Evolutionary conservation suggests a regulatory function of AUG triplets in 5′-UTRs of eukaryotic genes

By comparing sequences of human, mouse and rat orthologous genes, we show that in 5′-untranslated regions (5′-UTRs) of mammalian cDNAs but not in 3′-UTRs or coding sequences, AUG is conserved to a significantly greater extent than any of the other 63 nt triplets. This effect is likely to reflect, primarily, bona fide evolutionary conservation, rather than cDNA annotation artifacts, because the excess of conserved upstream AUGs (uAUGs) is seen in 5′-UTRs containing stop codons in-frame with the start AUG and many of the conserved AUGs are found in different frames, consistent with the location in authentic non-coding sequences. Altogether, conserved uAUGs are present in at least 20–30% of mammalian genes. Qualitatively similar results were obtained by comparison of orthologous genes from different species of the yeast genus Saccharomyces. Together with the observation that mammalian and yeast 5′-UTRs are significantly depleted in overall AUG content, these findings suggest that AUG triplets in 5′-UTRs are subject to the pressure of purifying selection in two opposite directions: the uAUGs that have no specific function tend to be deleterious and get eliminated during evolution, whereas those uAUGs that do serve a function are conserved. Most probably, the principal role of the conserved uAUGs is attenuation of translation at the initiation stage, which is often additionally regulated by alternative splicing in the mammalian 5′-UTRs. Consistent with this hypothesis, we found that open reading frames starting from conserved uAUGs are significantly shorter than those starting from non-conserved uAUGs, possibly, owing to selection for optimization of the level of attenuation

Evolutionary conservation suggests a regulatory function of AUG triplets in 50 -UTRs of eukaryotic genes

By comparing sequences of human, mouse and rat orthologous genes, we show that in 50 -untranslated regions (50 -UTRs) of mammalian cDNAs but not in 30 - UTRs or coding sequences, AUG is conserved to a significantly greater extent than any of the other 63 nt triplets. This effect is likely to reflect, primarily, bona fide evolutionary conservation, rather than cDNA annotation artifacts, because the excess of conserved upstream AUGs (uAUGs) is seen in 50 -UTRs containing stop codons in-frame with the start AUG and many of the conserved AUGs are found in different frames, consistent with the location in authentic non-coding sequences. Altogether, conserved uAUGs are present in at least 20–30% of mammalian genes. Qualitatively similar results were obtained by comparison of orthologous genes from different species of the yeast genus Saccharomyces. Together with the observation that mammalian and yeast 50 -UTRs are significantly depleted in overall AUG content, these findings suggest that AUG triplets in 50 -UTRs are subject to the pressure of purifying selection in two opposite directions: the uAUGs that have no specific function tend to be deleterious and get eliminated during evolution, whereas those uAUGs thatdoserveafunctionareconserved.Mostprobably, the principal role of the conserved uAUGs is attenuation of translation at the initiation stage, which is often additionally regulated by alternative splicing in the mammalian 50 -UTRs. Consistent with this hypothesis, we found that open reading frames starting from conserved uAUGs are significantly shorter than those starting from non-conserved uAUGs, possibly, owing to selection for optimization of the level of attenuation

Evolution of alternative and constitutive regions of mammalian 5'UTRs

<p>Abstract</p> <p>Background</p> <p>Alternative splicing (AS) in protein-coding sequences has emerged as an important mechanism of regulation and diversification of animal gene function. By contrast, the extent and roles of alternative events including AS and alternative transcription initiation (ATI) within the 5'-untranslated regions (5'UTRs) of mammalian genes are not well characterized.</p> <p>Results</p> <p>We evaluated the abundance, conservation and evolution of putative regulatory control elements, namely, upstream start codons (uAUGs) and open reading frames (uORFs), in the 5'UTRs of human and mouse genes impacted by alternative events. For genes with alternative 5'UTRs, the fraction of alternative sequences (those present in a subset of the transcripts) is much greater than that in the corresponding coding sequence, conceivably, because 5'UTRs are not bound by constraints on protein structure that limit AS in coding regions. Alternative regions of mammalian 5'UTRs evolve faster and are subject to a weaker purifying selection than constitutive portions. This relatively weak selection results in over-abundance of uAUGs and uORFs in the alternative regions of 5'UTRs compared to constitutive regions. Nevertheless, even in alternative regions, uORFs evolve under a stronger selection than the rest of the sequences, indicating that some of the uORFs are conserved regulatory elements; some of the non-conserved uORFs could be involved in species-specific regulation.</p> <p>Conclusion</p> <p>The findings on the evolution and selection in alternative and constitutive regions presented here are consistent with the hypothesis that alternative events, namely, AS and ATI, in 5'UTRs of mammalian genes are likely to contribute to the regulation of translation.</p

AUG_hairpin: prediction of a downstream secondary structure influencing the recognition of a translation start site

<p>Abstract</p> <p>Background</p> <p>The translation start site plays an important role in the control of translation efficiency of eukaryotic mRNAs. The recognition of the start AUG codon by eukaryotic ribosomes is considered to depend on its nucleotide context. However, the fraction of eukaryotic mRNAs with the start codon in a suboptimal context is relatively large. It may be expected that mRNA should possess some features providing efficient translation, including the proper recognition of a translation start site. It has been experimentally shown that a downstream hairpin located in certain positions with respect to start codon can compensate in part for the suboptimal AUG context and also increases translation from non-AUG initiation codons. Prediction of such a compensatory hairpin may be useful in the evaluation of eukaryotic mRNA translation properties.</p> <p>Results</p> <p>We evaluated interdependency between the start codon context and mRNA secondary structure at the CDS beginning: it was found that a suboptimal start codon context significantly correlated with higher base pairing probabilities at positions 13 – 17 of CDS of human and mouse mRNAs. It is likely that the downstream hairpins are used to enhance translation of some mammalian mRNAs <it>in vivo</it>. Thus, we have developed a tool, <it>AUG_hairpin</it>, to predict local stem-loop structures located within the defined region at the beginning of mRNA coding part. The implemented algorithm is based on the available published experimental data on the CDS-located stem-loop structures influencing the recognition of upstream start codons.</p> <p>Conclusion</p> <p>An occurrence of a potential secondary structure downstream of start AUG codon in a suboptimal context (or downstream of a potential non-AUG start codon) may provide researchers with a testable assumption on the presence of additional regulatory signal influencing mRNA translation initiation rate and the start codon choice. <it>AUG_hairpin</it>, which has a convenient Web-interface with adjustable parameters, will make such an evaluation easy and efficient.</p

Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitude

BACKGROUND: In addition to studies of plant gene function and developmental analyses, plant biotechnological use is largely dependent upon transgenic technologies. The moss Physcomitrella patens has become an exciting model system for studying plant molecular processes due to an exceptionally high rate of nuclear gene targeting by homologous recombination compared with other plants. However, its use in transgenic approaches requires expression vectors that incorporate sufficiently strong promoters. To satisfy this requirement, a set of plant expression vectors was constructed and equipped with either heterologous or endogenous promoters. RESULTS: Promoter activity was quantified using the dual-luciferase reporter assay system. The eight different heterologous promoter constructs tested exhibited expression levels spanning three orders of magnitude. Of these, the complete rice actin1 gene promoter showed the highest activity in Physcomitrella, followed by a truncated version of this promoter and three different versions of the cauliflower mosaic virus 35S promoter. In contrast, the Agrobacterium tumefaciens nopaline synthase promoter induced transcription rather weakly. Constructs including promoters commonly used in mammalian expression systems also proved to be functional in Physcomitrella. In addition, the 5' -regions of two Physcomitrella glycosyltransferases (i.e. α1,3-fucosyltransferase and β1,2-xylosyltransferase) were identified and functionally characterised in comparison to the heterologous promoters. Furthermore, motifs responsible for enhancement of translation efficiency – such as the TMV omega element and a modified sequence directly prior the start codon – were tested in this model. CONCLUSION: We developed a vector set that enables gene expression studies, both in lower and higher land plants, thus providing valuable tools applicable in both basic and applied molecular research

Synthesis and subcellular fate of proteins encoded by the mouse int-2 (FGF3) gene

The int-2 gene encodes a member of the FGF family and was discovered as a proto-oncogene transcriptionally activated in tumours induced by Mouse Mammary Tumour Virus. Int-2 transcription is rarely detected in adult mouse tissues apart from low levels in brain and testis, but in situ hybridisation has revealed widespread expression during embryogenesis. The predicted int-2 gene product has a molecular mass of 27 kD and a hydrophobic sequence at the N-terminus which acts as a signal peptide for vectorial synthesis into the endoplasmic reticulum. The products have proved difficult to detect in natural sources known to contain int-2 RNA such as mammary tumours and embryo-derived cell lines. Consequently the protein has been characterised by expression of cloned cDNAs in COS-1 cells using an SV40-based plasmid vector, in insect cells infected with recombinant baculoviruses and by translation of synthetic sense RNA in cell-free systems supplemented with canine pancreatic microsomes. These studies identified four major int-2 products ranging in size from 27.5 kD to 31.5 kD which arise by post-translational modification of a 28.5 kD primary translation product. Although int-2 proteins are targeted to the secretory pathway, they have only been detected at very low levels in the medium and the associated extracellular matrix of transfected COS-1 cells. Cell-free translation systems programmed with synthetic int-2 RNA identified an additional N-terminally extended product which initiates from an in-frame CUG codon located upstream of the first AUG. Immunofluorescent staining of transfected COS-1 cells showed that a substantial proportion of this extended product localised in the cell nucleus, while a truncated int-2 protein lacking both the N-terminal extension and the signal peptide was exclusively nuclear. These observations suggested that the signals required for nuclear localisation of int-2 were encoded in the body of the molecule, but only functioned when entry into the secretory pathway was compromised. Two nuclear targeting sequences were identified by mutagenesis. Fusion of these motifs to the cytosolic protein pyruvate kinase did not result in effective nuclear localisation. However chimaeras containing int-2 and hst, an exclusively secreted member of the FGF-family, identified a targeting signal sequence with superficial resemblance to the bipartite nuclear localisation sequence of Xenopus nucleoplasmin. Initiation at alternative codons changes the sub-cellular fate of the protein and in principle provides a means imparting distinct functions upon the different int-2 products. Although a biological activity has yet to be assigned to the nuclear forms, the secreted protein is transforming in a NIH3T3 focus assay. The efficiency of focus formation was augmented by mutations which prevented initiation at the CUG codon or at an upstream AUG in the +1 reading frame, resulting in elevated levels of secreted int-2. Thus transformation was shown to require a high level of int-2 synthesis and secretion. In agreement with these findings, mutations which reduce the efficiency of secretion reduce the focus forming ability of NIH3T3 cells transfected with int-2 cDNAs, substantiating the notion that transformation is effected through an autocrine loop mechanism that involves a cell surface receptor

Evidence for conservation and selection of upstream open reading frames suggests probable encoding of bioactive peptides

BACKGROUND: Approximately 40% of mammalian mRNA sequences contain AUG trinucleotides upstream of the main coding sequence, with a quarter of these AUGs demarcating open reading frames of 20 or more codons. In order to investigate whether these open reading frames may encode functional peptides, we have carried out a comparative genomic analysis of human and mouse mRNA 'untranslated regions' using sequences from the RefSeq mRNA sequence database. RESULTS: We have identified over 200 upstream open reading frames which are strongly conserved between the human and mouse genomes. Consensus sequences associated with efficient initiation of translation are overrepresented at the AUG trinucleotides of these upstream open reading frames, while comparative analysis of their DNA and putative peptide sequences shows evidence of purifying selection. CONCLUSION: The occurrence of a large number of conserved upstream open reading frames, in association with features consistent with protein translation, strongly suggests evolutionary maintenance of the coding sequence and indicates probable functional expression of the peptides encoded within these upstream open reading frames

Untranslated regions of mRNAs

Gene expression is finely regulated at the post-transcriptional level. Features of the untranslated regions of mRNAs that control their translation, degradation and localization include stem-loop structures, upstream initiation codons and open reading frames, internal ribosome entry sites and various cis-acting elements that are bound by RNA-binding proteins

Ectopic expression of a truncated CD40L protein from synthetic post-transcriptionally capped RNA in dendritic cells induces high levels of IL-12 secretion

<p>Abstract</p> <p>Background</p> <p>RNA transfection into dendritic cells (DCs) is widely used to achieve antigen expression as well as to modify DC properties. CD40L is expressed by activated T cells and interacts with CD40 receptors expressed on the surface of the DCs leading to Th1 polarization. Previous studies demonstrated that ectopic CD40L expression via DNA transfection into DCs can activate the CD40 receptor signal transduction cascade. In contrast to previous reports, this study demonstrates that the same effect can be achieved when RNA encoding CD40L is electroporated into DCs as evidenced by secretion of IL-12. To achieve higher levels of IL-12 secretion, a systematic approach involving modification of coding and noncoding regions was implemented to optimize protein expression in the DCs for the purpose of increasing IL-12 secretion.</p> <p>Results</p> <p>Site-directed mutagenesis of each of the first five in-frame methionine codons in the CD40L coding sequence demonstrated that DCs expressing a truncated CD40L protein initiated from the second methionine codon secreted the highest levels of IL-12. In addition, a post-transcriptional method of capping was utilized for final modification of the CD40L RNA. This method enzymatically creates a type I cap structure identical to that found in most eukaryotic mRNAs, in contrast to the type 0 cap incorporated using the conventional co-transcriptional capping reaction.</p> <p>Conclusion</p> <p>The combination of knocking out the first initiation methionine and post-transcriptional capping of the CD40L RNA allowed for approximately a one log increase in IL-12 levels by the transfected DCs. We believe this is a first report describing improved protein expression of post-transcriptionally capped RNA in DCs. The post-transcriptional capping which allows generation of a type I cap may have broad utility for optimization of protein expression from RNA in DCs and other cell types.</p

Translation of the first upstream ORF in the hepatitis B virus pregenomic RNA modulates translation at the core and polymerase initiation codons

The human hepatitis B virus (HBV) has a compact genome encoding four major overlapping coding regions: the core, polymerase, surface and X. The polymerase initiation codon is preceded by the partially overlapping core and four or more upstream initiation codons. There is evidence that several mechanisms are used to enable the synthesis of the polymerase protein, including leaky scanning and ribosome reinitiation. We have examined the first AUG in the pregenomic RNA, it precedes that of the core. It initiates an uncharacterized short upstream open reading frame (uORF), highly conserved in all HBV subtypes, we designated the C0 ORF. This arrangement suggested that expression of the core and polymerase may be affected by this uORF. Initiation at the C0 ORF was confirmed in reporter constructs in transfected cells. The C0 ORF had an inhibitory role in downstream expression from the core initiation site in HepG2 cells and in vitro, but also stimulated reinitiation at the polymerase start when in an optimal context. Our results indicate that the C0 ORF is a determinant in balancing the synthesis of the core and polymerase proteins