The search for mechanisms of translational regulation has yielded many experimentally identified internal ribosome entry sites (IRES). Because of the lack of sequence similarity among the experimentally IRESs, it is widely assumed that IRESs posses stable secondary structure allowing them to interact with the components of the translation machinery. Contrary to this view, here we show that IRES activity in nine yeast IRESs, mapped to 60 nt immediately upstream of the initiation AUG, is strongly associated with the lack of secondary structure of IRESs. Furthermore, the reverse complements of these IRESs, with their secondary structure more stable than those of the IRESs, exhibit little IRES activity. The generality of this association is exemplified by the observation that, in the natural _vpu-env_ bicistronic mRNA in HIV-1, the mRNA segment (60 nt) immediately upstream of the initiation AUG of _env_ has the weakest secondary structure among all dominant HIV-1 mRNA species. These results suggest a unified model of alternative translation initiation

Xuhua Xia

English

Nature Precedings

 1
Internal ribosomal entry site lacks secondary structure 
 
Xuhua Xia  
Department of Biology, University of Ottawa and 
Ottawa Institute of Systems Biology 
Correspondence should be addressed to: Department of Biology, University of Ottawa, 
30 Marie Curie, Ottawa, Canada K1N 6N5. E-mail: xxia@uottawa.ca, Telephone: 1-613-
562-5800 ext 6886, Fax: 1-613-562-5486. 
 
Keywords:  translation initiation, 5'-UTR, internal ribosomal entry site, minimum free 
energy, RNA secondary structure, HIV-1. 
 
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The search for mechanisms of translational regulation has yielded many 
experimentally identified internal ribosome entry sites (IRES). Because of the lack 
of sequence similarity among the experimentally IRESs 1,2, it is widely assumed that 
IRESs posses stable secondary structure allowing them to interact with the 
components of the translation machinery 1-4. Contrary to this view, here we show 
that IRES activity in nine yeast IRESs, mapped to 60 nt immediately upstream of 
the initiation AUG, is strongly associated with the lack of secondary structure of 
IRESs. Furthermore, the reverse complements of these IRESs, with their secondary 
structure more stable than those of the IRESs, exhibit little IRES activity. The 
generality of this association is exemplified by the observation that, in the natural 
vpu-env bicistronic mRNA in HIV-1, the mRNA segment (60 nt) immediately 
upstream of the initiation AUG of env has the weakest secondary structure among 
all dominant HIV-1 mRNA species. These results suggest a unified model of 
alternative translation initiation. 
In contrast to the overwhelming evidence of transcriptional regulation, little experimental 
support is available for translational regulation 5. Recent studies on viral genes translated 
only at specific infectious stage and on eukaryotic genes translated at specific 
developmental stage prompted an intensive search of translational regulation 
mechanisms. These viral and eukaryotic genes typically have complex and stable 
secondary structure at their 5’-UTR preventing the regular cap-dependent scanning of 
translation initiation, and are typically translated when the essential components of cap-
dependent translation initiation complex are missing in the cell, leading to the proposal of 
internal ribosome entry site (IRES) 1,2. However, although many IRESs have been 
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experimentally identified and an IRES database has been created 6, there has been no 
sequence similarity among the IRESs 1,2. The lack of sequence similarity has resulted in a 
widely held view that IRESs posses stable secondary structure allowing them to interact 
with the components of the translation machinery 1-4, but this view has never been 
critically evaluated.  
We tested whether IRES activity (measured by the protein production of the downstream 
gene in unit of protein/mRNA) depends on stable secondary structure by studying 12 
yeast genes (NCE102, GPR1, YMR181C, GIC1, FLO8, BOI1, MSN1, PAB1, eIF4G2, 
TPS2, HMS2, and YEL033W) whose 5’-UTRs differ dramatically in IRES activity 7. The 
IRES activity was mapped to 60 nt immediately upstream of the initiation AUG 7. Also 
included in the analysis are the reverse complements of four of the experimentally 
identified yeast IRESs in YMR181C, GPR1, FLO8, and BOI1 (designated as 
YMR181Crc, GPR1rc, FLO8rc, and BOI1rc, respectively). 
We found that the IRES activity of yeast IRESs is strongly associated with the lack of 
secondary structure measured by the minimum free energy of the 60 nt immediately 
upstream of the initiation AUG (Fig. 1, r = -0.7756, p = 0.0001), contrary to the 
conventional belief that IRESs should have complex and stable secondary structure 1-4. 
This result suggest that structure-less RNA segments immediately upstream of the 
initiation AUG can facilitate internal ribosome entry (IRE) in yeast. It is remarkable that 
the reverse complements of four of the identified IRESs have little IRES activity and 
feature relatively stable secondary structure.  
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Figures 
YEL033W
HMS2
TPK2
BOI1
GPR1
FLO8
GIC1
YMR181C
MSN1
NCE102
PAB1
TIF4632
BOI1rc
GPR1rc
FLO8rc
YMR181Crc
y = 14.578e0.1238x
R2 = 0.6016
0
2
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12
14
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18
-20-18-16-14-12-10-8-6-4-20
Structural stability (MFE)
IR
ES
 a
ct
iv
ity
 (r
an
k)
Fig. 1. Negative correlation between IRES activity, measured by the protein production 
of the downstream gene (protein/mRNA), and structural stability, measured by the 
minimum free energy (MFE). The MFE is shown in reverse order because greater 
stability is associated with more negative MFE values. The ranking of IRES activity 
(protein/mRNA) is based on Gilbert et al. 7 and verified by the authors. The reverse 
complements of four IRES-containing genes are colored in red. TIF4632 is the name in 
GenBank for gene eIF4G2. 
 
Past effort in searching for structure conservation among IRESs, especially among 
eukaryotic IRESs has essentially come to nothing 1. The lack of sequence and structure 
conservation among reported IRESs has become one of the main reasons for IRES to be 
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the target of ridicule 5. Our finding that yeast IRESs are sequence segments devoid of 
secondary structure explains the lack of sequence or structural similarity among IRESs, 
i.e., sequence segments devoid of secondary structure do not need to have any sequence 
or structural similarity. 
To test the generality of the observation that structure-less sequence segments 
immediately upstream of the initiation AUG can facilitate internal ribosome entry, we 
examined the natural vpu-env bicistronic mRNA in HIV-1 to see whether the 60 nt 
immediately upstream of the initiation AUG of the downstream env gene lacks secondary 
structure relative to other dominant HIV-1 mRNA species. The translation of the 
downstream env gene does not follow the model of leaking scanning because mutations 
that removed, altered the strength of, or introduced upstream vpu AUG codons, while 
dramatically altering the upstream Vpu expression, had little impact on the consistent 
expression of Env protein of the downstream env gene 8. This suggests that Env 
translation is mediated by a cap-independent translation initiation mechanism 8. 
Remarkably, the 60 nt immediately upstream of the env initiation AUG is devoid of 
secondary structure with MFE close to 0 (Table 1). In contrast, the 60 nt immediately 
upstream of the initiation AUG of other major mRNA species all have more stable 
secondary structure (Table 1). These results suggest a unified model of alternative 
translation initiation from yeast to mammalian cells. 
 
 
 
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Table 1. Minimum free energy (MFE) of the 60 nt immediately upstream of the initiation 
AUG among dominant HIV-1 splicing variants. 
mRNA Exon(1) Length AUGi(2) MFE
Nef2 1/5/7 1615  777 -9.52
Rev2 1/4a/7 1637  305 -16.2
Vpr1 1/3a/7 2202  459 8.1
Vpr3 1/3E 4535  459 8.1
Vpu 1/5E 3948  375 -4.1
Env 1/5E 3948 538 -0.1
Tat1 1/4/7 1814  343 -9.9
Tat5 1/4E 4147  343 -9.9
Vif2 1/2E 5012  418 -2.16
(1) According to Purcell and Martin 9. 
(2) Site of nucleotide A in the initiation AUG 
 
In summary, the experimentally identified yeast IRESs are sequence segments with no or 
weak secondary structure. The association between IRES activity and the lack of 
secondary structure in both yeast genes and HIV1 genes suggests a possibly ubiquitous 
alternative mechanism of translation initiation in eukaryotes. 
 
Methods 
The annotated Saccharomyces cerevisiae genome, dated Sept. 17, 2007, was downloaded 
from ftp.ncbi.nih.gov/genomes/Saccharomyces_cerevisiae. The coding sequences (CDSs) 
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and the 60 nt immediately upstream of the initiation AUG is extracted by using DAMBE 
10,11.  
The minimum free energy (MFE) is computed by using DAMBE which incorporates the 
function library of the Vienna RNA package 12, at 37°C, with no lonely pairs and with no 
GU pairs at the end of helices. The result is similar to that from the MFold server 
(http://www.bioinfo.rpi.edu/applications/hybrid/zipfold.php) 13, but the latter sometimes 
produce positive MFE values that are difficult to interpret. The relative rank of MFE 
remains the same when computed at higher or lower temperatures. 
The most abundant mRNA species of HIV-1 genes were constructed according to an 
extensive identification and quantification of HIV-1 splicing variants 9. The 5'UTRs, 
together with the coding sequences of these abundant mRNA species are collected in the 
first supplemental FastA file (HIV_NC_001802_mRNAs.FAS). The supplemental FastA 
file (HIV1Upstream.fas) contains the 60 nt immediately upstream of the initiation AUG 
of these mRNAs.  
 
 
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Acknowledgements 
We thank S. Aris-Brosou, S. Khalouei and X. Yao for discussion. S. Khalouei assembled 
HIV-1 splicing variants. J. A. Doudna and her colleagues verified the ranking of IRES 
activities of yeast genes. This work is supported by the Discovery, RTI and Strategic 
Research Grants from Natural Science and Engineering Research Council (NSERC) of 
Canada to XX. NSERC is not involved in any part of the study other than providing 
funds.
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Internal ribosomal entry site lacks secondary structure

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