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.
Nature Precedings : hdl:10101/npre.2007.1248.1 : Posted 23 Oct 2007
2
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
Nature Precedings : hdl:10101/npre.2007.1248.1 : Posted 23 Oct 2007
3
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.
Nature Precedings : hdl:10101/npre.2007.1248.1 : Posted 23 Oct 2007
4
Figures
YEL033W
HMS2
TPK2
BOI1
GPR1
FLO8
GIC1
YMR181C
MSN1
NCE102
PAB1
TIF4632
BOI1rc
GPR1rc
FLO8rc
YMR181Crc
y = 14.578e
0.1238x
R
2
= 0.6016
0
2
4
6
8
10
12
14
16
18
-20
-18
-16
-14
-12
-10
-8
-6
-4
-2
0
Structural stability (MFE)
I
R
ES act
i
v
i
t
y
(
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
Nature Precedings : hdl:10101/npre.2007.1248.1 : Posted 23 Oct 2007
5
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.
Nature Precedings : hdl:10101/npre.2007.1248.1 : Posted 23 Oct 2007
6
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 AUG
i
(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)
Nature Precedings : hdl:10101/npre.2007.1248.1 : Posted 23 Oct 2007
7
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.
Nature Precedings : hdl:10101/npre.2007.1248.1 : Posted 23 Oct 2007
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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.
Nature Precedings : hdl:10101/npre.2007.1248.1 : Posted 23 Oct 2007
9
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