Isolation and identification of short nucleotide sequences that affect translation initiation in
            <i>Saccharomyces</i>
            <i>cerevisiae</i>

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We previously identified an internal ribosome entry site (IRES) within the 5 leader of the mRNA encoding the Gtx homeodomain protein and showed that shorter nonoverlapping segments of this 5 leader could enhance the translation of a second cistron in a dicistronic mRNA. One of these segments was 9 nt in length, and when multiple copies of this IRES module were linked together, IRES activity was greatly enhanced. To further expand the potential uses of these synthetic constructs and facilitate analyses of the mechanism by which they affect translation, we show here that an IRES containing five linked copies of the 9-nt sequence can also enhance translation in the 5 leader of a monocistronic mRNA. Moreover, a search for interactions of the IRES module with cellular factors revealed specific binding to 40S ribosomal subunits but not to other cellular components. Based on the results of earlier studies suggesting that this sequence could bind to a complementary segment of 18S rRNA, we tested various sequences for possible links between the length of the complementary match, their binding to ribosomes, and their influence on translational efficiency. We found that the length of the complementary match was directly correlated with the ability of RNA probes to bind to ribosomes. In addition, translation was maximally enhanced (Ϸ8-fold) by a 7-nt segment of the 9-nt element; the enhancement declined progressively as the complementary stretches became progressively longer or shorter. The results suggest that the Gtx 9-nt sequence affects translation efficiency by a mechanism that involves base pairing to 18S rRNA.I n earlier studies, we identified cis-acting sequences within the 5Ј leaders of the mRNAs for the homeodomain protein Gtx and the cold-stress-induced protein Rbm3 that appeared to function as internal ribosome entry sites (IRESs). When tested in isolation these relatively short sequences enhanced the translation of a second cistron in a dicistronic mRNA (1, 2). In addition, we found that RNA sequences with IRES activity could be selected from libraries of random 9-to 18-nt sequences (3, 4). Although in most cases, IRES activity mediated by an individual IRES element was relatively weak, it could be greatly enhanced when multiple copies of some of the individual IRES elements were linked together in tandem (1, 3).Synthetic IRESs based on the 9-nt Gtx IRES module have proven to be particularly useful in various vector systems for generating high levels of expression from multicistronic mRNAs (e.g., refs. 5-7). Moreover, they are shorter and are more active than viral IRESs, such as the encephalomyocarditis virus IRES, that are typically used for these applications, and synthetic IRES activity can be enhanced or diminished by increasing or decreasing the number of IRES modules.The question arises whether, in addition to enhancing translation in a dicistronic context, such constructs would also enhance translation in a monocistronic context, i.e., in the 5Ј leader of a reporter mRNA. There are a number of reasons p...

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confidence: 89%

Biochemical and functional analysis of a 9-nt RNA sequence that affects translation efficiency in eukaryotic cells

Chappell

Edelman

Mauro

2004

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“…RNA folding programs such as MFOLD (31) predict that this sequence exhibits little, if any, structure. There have been reports of short sequences of between 9 and 22 nt that can serve as IRESs (32)(33)(34)(35). Unlike the tk IRES, these sequences were analyzed in reporter constructs that placed the test sequence in close proximity to the AUG of the downstream reporter gene.…”

Section: Discussionmentioning

confidence: 99%

An unusual internal ribosome entry site in the herpes simplex virus thymidine kinase gene

Griffiths

Coen

2005

We have investigated a herpes simplex virus mutant that expresses low levels of thymidine kinase (TK), a phenotype associated with drug resistance and pathogenicity, despite a single-base deletion in the gene. Using a dual-reporter system, a 39-nt sequence including the mutation was shown to direct expression of the downstream reporter gene in reticulocyte lysate. Translation of the downstream reporter was not impaired when the mRNA lacked a 5 cap or had a stable stem loop 5 of the upstream reporter and was relatively resistant to edeine, an antibiotic that prevents AUG codon recognition by the 40S-eIF2-GTP͞Met-tRNAi complex. Twelve nucleotides were as active as the original sequence for translation of the downstream reporter. Surprisingly, this sequence lacks an AUG codon. Analysis of point mutations showed that a CUG codon in the sequence was important. However, many single-base changes had only limited effects, and introduction of AUG codons did not increase translation. A mutant virus containing both the singlebase deletion and a mutation that reduced downstream translation in vitro had significantly less TK activity than a virus with the single-base deletion alone. Thus, a remarkably short internal ribosome entry site (IRES) that lacks an AUG codon resides in the viral tk gene. The IRES appears to be responsible for TK expression from a drug-resistant mutant that would otherwise express no TK, which may contribute to pathogenicity. Because we found numerous short sequences with IRES activity, there might be many hitherto unrecognized polypeptides expressed at low levels from eukaryotic mRNAs. drug resistance ͉ pathogenesis ͉ proteomics ͉ translation ͉ acyclovir

“…The present development of a feedback method to identify individual IRES elements is an extension of our earlier studies (20,22) and is motivated by the possibility that short sequences, such as the 7-nt Gtx IRES module are widespread within the mRNA population and that combinations of these mRNA sequences may have important global effects on the proteome. The development of an efficient selection method to identify IRES elements will enable their sequences to be characterized on the basis of sequence motifs and expression properties; it will also enable an assessment of the frequency of these elements in the messenger population.…”

Section: Discussionmentioning

confidence: 99%

“…In earlier studies, we generated synthetic IRESes containing multiple individual IRES elements and showed that this multimerization led to higher, and in some cases exponential, increases in IRES activity. To facilitate the discovery process, we and others have developed a number of methods to screen for IRES elements in mammalian cells (20,21) and in yeast (22). In all of these studies, dicistronic mRNAs containing a library of random nucleotide sequences in the intercistronic sequence (ICS) were expressed in cells, and those cells containing IRES elements were identified on the basis of the expression of the second cistron.…”

mentioning

confidence: 99%

A positive feedback vector for identification of nucleotide sequences that enhance translation

Zhou

Edelman²,

Mauro³

2005

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In earlier studies, we identified short (6-to 22-nt) sequences that functioned as internal ribosome entry sites (IRESes) and enhanced translation. The size of these IRES elements suggested that they might be prevalent within the messenger population and that individual elements might affect the translation of different groups of mRNAs. To begin to assess the number of different IRES elements in mammalian cells, we have developed a powerful method that uses a positive feedback mechanism to amplify the activities of individual IRES elements. This method uses a vector that encodes a dicistronic mRNA with a reporter gene (Renilla luciferase or the EGFP) as the first cistron and the yeast Gal4͞viral protein 16 (VP16) transcription factor as the second cistron. Transcription of this mRNA is driven by a minimal promoter containing four copies of the Gal4 upstream activation sequence. In this method, the presence of an IRES in the intercistronic region facilitates the translation of Gal4͞VP16, which binds to the upstream activation sequences and triggers a positive feedback loop that escalates the production of dicistronic mRNA and Gal4͞VP16. A corresponding increase in the translation of the first cistron (luciferase or EGFP) is monitored either by measuring luciferase activity or by using FACS. The latter enables IRES-positive cells to be isolated. We present tests of the feedback mechanism by using an IRES module from Gtx homeodomain mRNA and an IRES from hepatitis C virus and demonstrate the utility of this vector system for the screening, identification, and analysis of IRES elements.internal ribosome entry site ͉ selection