Translational control via protein-regulated upstream open reading frames

Analysis of the regulation of msl-2 mRNA by Sex lethal (SXL), which is critical for dosage compensation in Drosophila, has uncovered a mode of translational control based on common 5' untranslated region elements, upstream open reading frames (uORFs), and interaction sites for RNA-binding proteins. We show that SXL binding downstream of a short uORF imposes a strong negative effect on major reading frame translation. The underlying mechanism involves increasing initiation of scanning ribosomes at the uORF and augmenting its impediment to downstream translation. Our analyses reveal that SXL exerts its effect controlling initiation, not elongation or termination, at the uORF. Probing the generality of the underlying mechanism, we show that the regulatory module that we define experimentally functions in a heterologous context, and we identify natural Drosophila mRNAs that are regulated via this module. We propose that protein-regulated uORFs constitute a systematic principle for the regulation of protein synthesis.     

Alterations of the three short open reading frames in the Rous sarcoma virus leader RNA modulate viral replication and gene expression.

The Rous sarcoma virus (RSV) leader RNA has three short open reading frames (ORF1 to ORF3) which are conserved in all avian sarcoma-leukosis retroviruses. Effects on virus propagation were determined following three types of alterations in the ORFs: (i) replacement of AUG initiation codons in order to prohibit ORF translation, (ii) alterations of the codon context around the AUG initiation codon to enhance translation of the normally silent ORF3, and (iii) elongation of the ORF coding sequences. Mutagenesis of the AUG codons for ORF1 and ORF2 (AUG1 and AUG2) singly or together delayed the onset of viral replication and cell transformation. In contrast, mutagenesis of AUG3 almost completely suppressed these viral activities. Mutagenesis of ORF3 to enhance its translation inhibited viral propagation. When the mutant ORF3 included an additional frameshift mutation which extended the ORF beyond the initiation site for the gag, gag-pol, and env proteins, host cells were initially transformed but died soon thereafter. Elongation of ORF1 from 7 to 62 codons led to the accumulation of transformation-defective virus with a delayed onset of replication. In contrast, viruses with elongation of ORF1 from 7 to 30 codons, ORF2 from 16 to 48 codons, or ORF3 from 9 to 64 codons, without any alterations in the AUG context, exhibited wild-type phenotypes. These results are consistent with a model that translation of the ORFs is necessary to facilitate virus production.     

The positive regulatory function of the 5''-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function.     

Small open reading frames in 5' untranslated regions of mRnas

Using the 5'-end sequence data from 'oligo-capped' cDNAs, we generated a representative full-length cDNA dataset for 4870 RefSeq entries, and analyzed the 5' untranslated region (UTR) of these genes. To our surprise, about half of the 4870 genes had an upstream ATG before the ATG that starts the longest open reading frame (ORF), suggesting that about half of them have small ORFs in their 5' UTR of average length of 31 amino acids. They require attention for further analysis to identify their biological role.     

Potential open reading frames within 5'-untranslated regions of eukaryotic mRNAs

It is known that 5'-untranslated sequences of eukaryotic mRNA often contain AUG triplets, which may serve as the sites of translation initiation. It is thought that these leader open reading frames can fulfil the regulatory functions and encode functionally active proteins. However, they have been incompletely characterized. The article deals with the context organization of leader reading frames of eukaryotic mRNAs. It has been shown that their characteristics correlate with the position relative to the protein-encoding sequence, which may be associated with the efficiency of initiation of translation.     

Simultaneous cloning of open reading frames into several different expression vectors

Genomic sequencing has enabled the prediction of thousands of genes, most of which either cannot be assigned a function or can be only broadly categorized on the basis of sequence alone. High-throughput strategies for elucidating protein function are of high priority, and numerous approaches are being developed. Many of these approaches require the cloning of open reading frames (ORFs) into expression vectors that enable the encoded proteins to be tested for biological and biochemical activities. Typically, more than one type of vector must be employed, as different experiments require different conditions of protein production. Here we show that it is possible to simultaneously transfer a single ORF from a source vector to four target vectors using a commercially available in vitro recombination system. To test the approach, we constructed new vectors for expression of fusion proteins in yeast, including vectors for the LexA two-hybrid system. We show that individual ORFs can be efficiently transferred to four different vectors in a single in vitro reaction. The resulting expression plasmids can be separated using prototrophic markers specific to each vector. Using this system to produce multiple expression constructs simultaneously could greatly facilitate high-throughput subcloning and proteomic studies.     

A high-throughput drug screen targeted to the 5'untranslated region of Alzheimer amyloid precursor protein mRNA

The authors employed a novel approach to identify therapeutics effective in Alzheimer disease (AD). The 5'untranslated region (5'UTR) of the mRNA of AD amyloid precursor protein (APP) is a significant regulator of the levels of the APP holoprotein and amyloid beta (Abeta) peptide in the central nervous system. The authors generated stable neuroblastoma SH-SY5Y transfectants that express luciferase under the translational control of the 146-nucleotide APP mRNA 5'UTR and green fluorescent protein (GFP) driven by a viral internal ribosomal entry site. Using a high-throughput screen (HTS), they screened for the effect of 110,000 compounds obtained from the library of the Laboratory for Drug Discovery on Neurodegeneration (LDDN) on the APP mRNA 5'UTR-controlled translation of the luciferase reporter. This screening yielded several nontoxic specific inhibitors of APP mRNA 5'UTR-driven luciferase that had no effect on the GFP expression in the stable SH-SY5Y transfectants. Moreover, these compounds either did not inhibit or inhibited to a much lower extent the expression of the luciferase reporter regulated by a prion protein (PrP) mRNA 5'UTR, used as an alternative mRNA structure to counterscreen APP mRNA 5'UTR in stably transfected SH-SY5Y cell lines. The hits obtained from this robust, specific, and highly quantitative HTS will be characterized to identify agents that may be developed into useful future therapeutic agents to limit APP translation and Abeta production for AD.     

Sendai virus P gene produces multiple proteins from overlapping open reading frames

The Sendai virus P gene contains overlapping open reading frames (ORFs), and several unusual mechanisms are used to produce multiple proteins from all three ORFs of this gene. These include the use of a non-AUG start codon, leaky ribosomal scanning, what appears to be scanning-independent ribosomal initiation and/or ribosomal jumping, and a form of mRNA editing.     

Upstream open reading frames regulate the translation of the multiple mRNA variants of the estrogen receptor alpha

It is by now well established that the estrogen receptor alpha (ER alpha) is transcribed from multiple promoters. One direct consequence of multiple promoters is the generation of mRNA variants with different 5'-untranslated regions (5'-UTRs). However, the potential roles of these individual mRNA variants are not known. All 5'-UTRs of ER alpha contain between one and six upstream open reading frames. In this study the effect of the 5'-UTRs of major human and mouse ER alpha mRNA variants on translation was evaluated. Some of the 5'-UTRs were found to strongly inhibit translation of the downstream open reading frame. Mutation of the upstream AUG codons partially or completely restored translation efficiency. A toeprinting analysis and assessment of the contribution of each AUG codon to the inhibitory effect on translation showed that leaky scanning and reinitiation occurs with these mRNAs. In conclusion, the upstream open reading frames in the 5'-UTRs of ER alpha mRNAs have the potential to regulate estrogen receptor alpha expression.     

Evolutionary dynamics of introns and their open reading frames in the U7 region of the mitochondrial rnl gene in species of Ceratocystis

The mtDNA rnl-U7 region has been examined for the presence of introns in selected species of the genus Ceratocystis. Comparative sequence analysis identified group I and group II introns encoding single and double motif LAGLIDADG open reading frames (ORFs) at the following positions L1671, L1787, and L1923. In addition downstream of the rnl-U7 region group I introns were detected at positions L1971 and L2231, and a group II intron at L2059. A GIY-YIG type ORF was located within one mL1923 LAGLIDADG type ORF and a degenerated GIY-YIG ORF fused to a nad2 gene fragment was found in association with the mL1971 group I intron. The diversity of composite elements that appear to be sporadically distributed among closely related species of Ceratocystis illustrates the potential for homing endonucleases and their associated introns to invade new sites. Phylogenetic analysis showed that single motif LADGLIDADG ORFs related to the mL1923 ORFs have invaded the L1787 group II intron and the L1671 group I intron. Phylogenetic analysis of intron encoded single and double motif LAGLIDADG ORFs also showed that these ORFs transferred four times from group I into group II B1 type introns.     

Identification of functional open reading frames in chloroplast genomes

We have used a rapid computer dot-matrix comparison method to identify all DNA regions which have been evolutionarily conserved between the completely sequenced chloroplast genomes of tobacco and a liverwort. Analysis of these regions reveals 74 homologous open reading frames (ORFs) which have been conserved as to length and amino acid sequence; these ORFs also have an excess of nucleotide substitutions at silent sites of codons. Since the nonfunctional parts of these genomes have become saturated with mutations and show no sequence similarity whatsoever, the homologous ORFs are almost certainly functional. A further four pairs of ORFs show homology limited to only a short part of their putative gene products. Amino acid sequence identities range between 50 and 99%; some chloroplast proteins are seen to be among the most slowly evolving of all known proteins. A search of the nucleotide and amino acid sequence databanks has revealed several previously unidentified genes in chloroplast sequences from other species, but no new homologies to prokaryotic genes.