with a plasmid expressing a reporter TOP mRNA in a human cell line. Our results suggest that in vivo La protein plays a positive role in the translation of TOP mRNA. They also suggest that the function of La is to counteract translational repression exerted by a negative factor, possibly cellular nucleic acid binding protein (CNBP), which has been previously shown to bind the 5'-UTR downstream from the 5'-TOP sequence

Effect of 3 ' UTR length on the translational regulation of 5 '-terminal oligopyrimidine mRNAs

In Vertebrates, all genes coding for ribosomal proteins, as well as those for other proteins implicated in the production and function of translation machinery, are regulated by mitogenic and nutritional stimuli, at the translational level. A cis-regulatory element necessary for this regulation is the typical 5' UTR, common to all ribosomal protein mRNAs, which always starts at the 5' end with several pyrimidines. Having noticed that the 3' UTR of all ribosomal protein mRNAs is much shorter than most cellular mRNAs, we have now studied the possible implication of this 3' UTR feature in the translational regulation. For this purpose, we constructed a number of chimeric genes whose transcribed mRNAs contain: (1) the 5' UTR of ribosomal protein S6 mRNA or, as a control, of betaactin mRNA; (2) the EGFP reporter coding sequence from the starting AUG to the stop codon; (3) different 3' UTRs of various lengths. These constructs have been stably transfected in human HEK293 cells, and the translation regulation of the expressed chimeric mRNAs has been analyzed for translation efficiency, in growing and in serum starved cells, by the polysome association assay. The results obtained indicate that, while the typical growth-associated translational regulation is bestowed on an mRNA by the pyrimidine sequence containing 5' UTR, the stringency of regulation depends on the short size of the 3' UTR. (C) 2004 Elsevier B.V. All rights reserved

Structure of human succinic semialdehyde dehydrogenase gene: Identification of promoter region and alternatively processed isoforms

Mitochondrial NAD+-dependent succinic semialdehyde dehydrogenase (ALDH5A1, SSADH) represents the last enzyme in the GABA catabolism and irreversibly oxidizes SSA to succinate. In human, SSADH deficiency results in 4-hydroxybutyric aciduria, an autosomal recessive disorder due to an accumulation of GABA and 4-hydroxybutyric acid in the CNS. We already identified SSADH gene on human chromosome 6p22 and characterized the coding region. Furthermore, we described the first two mutations causing the disease. We report here the complete cDNA and genomic structure of the gene. A single transcription start site was identified by RNase protection 122 bp upstream of the ATG. EST database search and reporter gene constructs of the 3′ genomic region showed that the two major SSADH mRNA isoforms are due to alternative polyadenylation sites. The two mRNAs of 1827 and 5225 nt were analyzed for differential stability and translation efficiency. The analysis of mRNA turnover showed that both SSADH transcripts are equally stable. Similarly, a measurement of polysomal association capability of the two GFP-SSADH reporter mRNAs (containing the 3′ UTR regions of the two SSADH mRNAs) did not reveal any difference. However, we cannot exclude the fact that differential properties could be restricted to particular physiological conditions and/or specific tissues. We have also identified an alternatively spliced small exon, which may lead to a novel isoform of the enzyme. Furthermore, we report here on naturally occurring missense variants, which may significantly contribute to inter-individual variation of SSADH activity, possibly influencing GABA and GHB endogenous levels. © 2002 Elsevier Science (USA). All rights reserved

Expression of the Homeobox Genes OTX2 and OTX1 in the Early Developing Human Brain

In rodents, the Otx2 gene is expressed in the diencephalon, mesencephalon, and cerebellum and is crucial for the development of these brain regions. Together with Otx1, Otx2 is known to cooperate with other genes to develop the caudal forebrain and, further, Otx1 is also involved in differentiation of young neurons of the deeper cortical layers. We have studied the spatial and temporal expression of the two homeobox genes OTX2 and OTX1 in human fetal brains from 7 to 14 weeks postconception by in situ hybridization and immunohistochemistry. OTX2 was expressed in the diencephalon, mesencephalon, and choroid plexus, with a minor expression in the basal telencephalon. The expression of OTX2 in the hippocampal anlage was strong, with no expression in the adjacent neocortex. Contrarily, the OTX1 expression was predominantly located in the proliferative zones of the neocortex. At later stages, the OTX2 protein was found in the subcommissural organ, pineal gland, and cerebellum. The early expression of OTX2 and OTX1 in proliferative cell layers of the human fetal brain supports the concept that these homeobox genes are important in neuronal cell development and differentiation: OTX1 primarily in the neocortex, and OTX2 in the archicortex, diencephalon, rostral brain stem, and cerebellum. (J Histochem Cytochem 58:669–678, 2010

The molecular elements that underlie developmental evolution

Abundant evidence indicates that developmental evolution, the foundation of morphological evolution, is based on changes in gene function. Over the past decade a consensus has developed that transcriptional regulation, acting through enhancer sequences, is the primary level of evolutionary significant change. Here we propose that other regulatory levels are probably as important as enhancers in developmental evolution. We also explain why these alternative regulatory levels might have been neglected, and briefly discuss ways to test our hypothesis