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 condition

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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

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

Translational regulation of Elk-1 and its neuronal specific isoform, sElk-1

La synthèse des protéines est un processus complexe impliquant de nombreux mécanismes biologiques. En particulier, la régulation de la traduction des ARNm permet aux cellules de répondre rapidement à un stimulus extracellulaire ou à un changement dans la composition de leur environnement. Ces signaux peuvent être transmis par l'intermédiaire des récepteurs tyrosine kinases, d'intégrines, ou indirectement, via l'entrée de calcium ou de la disponibilité d'acides aminés. Il est maintenant bien connu que la plupart des voies de signalisation sont impliquées dans le contrôle de la traduction. Elles peuvent induire la phosphorylation de facteurs comme eIF4E, 4EBP1, eIF4B et la protéine ribosomale S6 et ainsi avoir un impact sur l'initiation de la traduction. Il est maintenant largement admis que la région non traduite avant (5'UTR) et après (3'UTR) la région codante, sont impliquées dans la modulation de l'expression des protéines. En effet, des éléments structurels, des sites de liaison pour des protéines ou des ARNs, ainsi que des AUGs dans le 5'UTR peuvent influencer la traduction. En outre, d'autres modes d'initiation (par exemple, les IRES) permettent d'assurer la traduction des ARNm lors de contextes cellulaires particuliers. En ce qui concerne ce dernier point, nous avons développé une technique permettant de suivre le re-recrutement polysomal suite à une courte exposition à une drogue..

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

elk-1 mRNA within the 5 0 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 0 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 0 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 0 UTRs, a situation which may reflect the need for tight translational control of ELK-1 in different tissues and under changing physiological conditions

The chimeric gene CHRFAM7A, a partial duplication of the CHRNA7 gene, is a dominant negative regulator of a7*nAChR function

The human a7 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 a7*nAChR, results in opening of the channel and entry of mono- and divalent cations, including Ca2+, that presynaptically participates to neurotransmitter release and postsynaptically to down-stream changes in gene expression. Schizophrenic patients have low levels of a7*nAChR, as measured by binding of the ligand [125I]-a-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 2 bp 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 a7 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 a7 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. © 2011 Elsevier Inc

An AUG Codon Conserved for Protein Function Rather than Translational Initiation: The Story of the Protein sElk1

<div><p>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 <i>ELK1</i> 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 <i>de-novo</i> 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 AUG^Elk1 serve to limit 40S access to the AUG^sElk1 codon.</p></div

Function of the methionine 55.

<p>(A). Organisation of the Elk1 protein. Upper panel: Protein functional domains (ETS  =  DNA binding, B =  SRF interaction, D =  MAPK docking site, TAD  =  transactivation domain). Middle panel: Alignment of the Elk1 ETS domain from mouse, rat and human. The residues that make conserved DNA backbone and base contacts and are common to both Elk1 and SAP1 are indicated by the inverted triangles. The methionine corresponding to the AUG^sElk1 is boxed in grey <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102890#pone.0102890-Mo1" target="_blank">[10]</a>. Lower panel: A consensus ETS domain based upon the alignment of 12 family members with DBDs both N- and C-terminal. Tightly conserved amino acids are indicated in bold capitol. The methionine corresponding to the AUG^sELK1 is boxed in grey (adapted from <a href="http://content.lib.utah.edu/utils/getfile/collection/etd1/id/1528/filename/1687.pdf" target="_blank">http://content.lib.utah.edu/utils/getfile/collection/etd1/id/1528/filename/1687.pdf</a>) (B). Chip analysis at the c-fos promoter. HEK293T cells were transfected with plasmids expressing ELK1^HA, sElk1^HA or the Elk1^HA M55S mutant. ChIP analysis coupled to qPCR was performed as outlined in the supplemental section. The assay was performed twice (the duplicate columns) and analysed by qPCR. The ΔCt values (relative to input) were then normalised relative to Elk1^HA which was given a value of 1 in each experiment. (C). Immunoblot confirming expression from the transfected plasmids. Mock indicates non-transfected cells.</p