A common RNA structural motif involved in the internal initiation of translation
of cellular mRNAs
A common RNA structural motif involved in the internal initiation of translation of cellular mRNAs
Shu-Yun
Le*
and
Jacob V.
Maizel Jr
Laboratory of Mathematical Biology, Division of Basic Sciences, National Cancer
Institute, NIH, Building 469, Room 151,
Frederick
, MD 21702,
USA
Received September 11, 1996;
Revised and Accepted November 11, 1996
ABSTRACT
The 5'-non-translated regions (5'NTR) of human immunoglobulin heavy chain binding protein (BiP), Antennapedia (Antp) of Drosophila and human fibroblast growth factor 2 (FGF-2) mRNAs are reported to mediate translation initiation by an internal ribosome binding mechanism. In this study, we investigate predicted features of the higher order structures folded in these 5'NTR sequences. Statistical analyses of RNA folding detected a 92 nt unusual folding region (UFR) from 129 to 220, close to the initiator AUG in the BiP mRNA. Details of the structural analyses show that the UFR forms a Y-type stem-loop structure with an additional stem-loop in the 3'-end resembling the common structure core found in the internal ribosome entry site (IRES) elements of picornavirus. The Y-type structural motif is also conserved among a number of divergent BiP mRNAs. We also find two RNA elements in the 5'-leader sequence of human FGF-2. The first RNA element (96 nt) is 2 nt upstream of the first CUG start codon located in the reported IRES element of human FGF-2. The second (107 nt) is immediately upstream of the authentic initiator AUG of the main open reading frame. Intriguingly, the folded RNA structural motif in the two RNA elements is conserved in other members of FGF family and shares the same structural features as that found in the 5'NTR of divergent BiP mRNAs. We suggest that the common RNA structural motif conserved in the diverse BiP and FGF-2 mRNAs has a general function in the internal ribosome binding mechanism of cellular mRNAs.
INTRODUCTION
Human fibroblast growth factor 2 (FGF-2) belongs to a family of potent mitogens that are implicated in many
aspects of cell growth and differentiation, both in normal and neoplastic
settings. The intracellular distribution of FGF-2 plays an important role in cell behavior. Four forms of human FGF-2, a small protein of 18 kDa and three larger proteins of 21, 21.5 and 22.5 kDa, are synthesized
from the same mRNA (
1
). These different proteins result from alternative initiation of translation by using a canonical initiator AUG and
three in-frame, upstream, non-canonical CUG initiators (
2
).
Most mRNAs encoding oncoproteins and cell factors related to cell proliferation
possess a long, GC-rich and structured 5'-leader sequence with one or more AUG triplets. The leader
sequence of human FGF-2 contains a 301 nt long 5'-non-translated region (5'NTR) with two AUG triplets that is located
upstream of the first start codon (CUG) and a 165 nt alternatively translated
region (ATR) from the first start codon CUG to the major translational
initiator AUG of the FGF-2 mRNA. The base composition of the ATR sequence is up to 90% GC. Kozak (
3
) suggested that the expression of this type of gene, with a GC-rich and highly structured leader sequence, is translationally regulated. Recently, Vagner
et al
. (
4
) demonstrated that the alternative translation of human FGF-2 mRNA was mediated by an internal ribosome binding mechanism. Furthermore, a 165 nt RNA element just upstream of the first start codon CUG is proposed as an internal
ribosome entry site (IRES). These results indicate that the regulation of
translational initiation plays a crucial role in the control of cell
proliferation.
The internal ribosome binding mechanism, independent of the 5'-cap structure and of the cap binding protein complex eIF-4E, was first observed in poliovirus (
5
) and subsequently in cardiovirus (
6
), aphthovirus (
7
-
9
), rhinovirus (
10
) and hepatitis A virus (
11
,
12
). The phenomenon is of general importance as it is not restricted to
picornaviruses. It is also found in other types of viruses, including hepatitis
C virus (HCV) (
13
,
14
) and bovine viral diarrhea virus (
15
), as well as some cellular mRNAs, such as human immunoglobulin heavy chain
binding protein (BiP) mRNA (
16
), Antennapedia (Antp) mRNA of
Drosophila
(
17
) and FGF-2 mRNA (
4
). Capped eukaryotic mRNAs can be translated without significant amounts of the
intact protein complex eIF-4F. Initiation and translation by an internal ribosome binding mechanism
independent of an intact eIF-4F holoenzyme complex may be advantageous for those mRNAs whose translation is important at mitosis in the cell cycle, because of the presence of
underphosphorylated eIF-4F.
Internal ribosome binding is dependent on interactions between a
cis
-acting IRES and a
trans
-acting, cell type-specific factor, such as p57/PTB (polypyrimidine tract binding
protein) required in the internal initiation of enteroviruses (
18
). The RNA-protein interaction involves the specific recognition of sequences and/or
structural elements of the IRES element within the 5'NTR by the cellular factors. Thus, it is important to determine the
common structural motif folded within these IRES elements in BiP, Antp and FGF-2 mRNAs. Recently, we proposed a common RNA structural motif (
19
) that is conserved in all IRES elements examined to date involved in the internal ribosome binding mechanism, from picornavirus
to pestivirus and HCV. The common structural motif shares a structural feature
similar to that observed in the catalytic core of group I introns.
Sequence comparison of the 221 nt 5'NTR of BiP mRNA with the IRES of picornaviruses did not reveal any
notable homology. Up to now, we do not know whether there is any significant
relationship between the viral and cellular IRES. Does the functional IRES in
the 5'NTR of cellular mRNAs comprise an undiscovered, conserved structural
motif that could be correlated with the mediation of internal ribosome entry in
BiP and FGF-2 mRNAs? We address this question in this study.
MATERIALS AND METHODS
RESULTS
The UFR and common RNA structural motif of BiP IRES elements
A UFR from nt 129 to 220 is detected in the segment including the 5'NTR and 50 nt downstream of the AUG start codon. This is one of the most
stable regions in the 5' portion of BiP mRNA. Although the segment 129-220 has the lowest stability scores computed by both the Tinoco (
59
) and Turner (
41
) energy rules, its scores related to the random shuffling sequences are not
statistically significant (Fig.
2
). This means that thermodynamic stability alone may not be a good predictor for
representing the functional form of this RNA segment.
Structural features of IRES elements in BiP and FGF-2
The sequence alignment of BiP and FGF-2 IRES elements and the 5'UTR of other BiP and FGF family members is shown in Figure
6
. The alignment was gradually refined by means of their common structural
information. In the common Y-type structure, stem A is usually interrupted by an internal loop and its
size ranges from 8 to 13 bp. There is an intervening unpaired base between
stems A and B in some cases. Stem B and stem C contain 4-8 bp and a hairpin loop of 3-8 unpaired bases. In some cases, stems B and C are broken by a
small bulge or internal loop. Stem C is frequently connected to stem A by a
loop of 3-5 nt. The Y-type structure is followed by an additional stem-loop D, in which there are differences in sequence length.
One remarkable property is that stem-loop D is just a few nucleotides upstream from the authentic initiator.
The proposed common structural motif of these IRES elements is confirmed by the
intra- and/or interphylum covariance seen in Figure
6
and Table
2
that maintains the base pairing potential in stems A-D. The structural conservation of these divergent sequences indicates
that the predicted common RNA structural motif involved in internal initiation
of the translation of cellular BiP and FGF-2 mRNAs could play an important role in internal ribosome binding control
of cellular mRNAs.
DISCUSSION
A common RNA structural motif, including a Y-type stem-loop structure followed by a small stem-loop structure just upstream of the translational initiator, is a conserved property
found in IRES elements identified in the cellular BiP and FGF-2 mRNAs. Although the Antp gene of
D.melanogaster
contains an exceptionally long 5'NTR (>1500 nt), the 5'-border of exon D functions as the IRES element (
17
). In the 5'-border of exon D there is a 55 nt RNA sequence highly conserved
among
D.melanogaster
,
D.virilis
and
D.subobscura
. Preliminary experiments from Sarnow's laboratory (
17
,
46
) indicated that the highly conserved 55 nt sequence (nt 1-55 in Fig.
7
) was required for Antp IRES function. The conserved 55 nt sequence is one of
the smallest IRES elements reported, which is located over 350 nt upstream of
the AUG translational start codon. A Y-type stem-loop structure similar to that observed in other IRES elements of
cellular mRNAs can be formed in the RNA fragment that includes the conserved 55
nt and the following 8 nt. However, these folded RNA structures are not highly
stable. One possibility is that IRES-dependent translation of Antp requires additional protein factors to
stabilize the folded RNA common structure. Ribosome movement of the 350 nt from
the IRES to the initiating AUG may occur by conventional scanning.
Among the common RNA structural motifs of the IRES elements of BiP, FGF-2 and Antp mRNAs, the most striking structural property may be represented
by the conserved, conceivably coaxial stacking between stem B and stem C within
the common Y-type stem-loop structure. Sequence comparison (Table
1
) indicated that while these suggested RNA functional elements diverge in
sequence similarity, the folded structures have conserved folding shapes and
positions, just a few nucleotides (except for Antp mRNAs) upstream of the
authentic initiation codon. The conservation of these structural motifs among
these divergent BiP sequences, from protozoa to human, strongly supports their
importance. Moreover, the predicted structural motifs of BiP IRES elements are
also conserved in FGF-2 IRES elements, although their sequence similarities are <= 35%. The evolutionary stability of this structural motif is consistent
with a crucial role in the IRES-dependent translation of cellular mRNAs.
Previously, we proposed a structural core of secondary and tertiary structures
(consisting of stems E-H) that is common to all the IRES elements of picornaviruses, HCV and
pestiviruses (
19
). This conserved structural motif (Fig.
4
), found among a large number of dissimilar sequences, is likewise a candidate
structural core essential to the function of these viral IRES elements.
Interestingly, the proposed conserved superstructures for these viral IRES elements share a striking structural resemblance to the higher
ordered structure of group I introns. The common structural core (Fig.
4
) of these viral IRES, composed of stems E-H, shares tertiary structural features analogous to the core structure
centered on helical regions P3, P4, P6 and P7 in group I introns (
48
). The stacking between stems E and H corresponds to the stacking of helices P3
and P7 and the possible coaxial stacking of stems F and G corresponds to the
stacking between P4 and P6. By means of such coaxial stem stacking and other
tertiary interactions, the group I introns form a compact three-dimensional structure.
A comparison of the two structural motifs of the viral and cellular IRES elements indicated that they share some resemblance in the folding shape, stem stacking and sequence location of the 5'NTR region (Fig.
4
). For the two common structural motifs, stems A-D in cellular IRES correspond to stems E, F, G and I/J of viral IRES
elements respectively. Possible coaxial stacking between stems B and C in the
cellular IRES corresponds to the stacking of viral stems F and G. The coaxial
stacking between two adjacent stems may be one of the most important properties
observed in cellular and viral IRES elements. Stem stacking is an important tertiary structural interaction, as shown in group I introns (
49
,
50
), rRNAs (
51
,
52
) and tRNAs (
53
). The newly developed energy parameters for coaxial stacking between two
adjacent stems (
54
) indicate that such a distinct structural element is quite favorable to the
stabilization of RNA folding. Similar to group I introns, the common core
structures of the viral and cellular IRES can form a compact tertiary structure
by means of stem stacking. However, the new energy parameters contributed by the stem stacking have not
been included in our current Monte Carlo simulation procedure.
Comparatively little is known about IRES elements in cellular mRNAs relative to
the viral IRES. Limited information on the boundaries of cellular IRES elements has been determined by deletion analysis. However, the exact positions of these IRES elements have not been determined by extensive experimental testing. We suggest that the function of both viral and cellular IRES elements is
correlated with the distinct, conserved RNA higher order structures folded in
these elements.
The difference between the two structural motifs of the viral and cellular IRES
is also clear. The conserved RNA pseudoknot composed of stems E and H in all
viral IRES elements was not observed in the cellular IRES. The RNA pseudoknot
has been demonstrated to be essential for HCV IRES function (
55
). If the pseudoknot is an important structural property of the viral IRES, then
there is a difference in the IRES-dependent mechanism that occurs in cellular and viral internal initiation.
Currently, two cellular
trans
-acting factors, the La antigen (
56
) and PTB (
57
,
58
), have been found to bind to picornavirus IRES elements and to be essential for
their internal initiation of translation. However, PTB binds extremely poorly
to the BiP IRES and two other proteins, ~60 and 95 kDa in size, can bind specifically to the BiP IRES (
46
). It is possible that different
trans
-acting factors are required which are dependent on the specific IRES.
Few IRES elements have been found in cellular mRNAs. It is not clear what
advantage the IRES-dependent mechanism offers relative to the conventional ribosome scanning
mechanism in translational initiation of cellular mRNAs. Although the proposed common structural motif of the cellular IRES requires confirmation by experimental data, our model is supported by phylogenetic analysis of
divergent sequences from BiP, FGF-2 and the FGF family. The structural conservation is consistent with the
fact that these IRES elements are functionally related to each other. The
common structural motif of cellular IRES elements may provide useful
information on the relationship between viral and cellular IRES elements. It is
also helpful for searching more cellular mRNAs with an IRES. Knowledge of the
distribution of these structural motifs should further the discovery of the
underlying principles of cap-independent translation and internal initiation of translation in
eukaryotic cellular mRNAs.
ACKNOWLEDGEMENTS
The contents of this publication do not necessarily reflect the views or
policies of the Department of Health and Human Services, nor does mention of
trade names, commercial products or organizations imply endorsement by the US
Government.
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