An AUG codon conserved for protein function rather than translational initiation: the story of the protein sElk1.

Elk1 belongs to the ternary complex (TCF) subfamily of the ETS-domain transcription factors. Several studies have implicated an important function for Elk1 in the CNS including synaptic plasticity and cell differentiation. Whilst studying ELK1 gene expression in rat brain a 54 aa N-terminally truncated isoform lacking the DBD was observed on immunoblots. A similar protein was also detected in NGF differentiated PC12 cells. It was proposed that this protein, referred to as sElk1, arose due to a de-novo initiation event at the second AUG codon on the Elk1 ORF. Transient over-expression of sElk1 potentiated neurite growth in the PC12 model and induced differentiation in the absence of NGF, leading to the proposition that it may have a specific function in the CNS. Here we report on the translational expression from the mouse and rat transcript and compare it with our earlier published work on human. Results demonstrate that the previously observed sElk1 protein is a non-specific band arising from the antibody employed. The tight conservation of the internal AUG reported to drive sElk1 expression is in fact coupled to Elk1 protein function, a result consistent with the Elk1-SRE crystal structure. It is also supported by the observed conservation of this methionine in the DBD of all ETS transcription factors independent of the N- or C-terminal positioning of this domain. Reporter assays demonstrate that elements both within the 5'UTR and downstream of the AUGElk1 serve to limit 40S access to the AUGsElk1 codon.

Alternative splicing within the elk-1 5' untranslated region serves to modulate initiation events downstream of the highly conserved upstream open reading frame 2.

The 5' untranslated region (UTR) plays a central role in the regulation of mammalian translation initiation. Key components include RNA structure, upstream AUGs (uAUGs), upstream open reading frames (uORFs), and internal ribosome entry site elements that can interact to modulate the readout. We previously reported the characterization of two alternatively spliced 5' UTR isoforms of the human elk-1 gene. Both contain two uAUGs and a stable RNA stem-loop, but the long form (5' UTR(L)) was more repressive than the short form (5' UTR(S)) for initiation at the ELK-1 AUG. We now demonstrate that ELK-1 expression arises by a combination of leaky scanning and reinitiation, with the latter mediated by the small uORF2 conserved in both spliced isoforms. In HEK293T cells, a considerable fraction of ribosomes scans beyond the ELK-1 AUG in a reinitiation mode. These are sequestered by a series of out-of-frame AUG codons that serve to prevent access to a second in-frame AUG start site used to express short ELK-1 (sELK-1), an N-terminally truncated form of ELK-1 that has been observed only in neuronal cells. We present evidence that all these events are fine-tuned by the nature of the 5' UTR and the activity of the α subunit of eukaryotic initiation factor 2 and provide insights into the neuronal specificity of sELK-1 expression.

The chimeric gene CHRFAM7A, a partial duplication of the CHRNA7 gene, is a dominant negative regulator of α7*nAChR function.

The human α7 neuronal nicotinic acetylcholine receptor gene (CHRNA7) is a candidate gene for schizophrenia and an important drug target for cognitive deficits in the disorder. Activation of the α7*nAChR, results in opening of the channel and entry of mono- and divalent cations, including Ca(2+), that presynaptically participates to neurotransmitter release and postsynaptically to down-stream changes in gene expression. Schizophrenic patients have low levels of α7*nAChR, as measured by binding of the ligand [(125)I]-α-bungarotoxin (I-BTX). The structure of the gene, CHRNA7, is complex. During evolution, CHRNA7 was partially duplicated as a chimeric gene (CHRFAM7A), which is expressed in the human brain and elsewhere in the body. The association between a 2bp deletion in CHRFAM7A and schizophrenia suggested that this duplicate gene might contribute to cognitive impairment. To examine the putative contribution of CHRFAM7A on receptor function, co-expression of α7 and the duplicate genes was carried out in cell lines and Xenopus oocytes. Expression of the duplicate alone yielded protein expression but no functional receptor and co-expression with α7 caused a significant reduction of the amplitude of the ACh-evoked currents. Reduced current amplitude was not correlated with a reduction of I-BTX binding, suggesting the presence of non-functional (ACh-silent) receptors. This hypothesis is supported by a larger increase of the ACh-evoked current by the allosteric modulator 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596) in cells expressing the duplicate than in the control. These results suggest that CHRFAM7A acts as a dominant negative modulator of CHRNA7 function and is critical for receptor regulation in humans.

Associated proteins: The universal toolbox controlling ligand gated ion channel function.

Ligand gated ion channels are integral multimeric membrane proteins that can detect with high sensitivity the presence of a specific transmitter in the extracellular space and transduce this signal into an ion flux. While these receptors are widely expressed in the nervous system, their expression is not limited to neurons or their postsynaptic targets but extends to non-neuronal cells where they participate in many physiological responses. Cells have developed complex regulatory mechanisms allowing for the precise control and modulation of ligand gated ion channels. In this overview the roles of accessory subunits and associated proteins in these regulatory mechanisms are reviewed and their relevance illustrated by examples at different ligand gated ion channel types, with emphasis on nicotinic acetylcholine receptors. Dysfunction of ligand gated ion channels can result in neuromuscular, neurological or psychiatric disorders. A better understanding of the precise function of associated proteins and how they impact on ligand gated ion channels will provide new therapeutic opportunities for clinical intervention.

Cooperation between PU.1 and CAAT/enhancer-binding protein beta is necessary to induce the expression of the MD-2 gene.

Myeloid differentiation factor 2 (MD-2) binds Gram-negative bacterial lipopolysaccharide with high affinity and is essential for Toll-like receptor 4-dependent signal transduction. MD-2 has recently been recognized as a type II acute phase protein. Plasma concentrations of the soluble form of MD-2 increase markedly during the course of severe infections. Its production is regulated in hepatocytes and myeloid cells by interleukin-6 (IL-6) but not IL-1beta. In the present work we show that two transcription factors (TF), PU.1 and CAAT/enhancer-binding protein beta (C/EBPbeta), participate in the activation of the human MD-2 gene in hepatocytic cells after stimulation with IL-6. PU.1 TF and proximal PU.1 binding sites in the MD-2 promoter were shown to be critical for the basal activity of the promoter as well as for IL-6-induced soluble MD-2 production. Deletions of proximal portions of the MD-2 promoter containing PU.1 and/or NF-IL-6 consensus binding sites as well as site-directed mutagenesis of these binding sites abrogated IL-6-dependent MD-2 gene activation. We show that the cooperation between C/EBPbeta and PU.1 is critical for the transcriptional activation of the MD-2 gene by IL-6. PU.1 was essentially known as a TF involved in the differentiation of myeloid precursor cells and the expression of surface receptors of the innate immunity. Herein, we show that it also participates in the regulation of an acute phase protein, MD-2, in nonmyeloid cells cooperatively with C/EBPbeta, a classical IL-6-inducible TF.

Transcriptional and post-transcriptional profile of human chromosome 21.

Recent studies have demonstrated extensive transcriptional activity across the human genome, a substantial fraction of which is not associated with any functional annotation. However, very little is known regarding the post-transcriptional processes that operate within the different classes of RNA molecules. To characterize the post-transcriptional properties of expressed sequences from human chromosome 21 (HSA21), we separated RNA molecules from three cell lines (GM06990, HeLa S3, and SK-N-AS) according to their ribosome content by sucrose gradient fractionation. Polyribosomal-associated RNA and total RNA were subsequently hybridized to genomic tiling arrays. We found that approximately 50% of the transcriptional signals were located outside of annotated exons and were considered as TARs (transcriptionally active regions). Although TARs were observed among polysome-associated RNAs, RT-PCR and RACE experiments revealed that approximately 40% were likely to represent nonspecific cross-hybridization artifacts. Bioinformatics discrimination of TARs according to conservation and sequence complexity allowed us to identify a set of high-confidence TARs. This set of TARs was significantly depleted in the polysomes, suggesting that it was not likely to be involved in translation. Analysis of polysome representation of RefSeq exons showed that at least 15% of RefSeq transcripts undergo significant post-transcriptional regulation in at least two of the three cell lines tested. Among the regulated transcripts, enrichment analysis revealed an over-representation of genes involved in Alzheimer's disease (AD), including APP and the BACE1 protease that cleaves APP to produce the pathogenic beta 42 peptide. We demonstrate that the combination of RNA fractionation and tiling arrays is a powerful method to assess the transcriptional and post-transcriptional properties of genomic regions.

An approach to analyse the specific impact of rapamycin on mRNA-ribosome association.

BACKGROUND: Recent work, using both cell culture model systems and tumour derived cell lines, suggests that the differential recruitment into polysomes of mRNA populations may be sufficient to initiate and maintain tumour formation. Consequently, a major effort is underway to use high density microarray profiles to establish molecular fingerprints for cells exposed to defined drug regimes. The aim of these pharmacogenomic approaches is to provide new information on how drugs can impact on the translational read-out within a defined cellular background. METHODS: We describe an approach that permits the analysis of de-novo mRNA-ribosome association in-vivo during short drug exposures. It combines hypertonic shock, polysome fractionation and high-throughput analysis to provide a molecular phenotype of translationally responsive transcripts. Compared to previous translational profiling studies, the procedure offers increased specificity due to the elimination of the drugs secondary effects (e.g. on the transcriptional read-out). For this pilot "proof-of-principle" assay we selected the drug rapamycin because of its extensively studied impact on translation initiation. RESULTS: High throughput analysis on both the light and heavy polysomal fractions has identified mRNAs whose re-recruitment onto free ribosomes responded to short exposure to the drug rapamycin. The results of the microarray have been confirmed using real-time RT-PCR. The selective down-regulation of TOP transcripts is also consistent with previous translational profiling studies using this drug. CONCLUSION: The technical advance outlined in this manuscript offers the possibility of new insights into mRNA features that impact on translation initiation and provides a molecular fingerprint for transcript-ribosome association in any cell type and in the presence of a range of drugs of interest. Such molecular phenotypes defined pre-clinically may ultimately impact on the evaluation of a particular drug in a living cell.

Alternatively spliced isoforms of the human elk-1 mRNA within the 5' UTR: implications for ELK-1 expression.

The expression of cellular proteins that play central roles in the regulation of cell growth and differentiation is frequently tightly controlled at the level of translation initiation. In this article, we provide evidence that the ETS domain transcription factor ELK-1 forms part of this class of genes. Its mRNA 5' UTR is composed of a complexed mosaic of elements, including uAUGs, uORFs and RNA structure, that interplay to modulate ribosomal access to the ELK-1 AUG start codon. Superimposed upon this is the generation of two different 5' UTRs via alternative splicing. The two spliced isoforms show altered cellular and tissue distributions and behave differently in polysomal recruitment assays in the presence of the drug rapamycin. We propose that repression is therefore the sum of a series of interplaying negative elements within the 5' UTRs, a situation which may reflect the need for tight translational control of ELK-1 in different tissues and under changing physiological conditions.