From the Department of Microbiology and Cell Biology
and the ¶ Molecular Biophysics Unit, Indian Institute of Science,
Bangalore 560012, India
Received for publication, October 8, 2002, and in revised form, December 11, 2002
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ABSTRACT |
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The human La autoantigen has been shown to
interact with the internal ribosome entry site (IRES) of hepatitis C
virus (HCV) in vitro. Using a yeast three-hybrid system, we
demonstrated that, in addition to full-length La protein, both N- and
C-terminal halves were able to interact with HCV IRES in
vivo. The exogenous addition of purified full-length and
truncated La proteins in rabbit reticulocyte lysate showed
dose-dependent stimulation of HCV IRES-mediated
translation. However, an additive effect was achieved adding the
terminal halves together in the reaction, suggesting that both might
play critical roles in achieving full stimulatory activity of the
full-length La protein. Using computational analysis, three-dimensional
structures of the RNA recognition motifs (RRM) of the La protein were
independently modeled. Of the three putative RRMs, RRM2 was predicted
to have a good binding pocket for the interaction with the HCV IRES
around the GCAC motif near the initiator AUG and RRM3 binds perhaps in
a different location. This observation was further investigated by the
filter-binding and toe-printing assays. The results presented here
strongly suggest that both the N- and C-terminal halves can interact
independently with the HCV IRES and are involved in stimulating
internal initiation of translation.
Hepatitis C virus (HCV)1
is a single-stranded, positive-sense enveloped RNA virus classified in
a separate genus of the family Flaviviridae (1). HCV has been shown to
be the primary causative agent of non-A, non-B viral hepatitis,
which often leads to development of chronic hepatitis, cirrhosis, or
hepatocellular carcinoma (2, 3). The HCV genome RNA is ~9.5 kb in
length and consists of a 5'-untranslated region (UTR), a long open
reading frame encoding the viral polyprotein, and a 3'-UTR (4). The
translation initiation of HCV occurs by cap-independent mechanism that
is directed by the highly structured 5'-UTR (341 nt long), consisting
of four stem-loops and a pseudoknot structure. The 5'-UTR contains
unique cis-acting element called "internal ribosome entry
site" (IRES) that mediates 5'-cap-independent internal initiation of
translation (5-9). The HCV IRES element requires most of the 5'-UTR,
except the first 40 nt, and extends to a short stretch (30-40 nt) of sequence downstream of the initiator AUG codon (6, 10). Hepatitis C
virus IRES is significantly different from that of picornaviruses (e.g. poliovirus) with respect to length, secondary and
tertiary structure of the 5'-UTR RNA (11). Also in contrast to
picornavirus IRES, HCV IRES does not have a strict requirement for the
canonical initiation factors (eIFs) other than eIF2 and eIF3 (12). A
number of cellular polypeptides have been shown to specifically
interact with the HCV 5'-UTR RNA, which include polypyrimidine-tract
binding protein (p57/60) (13), human La antigen (La, p50/52) (14), poly(rC)-binding protein 2 (15), heterogeneous nuclear
ribonucleoprotein L (p68) (16), and ribosomal protein factors S9 and S5
(12). Interaction of these proteins with the 5'-UTR may be important for translation and/or replication of the HCV genome, although the
precise roles are not known yet.
The La protein (also called SS-B) was originally identified as an
autoantigen that was recognized by sera from patients with systemic
lupus erythematosus and Sjogren's syndrome (17). Homologues of La
protein have been identified in fruit fly, (Drosophila
melanogaster) and in yeast (Saccharomyces cerevisiae)
(18). Human La antigen is an RNA-binding protein, belonging to the RNA
recognition motif (RRM) super family. It is a 50/52-kDa protein, which
is predominantly localized within the nucleus and which functions in
the maturation of RNA polymerase III transcripts and the unwinding of
double-stranded RNA (19-22). La protein has been shown to be
associated with U1RNA (23), telomerase RNA (24), adenovirus VA RNA
(25), vesicular stomatitis virus leader RNA (26), influenza virus (27),
Sindbis virus (28), and the HIV TAR element (29). The other targets of
the La protein binding include the 5'-UTRs of poliovirus (30), hepatitis C virus (14), encephalomyocarditis virus (31), and Bip
mRNA (32). Interestingly, it has been shown that La protein specifically interacts with both the 5'- and 3'-UTR of hepatitis C
virus RNA (33). La protein plays a functional role in internal initiation of translation where addition of purified La to RRL in the
in vitro translation assays using either HCV or poliovirus IRES resulted in stimulation of the translation activity (14, 30).
Sequestration of La in RRL inhibits HCV IRES-mediated translation, which can be rescued by exogenous addition of purified La protein. Different approaches of La depletion were employed to demonstrate the
functional requirement of La protein on HCV IRES-mediated translation.
A small RNA called I-RNA (60 nt) originally isolated from S. cerevisiae has been shown to bind to La protein and inhibit HCV
IRES-mediated translation (34). Similarly, a SELEX RNA generated against La protein has been used to compete with HCV 5'-UTR for the La
protein binding to block the IRES-mediated translation (35). However,
it has been noted that HCV IRES has a lower requirement of La protein,
compared with poliovirus IRES (36). Similarly, in absence of La
protein, X-linked inhibitor of apoptosis protein IRES-mediated
translation is shown to be severely affected (37) and Bip IRES activity
is enhanced severalfold in presence of La protein (32). Interestingly,
binding of La protein to the HIV TAR sequence alleviates the
translational repression exerted by the TAR sequence on a downstream
reporter gene (38) and, in the case of encephalomyocarditis virus, La
protein alleviates inhibitory activity of surplus polypyrimidine
tract-binding protein (31).
La protein has been shown to have three putative RRMs and a basic
region followed by a stretch of acidic region at the C terminus (39). A
truncated La protein (1-194), which still contained RRM and bound the
poliovirus 5'-UTR, failed to stimulate poliovirus translation or
correct the aberrant translation in RRL (40), suggesting that binding
of the La protein with the RNA is insufficient to explain the activity.
The C terminus also contains a homodimerization domain; deletion of
this domain has been shown to abrogate the ability of La protein to
enhance IRES-mediated translation of poliovirus (41).
In this report we have demonstrated that both N- and C-terminal halves
of La protein are able to interact with HCV IRES in vivo by
the yeast three-hybrid assay system. The relative binding affinities
have been determined using filter-binding assays. Furthermore, the
biological relevance of these interactions was studied by exogenous
addition of purified full-length and truncated La proteins in the
in vitro translation system using bicistronic constructs in
rabbit reticulocyte lysate. Interestingly, both the N- and C-terminal
halves of La proteins were able to stimulate the HCV IRES-mediated
translation unlike the case in poliovirus IRES, suggesting that the
deletion of the C terminus does not abrogate the ability of La protein
to enhance the translation of hepatitis C virus. To date, no
crystallographic or NMR structure information is available for either
the full-length or individual domains of La protein. Using
computational analysis, we have searched for traditional RRMs in the La
protein. We have generated the three-dimensional model of the RRM
regions of the La protein by comparative modeling on the basis of the
closest homologues of known structures determined using NMR or
crystallography. The predicted secondary structure of the region
105-208 residues of La (referred to as RRM2) showed similarity with
that of RRM-containing proteins of known structures. Additionally,
using comparative sequence and structural analysis, it is suggested
that the RRM2 within the N-terminal domain of La protein binds HCV RNA
at the GCAC motif near the initiator AUG. The prediction was further investigated by the filter-binding and toe-printing assays, which clearly demonstrated much higher RNA binding affinity of the RRM2 at
this region. The results constitute the first report to demonstrate physical interaction of La protein with the viral RNA in
vivo and provide direct evidence of the in vitro
interactions of RRM2 of La protein with the HCV IRES around the region
encompassing the initiator AUG and the GCAC motif.
Plasmids--
HCV 1b encoding plasmid, pCV, was generously given
by Dr. Akio Nomoto and Dr. Tsukiyama-Kohara (University of Tokyo,
Tokyo, Japan). HCV 5'-UTR along with 42-nt (18-383 nt) coding sequence was PCR-amplified from plasmid pCV and cloned between
HindIII and EcoRI sites of the mammalian
expression vector pcDNA3 (Invitrogen) to generate pcDHCV-383.
Similarly nt 1-341 of the HCV 5'-UTR were PCR amplified from the clone
pT7DC1-341 (a generous gift from Dr. Aleem Siddiqui, University of
Colorado, Denver, CO) and cloned into HindIII sites
of pcDNA3 vector to generate pcDHCV-341. The bicistronic reporter
vector, pRL HCV 1b, containing two reporter genes (Renilla
luciferase and firefly luciferase) separated by the HCV IRES was
generously provided by Dr. Richard M. Elliot (University of Glasgow,
Glasgow, Scotland, UK). Poliovirus 5'-UTR containing bicistronic
construct, p2-5', was a generous gift from the laboratory of Dr.
Nahum Sonenberg (McGill University, Montreal, Canada). The cDNA
clone encoding human La autoantigen, pETLa, was obtained from Dr.
Jack Keene (Duke University, Durham, NC). La coding sequence was
amplified by PCR using the following primers: 5'-GACCGGATCCATGGCTGAAAATGG-3' and
5'-CGTAGAATTCCTACTGGTCTCCAG-3'. The amplified product
was subcloned into BamHI and EcoRI sites of
pRSET-A vector (Invitrogen). The two deletion constructs pRSET-A La1-208 (La-N) and pRSET-A La209-408 (La-C) were constructed using the primers 5'-GACCGGATCCATGGCTGAAAATGG-3',
5'-GGCCGAATTCTTTAGCTCTTAATT-3', 5-ATATGGATCCCAGGAGCAAGAAGC-3', and
5'-CGTAGAATTCCTACTGGTCTCCAG-3'. The deletion
constructs pRSET-A La1-100 (RRM1) and pRSET-A La101-208 (RRM2) were
also constructed using the primers
5'-GACCGGATCCATGGCTGAAAATGG-3', 5'-GATCGAATTCACTTCA GGTAGGGG-3',
5'-GGCCGGATCCGAAGTGACTTGGGA-3', and
5'-CGTAGAATTCCTACTGGTCCCAG-3'. Similarly, pRSET-A
La209-300 (RRM3) was amplified by PCR and cloned in pRSET-A vector in
BamHI and EcoRI sites using the following
primers: 5'-ATATGGATCCCAGGAGCAAGAAGC-3' and
5'-TCTCGAATTCTCCCAAGTCACCTT-3'. All the PCRs were
carried out with 35 cycles, each cycle consisting of denaturation
(95 °C for 40 s), annealing (55 °C for 1 min), and extension
(68 °C for 1 min) using Pfx DNA polymerase (Invitrogen).
Purification of La Full-length and Truncated Proteins Using
Ni-NTA-Agarose Column--
Escherichia coli BL21 (DE3)
cells were transformed with pRSET-A vectors containing either the
full-length or the deletion mutants of La. Single colonies were
inoculated into 5 ml of LB broth containing 75 µg/ml ampicillin and
grown at 37 °C incubator shaker at 200 rpm speed until
A600 reached 0.6. The cultures were induced with
0.6 mM isopropyl-1-thio- In Vitro Transcription--
mRNAs were transcribed in
vitro from different linearized plasmid constructs under T7
promoters in run-off transcription reactions. The HCV bicistronic
construct, pRL HCV 1b, was linearized with HindIII, and
poliovirus bicistronic construct, p2-5', was linearized with
XhoI downstream of firefly luciferase to be used as
templates for run-off RNA synthesis. The linear DNA were
electrophoresed on agarose gels and extracted using silica beads
(Bangalore Genie) and then transcribed using a Ribomax large scale RNA
production system-T7 (Promega).
Radiolabeled mRNAs were transcribed in vitro using T7
RNA polymerase (Promega) and [ Filter-binding Assay--
The [ Yeast Transformation--
The RNA-hybrid and the protein-hybrid
vectors were co-transformed into yeast host strain, L40uraMS2, by
lithium acetate method. The overnight culture grown in 10 ml of YPD
medium (1% yeast extract, 2% peptone, and 2% dextrose) was diluted
into 50 ml of YPD such that A600 is 0.4 and
grown for an additional 2-4 h. The cells were pelleted, washed with 40 ml of 1× TE (10 mM Tris, pH 7.5, 1 mM EDTA),
resuspended in 2 ml of 1× LiAc/0.5× TE buffer (100 mM
lithium acetate, pH 7.5, and 0.5× TE) and incubated at 30 °C for 30 min. 1 µg of each plasmid and 100 µg of herring sperm DNA were
mixed with 100 µl of the yeast suspension and 700 µl of 1× LiAc,
40% polyethylene glycol 3350, 1× TE, and incubated at 30 °C for 30 min. 88 µl of Me2SO was added, and heat shock was given for 15 min at 42 °C. The cells were washed with 1× TE and plated onto His-selective plates and incubated at 30 °C. The transformants growing on His Liquid In Vitro Translation--
In vitro translation of the
capped bicistronic mRNAs were carried out in micrococcal
nuclease-treated rabbit reticulocyte lysates (RRL, Promega Corp.).
Briefly, 12.5-µl reaction mixtures contained 8.75 µl of RRL
containing 0.25 µl each of minus methionine and minus leucine amino
acid mixtures and 10 units of RNasin (Promega Corp.). The reaction
mixtures were incubated at 30 °C for 1 h and 30 min. 2 µl of
the reaction mixtures were assayed for both the Renilla and
firefly luciferase activity according to Promega protocol using the
Dual Luciferase reporter assay system. To assay the CAT reporter gene
activity, 2 µl of 1:10 dilution of the reactions mixtures were added
to 38 µl of 0.25 M Tris-HCl, pH 7.4, 2 µl of
[14C]chloramphenicol in total volume of 90 µl. After
incubation at 37 °C for 10 min, 8 µl of 10 mM
acetyl-CoA was added and incubated further for 1 h at 37 °C
followed by ethyl acetate (500 µl) extraction. The upper organic
phase was separated and evaporated to dryness. The residues were
resuspended in 25 µl of ethyl acetate and spotted onto silica gel TLC
plates, followed by chromatography using a mixture of
chloroform/methanol (95:5). The TLC plate was dried and exposed to
PhosphorImager, and bands corresponding to acetylated and unacetylated
forms were quantified. The relative CAT activity was expressed as a
ratio of acetylated form to the total.
Primer Extension Inhibition Analysis--
Primer extension
inhibition (toe-printing) assay was performed as described previously
(12). Briefly, increasing concentration of purified His-tagged La
full-length or deletion proteins (200, 400, and 600 ng) were incubated
with 2.5 pmol of in vitro transcribed RNA corresponding to
the HCV IRES (18-383), and binding reaction was performed in a final
volume of 20 µl at 30 °C for 20 min. To this reaction,
32P-end-labeled primer complimentary to 25 nucleotides of
the 3' end of the HCV-383 was added and allowed to extend using 3 units of avian myeloblastosis virus-reverse transcriptase (Promega) at
30 °C for 1 h. The cDNAs were alcohol-precipitated,
resuspended, and compared with the dideoxynucleotide sequence ladders
by electrophoresis on a 6% polyacrylamide, 7 M urea
denaturing gel.
Comparative Modeling--
Traditional RRMs in the La protein
were searched using the motif search tools such as PROSITE. The known
related crystal and NMR structures of RNA-bound complexes were used as
the basis in modeling the three-dimensional structure of RRM2-RNA
complex. At least six different proteins with complex crystal or NMR
structures of RRMs bound to RNA or DNA are available. The highest
sequence identity between any of the putative RRMs of La proteins and
the RRMs of known structure is on the order of 14%, which is very low.
Such sequence identities are possible even between two entirely unrelated proteins of completely different fold. Hence we identified the best homologues for comparative analysis and modeling on the basis
of highest compatibility of the sequences of putative RRMs in the La
protein with each of the known structures that are bound to RNA. The
structural features considered for compatibility analysis are solvent
accessibility, hydrogen bonding pattern, and secondary structures. It
is important to choose a minimal subset of known structures as the
basis under low sequence similarity situation, as it has been shown
earlier that choosing all the possible structures as basis can result
in high errors in the model (42).
Thus, two constraints were used in choosing the template structures:
(a) the RRM of known structure should be bound to RNA, and
(b) the compatibility of the sequences of RRMs in the La
protein with the chosen structures should be highest. Two different
known structures of RRMs of RNA-bound complexes used in the modeling were: "splicing factor U2B" from human and U1A spliceosomal protein.
The suite of programs encoded in COMPOSER (42) and
incorporated in SYBYL (Tripos Inc., St. Louis, MO) was used
to generate the three-dimensional models of RRMs in La protein. The
sequences of the known structures of RRM proteins were aligned on the
basis of their three-dimensional structures and refined using the
structural features such as solvent accessibility and secondary
structures and relationships such as hydrogen bonding pattern (43, 44). The optimal superposition of C Energy Minimization--
The COMPOSER-generated model
was energy minimized in SYBYL using the AMBER
force-field (47). During the initial cycles of energy minimization, the
backbone was kept rigid and side chains alone were moved. Subsequently,
all atoms in the structure were allowed to move during minimization.
This approach kept disturbance of the backbone structure to the
minimum. Energy minimization was performed until all short contacts and
inconsistencies in geometry were rectified. During the initial stages
of minimization, the electrostatic term was not included, as the main
objective was to relieve steric clashes and to rectify bad geometry.
Modeling of the RRM-RNA Complex--
The NMR structure of human
U1A protein bound to RNA (48) has been used as the template to model
the binding of RNA in the RRMs of La protein. The choice of 1aud is
made on the basis of the highest sequence similarity of 1aud with RRM2
and RRM3 of La. We have identified the regions of U1A protein that
interacts with RNA and placed the RNA in an identical orientation in
the RRM models of La protein. This has been achieved by optimally superimposing the RNA-interacting regions of U1A on the equivalent regions of the RRM model of La and carrying RNA from the U1A protein complex in the process.
La Protein Interacts with HCV IRES in Vivo in Yeast Three-hybrid
System--
Previously, it has been demonstrated that La autoantigen
interacts with the 5'-untranslated region of hepatitis C virus RNA in vitro and the C terminus effector domain modulates the
binding efficiency (35). In fact several trans-acting
factors have been shown to interact with the HCV 5'-UTR, but so far
none of the interactions have been shown in vivo. To
investigate whether La protein interacts with the HCV IRES RNA in
vivo, we took advantage of the genetic screening method by using
yeast three-hybrid system. The system allows detection of the target
RNA (bait) and potential protein (prey) interaction in a S. cerevisiae strain containing integrated reporters (histidine and
Similarly, to investigate whether the La-N or La-C can interact
independently with the HCV IRES RNA in vivo, either the
pYESTrp2La1-208 or pYESTrp2La209-408 vector was co-transformed with
the pRH5'-HCV RNA hybrid vector. Interestingly, both La-N and La-C
deletions demonstrated growth on selective plates (His Both N- and C-terminal Halves of La Protein Are Necessary to
Enhance HCV IRES-mediated Translation--
Human La autoantigen has
been shown to enhance HCV IRES-mediated translation. Previously, it has
been demonstrated that a mutant La protein comprising the C-terminal
half (La229-408) is capable of binding HCV 5'-UTR and stimulating the
HCV IRES-mediated translation in RRL, albeit to a lesser extent
compared with the full-length La protein (35). To further characterize
the La protein requirement for the HCV IRES function, the effect of
addition of purified N- and C-terminal halves of La proteins was
determined by in vitro translation reactions. The
bicistronic construct containing two reporter genes, Renilla
luciferase and firefly luciferase, flanked by the HCV IRES was used in
this experiment. The initiation of cap-independent translation,
occurring internally from HCV 5'-UTR, resulted in the synthesis of
firefly luciferase, whereas cap-dependent translation
produced the Renilla luciferase. The capped bicistronic RNA
was translated in the presence of increasing concentration of either
full-length or the truncated La proteins; the luciferase activities
were measured using the Stop and Glow dual luciferase assay system
(Promega) and plotted against La concentration (Fig.
2A). The results showed
gradual stimulation of luciferase activity up to ~4-fold with the
increase in concentration (25, 50, and 75 ng; Fig. 2A,
lanes 2-4) of full-length La protein. Furthermore, addition of either the N- or the C-terminal half of La
protein (12.5, 25, and 37.5 ng, Fig. 2A, lanes
5-7 and 8-10) showed modest increase (up to
2.5-3-fold) in HCV IRES-mediated translation. The result suggests that
the HCV IRES-mediated translation could be stimulated by specific
interaction of independent regions of the La protein. However, the
translation stimulation by either La-N or La-C was relatively less,
compared with the full-length. Thus, we investigated whether an
additive effect on HCV IRES-mediated translation could be achieved by
adding both La-N and La-C together in the same reaction. For this,
increasing concentrations of La-N and La-C proteins were added together
in equimolar amounts (6.25, 12.5, and 18.75 ng each) in the translation
reactions, and the luciferase activities were measured. The result
showed gradual increase of the HCV IRES-mediated translation up to
~6.5-fold (Fig. 2A, lanes 11-13),
which is equivalent to the level expected with the addition of
full-length La protein. However, no significant increase was observed
in the Renilla luciferase activity (Fig. 2A,
shaded bars). The results suggest that both N-
and C-terminal halves might play a critical role in achieving full
stimulatory activity of the full-length protein.
To further explore the role of N- and C-terminal halves of La protein
in poliovirus IRES-mediated translation, similar experiments were
carried out using poliovirus bicistronic construct (Fig. 2B). Addition of increasing concentrations of full-length La
protein (250, 500 and 750 ng; Fig. 2B, lanes
2-4) gradually enhanced poliovirus IRES-mediated
translation of firefly luciferase up to 2.5-fold. Interestingly,
addition of C-terminal half alone resulted in significant stimulation,
up to 2.2-fold (Fig. 2B, lanes 8-10).
However, the N-terminal half lacking the C-terminal effector domain
failed to stimulate the poliovirus IRES activity (Fig. 2B,
lanes 5-7). Cap-dependent
translations of CAT gene from the poliovirus bicistronic RNA were not
altered with the addition of either the full-length or the deleted La
proteins (Fig. 2B, shaded bars). Taken
together, the results strongly suggest that, indeed, La protein
enhances HCV IRES-mediated translation, and it appears that deletion of C-terminal effector domain does not abrogate the ability of La protein
to enhance HCV IRES-mediated translation.
Computational Analysis Revealed a Consensus RNA Recognition Pattern
in the RRMs of Known Structures--
To understand the structural
basis of the interaction of La protein with HCV RNA, we have searched
for traditional RRMs (49-52) in the La protein using the motif search
tools. The only region identified as RRM in the La protein ranges from
residue position 105 to 200, which is referred to as RRM2. However,
because the overall sequence similarity with proteins of known
structure is low, we confirmed the similarity by comparing predicted
secondary structure of RRM2 with the observed secondary structures in
the RRM-containing proteins of known structure. Correspondence between observed and predicted secondary structures is excellent (data not
shown), supporting the view that the region 105-200 could be a RRM
domain. We have assessed the capability of this predicted RRM2 to bind
RNA using three-dimensional structural models. We have used the known
related crystal and NMR structures of RNA bound complexes as the basis
in modeling the three-dimensional structure of RRM2-RNA complex. We
have also analyzed the other putative RRMs of La protein, RRM1 and
RRM3, in a similar way.
To start with, we analyzed by comparing the known three-dimensional
structural complexes of the RRM-containing proteins. As mentioned under
"Experimental Procedures," not all the known structures of RRMs
are available bound to RNA and the best sequence similarity between the
putative RRMs in La protein and the known structures is as low as 14%,
which is not significant. We have used the constraints that the
structures useful for modeling and analysis should be bound to RNA and
they should show best compatibility between sequence of RRMs in La with
the observed structural features such as solvent accessibility and
secondary structure. The known structures that passed these conditions
are: spliceosomal U2B"-U2A' protein (Ref. 53; code 1a9n), human U1A
protein (Ref. 48; code 1aud), hairpin ribozyme inhibitor (Ref.
54; code 1hp6), and U1A spliceosomal protein (Ref. 55; code 1urn).
However, it should be noted that 1aud, 1hp6, and 1urn practically refer
to the same protein, although the lengths and sequences of bases in the
RNA molecules bound to the known structural complexes vary
substantially. Despite being practically identical sequences, 1aud,
1hp6, and 1urn represent independent complex structure determination.
We had ensured in the comparative modeling that we take only one of
these three structures apart from 1a9n in generating the framework of
the model. Thus, although these three structures were considered for comparative analysis, we have ensured that our modeling and other results are not biased.
A detailed investigation of each of these complex structures revealed
that, apart from a short N-terminal region, two loops are involved in a
series of interactions with the RNA. These interactions are extremely
well conserved within the homologous protein-RNA complex structures
whose structure-based sequence alignment is shown in Fig.
3. Fig. 4A shows the
optimal superposition of the known
structures with a consensus stretch of RNA. One of the RNA-interacting loops is centered around residue position 50 (1aud numbering, Fig. 3),
which links two contiguous
Despite the fact that the bound RNA sequences and sizes vary
enormously, the region of RNA interacting with the two loops of the
protein is a stretch of four bases with a very well conserved conformation with an overall shape resembling an "L" shape (Fig. 4A). In three of the four structures, the sequence of this
region of four bases is GCAC, and in 1a9n the sequence is GCAG. It can be concluded that the known structures of RRM proteins with RNA bound
analyzed here represent a recognition pattern involving a highly
conserved sets of loops in the protein and a four-base stretch of
GCAC/G sequence in the RNA. Interestingly, a similar GCAC motif is
observed in the HCV IRES RNA as well.
Structure-based Analysis of RNA Binding Capability of RRMs in the
La Protein--
The overall sequence similarity as seen in Fig. 3 is
poor between RRM1 and the known structures with RRM. This feature could result in overall high structural variations in RRM1, even in case RRM1
adopts the overall fold as in the known structures. Significantly, in
the
From Fig. 3 it can be seen that the sequence of RRM2 of La protein
involves no insertion or deletion at the two critical loop regions
compared with RRM proteins of known structure. There are a few residue
changes in the loop regions compared with the known structures.
However, a detailed investigation of the model suggests that all the
residues in the two loops of RRM2 are comfortably placed in three
dimensions. Fig. 4B shows the modeled structure of RRM2
bound to RNA. Almost all the residues in the two loops are generally
well exposed to the surroundings in the known structures. The nature of
substitution of residues seen in RRM2 is conservative, involving good
accommodation of the loop sequences in the conformation seen in the
equivalent regions of the known structures. Thus, the backbone
conformations at the two RNA binding loops of RRM2 are likely to be
similar to those seen in the homologous proteins of known structure.
This will result in orientation of backbone amide and carbonyl groups
at the loop regions similar to that seen in the known structures
enabling RNA to bind at RRM2. Because of the predicted high similarity
in the backbone structure of RRM2 with the closest homologues, it is
suggested that RRM2 might bind to the GCAC motif in the HCV IRES RNA.
The overall sequence similarity of RRM3 of La protein with the RRMs of
known structure is significant and is an indication of the retention of
the fold. The sequence of the RRM3 could be comfortably modeled in the
RRM fold (Fig. 4C); however, there is a deletion of a
residue in the RRM2 of La Protein Has Greater Affinity for Binding with the HCV
IRES RNA--
RRM1 and RRM2 are located at the N-terminal half, and
the RRM3 is located at the beginning of the C-terminal half of La
protein (Fig. 1A). Our computational modeling data predict
that the RRM2 is the most plausible RRM for the interactions with the
HCV IRES. To test this hypothesis, we have performed filter-binding
assay using the full-length and the La deletion proteins corresponding to each RRM: La1-100 (RRM1), La101-208 (RRM2), and La209-300 (RRM3). Full-length and truncated La proteins were over-expressed in E. coli (BL21) and purified by passing through an Ni-NTA column. 32P-Labeled HCV IRES RNA (18-383 nt) probe (10 fmol) was
separately incubated with increasing concentration of each of the
deletions. The amount of bound RNA was determined by binding to
nitrocellulose filters and plotted to obtain the saturation curve (Fig.
5A). The linear portions of
each curve were subjected to regression analysis. The apparent
dissociation constant (Kd) was calculated in each
case as the protein concentration at which 50% of the RNA bound at
saturation was retained on the filter. Results showed that La protein
binds to HCV IRES RNA with relatively high affinity, with an apparent
Kd of 0.12 µM. As predicted by the
computer modeling data, the filter-binding assay results clearly
demonstrated that RRM2 protein binds to HCV IRES RNA with relatively
high affinity, with an apparent Kd of 0.14 µM, closely similar to that of full-length La protein.
However, the protein corresponding to RRM3 showed relatively lower
affinity with an apparent Kd of 0.23 µM. RRM1 protein alone failed to show significant binding
with the HCV IRES RNA, as demonstrated before in an earlier report.
The computational analysis of the three-dimensional structures of the
RRMs of the La protein predicted that RRM2 might interact with the HCV
IRES around the GCAC motif near the initiator AUG. To confirm that, we
have used HCV IRES mutant (HCV 5'-UTR 1-341) lacking the initiator AUG
and GCAC motif as probe RNA in the filter-binding assays. The
full-length La protein and RRM3 showed significant binding with the
mutant HCV IRES with the apparent Kd of 0.24 and
0.37 µM, respectively. However, the RNA binding with RRM2
was drastically reduced possibly because of lack of contact points on
the RNA (Fig. 5, C and D).
Toe-print Analysis Shows the Contact Points of RRM2 around GCAC
Motif near Initiator AUG of the HCV IRES RNA--
To precisely map the
RRM2 contact points on the HCV IRES RNA, toe-printing assays were
performed. Increasing concentrations of either purified full-length La
protein or deletions were incubated with 5 pmol of in vitro
transcribed RNA corresponding to the HCV IRES (18-383 nt). To this
complex, 32P-end-labeled primer complimentary to 24 nucleotides of the 3' end of the HCV-383 was added and extended using
avian myeloblastosis virus-reverse transcriptase. The resulting
extended products were analyzed on a 6% polyacrylamide, 7 M urea denaturing gel (Fig. 6, panels A and
B). For precise mapping of the contact points, a DNA
sequencing reaction, corresponding to the HCV 383 RNA and using the
same end-labeled primer for priming, was run alongside. The results
demonstrated specific reverse transcriptase pauses (toe-prints) with
the addition of increasing amount of proteins indicating possible
protein binding sites. Four specific toe-prints corresponding to
C-334, A-342, C-355, and A-364 were observed around the
initiator AUG, which showed dose dependence with the addition of
increasing concentrations of full-length La protein (Fig.
6A). RRM2 also showed similar toe-prints; however, prominent pauses were observed at A-342, C-355, and A-364, whereas, the contact
point C-334 was relatively lesser (Fig. 6B). RRM3 did not
show significant increase in the intensity of the toe-prints at these
points. The results suggest that the primer extension was inhibited at
several points around 342-364 nt, perhaps because of binding of RRM2
with this region encompassing the AUG (342-344 nt) and the GCAC motif
(346-349 nt).
La protein interaction with the 5'-UTR has been shown to play a
pivotal role in enhancing IRES-mediated translation of hepatitis C RNA.
In this report we have extended the study and further characterized the
interaction in relation to trans-activation of HCV IRES
function. Previously it was shown that the C-terminal domain of La
protein is responsible for binding with the HCV 5'-UTR, largely because of the presence of a stretch of basic amino acid residues in this region. In contrast, the N-terminal domain was indicated to play a
critical role in interaction with poliovirus 5'-UTR.
In this report, we demonstrate for the first time the in
vivo interaction of HCV IRES with La protein and also show that
both N- and C-terminal halves are able to interact independently with the RNA (Fig. 1). HCV IRES-mediated translation could be stimulated by
independent regions of La protein to some extent but an additive effect
was obtained by supplementing La-N and La-C proteins, suggesting that
both might play a critical role in achieving full stimulatory activity
(Fig. 2). By computational analyses we predicted that RRM2 of La
protein might bind HCV IRES at the GCAC motif near initiator AUG (Figs.
3 and 4), which was confirmed by filter-binding and toe-printing assays
(Figs. 5 and 6).
The binding affinity of full-length La protein for the HCV IRES
(18-383) was found to be significantly high. The major contributor to
the binding is apparently derived from RRM2, as suggested from the
computational modeling data and the filter-binding assay results. RRM2
showed highest affinity (Kd of 0.14 µM) compared with RRM3 (Kd of 0.23 µM). RRM1 failed to show considerable binding affinity
for the HCV IRES. This is consistent with our prediction made based on
structural modeling, as well as the earlier observation that the La
deletion 1-103 (comprising of RRM1 alone) did not interact with the
HCV 5'-UTR. However, the deletion La1-160, where part of RRM2 was
included, showed binding with the HCV 5'-UTR (35).
The region corresponding to amino acids 110-190 of human La protein
represents a canonical RRM designated as RRM2, which is conserved in
diverse organisms. The degree of conservation is consistent with the
idea that RRM2 might contribute to overall affinity of the HCV IRES
RNA. This is also supported by our observations in computational
modeling, filter-binding, and toe-printing assays. Among the three
putative RRMs in the La protein, the sequence pattern in RRM2 is
observed to be the closest to the RRMs of known crystal or NMR
structure. Highly conserved RNA binding regions of known structure
coupled with analysis of RRM-RNA interactions prompts us to predict
that RRM2 of La protein is likely to bind to GCAC motif, which is
located in the HCV IRES. A partially conserved sequence motif, CACAA,
has been observed to be present in several viral RNAs known to interact
with the La protein (35). Our computational analysis revealed the
presence of GCAC motif in all the known complex structures analyzed
that showed structural homology with RRM2 of human La protein.
Interestingly, a GCAC motif (346-349 nt) was detected immediate
downstream of the initiator AUG and a GCAU sequence (337-340 nt) in
the upstream. Primer extension inhibition assay (toe-printing) with the
HCV IRES and La full-length and RRM2 demonstrated specific toe-prints
at the AUG (342 nt) and few nucleotides downstream (355 and 364 nt).
Because this region encompasses the GCAC (346-349 nt), it is
conceivable that the RRM2 might contact the GCAC motif as predicted by
computational modeling. Additionally, in the filter-binding assay when
HCV IRES probe lacking the GCAC motif and AUG (1-341 nt) was used, the RRM2 binding was almost abrogated although it showed highest affinity for binding to HCV IRES RNA (18-383 nt). The results indicate that
perhaps RRM2 does not bind GCAU in absence of AUG. However, the binding
affinity of RRM3 with HCV IRES was only marginally affected in absence
of GCAC and AUG. Additionally, RRM3 did not show any specific pause
sites at the above region. It is possible that RRM3 binds to HCV 5'-UTR
in a location different from the site that binds RRM2. Consistent with
that fact, the full-length La protein was still able to bind to HCV
1-341 RNA probe (lacking AUG and GCAC motif) perhaps through RRM3. The
fact that RRM3 binds to a different region other than RRM2 attests to
the possible role of La protein as RNA chaperone to hold the
RNA-protein complex at the IRES for proper positioning of the 40 S
ribosome onto the initiator AUG.
Although RRM1 does not bind RNA on its own, it contributes
significantly in recognition of UUU-OH-containing RNAs (52). Additionally, it has been demonstrated earlier that deletion of the
N-terminal residues of RRM1 in mutants 22-408 and 26-408 decreased the affinity for binding to HIV leader RNA (29). Analysis of the known
structures of RRMs shows highly similar structures for RRMs bound to
RNA and unbound forms. Consistent with this observation, although RRM2
and RRM3 of La protein may not alter their domain structures
significantly as a result of binding to RNA, it is possible that the
relative orientation between the RRM domains of La could alter because
of gross structural changes to suit the orientation of bound RNA. Thus,
RRM1 might influence the access of RNA to RRM2 and RRM3. Model of the
three-dimensional structure of RRM3 reveals interesting differences in
the RNA binding region. These changes are predicted not to alter the
backbone conformation essential for RNA binding, but may alter the
specificity to the binding base sequence.
In yeast three-hybrid analysis, longer RNA baits sometimes
reduce the reporter gene signal. However, specific interactions have
been detected with RNA baits as long as 1600 nt (58). Keeping in mind
the multiple contact points of La protein within HCV 5'-UTR, to test
the interaction with the truncated La proteins, we preferred to use a
longer RNA (HCV 18-383) as bait rather than using smaller deletions.
We did observe significant increase in Our in vitro translation results showed
dose-dependent stimulation of both HCV and poliovirus
IRES-mediated translation with the addition of increasing concentration
of full-length purified La protein. However, we have deliberately used
a lower range of protein concentrations to monitor the marginal
differences (if any) on the HCV IRES-mediated translation upon addition
of the truncated La proteins. Perhaps because of this reason, in our assay we could observe stimulation only up to ~4-fold (below
saturation level) upon addition of full-length La protein as opposed to
earlier reports, where maximum 6-fold increase was demonstrated (35). Interestingly, addition of La-N or La-C protein alone could not stimulate the HCV IRES-mediated translation to the extent observed with
full-length La protein. However, addition of La-N and La-C together in
the same reaction could achieve ~6.5-fold stimulation of HCV IRES
activity, as expected with full-length La protein. This observation
indicates that perhaps both the terminal halves are involved in some
critical protein interactions. Deletion of either half loses the
contribution of some of the factors in stimulating HCV IRES activity.
The C-terminal half (termed as the effector domain) of La protein has
been shown to possess the dimerization domain, and La homodimerization
is essential for the trans-activation of poliovirus IRES
function (41). The fact that the N-terminal half alone was capable of
stimulating HCV IRES-mediated translation implies that perhaps La
protein dimerization may not be an essential prerequisite for the
HCV-RNA binding activity and trans-activation of HCV
IRES-mediated translation.
HCV IRES requires a limited number of trans-acting factors
as compared with the picornavirus IRES. The dimerization domain in the
C-terminal half might be involved in protein-protein interaction with
other trans-acting factors that could be critical for
poliovirus IRES-mediated translation but may not be required for HCV
IRES-mediated translation. Alternatively, a long helix-rich region has
been predicted near the center of the La protein (59), which is almost equally divided into the N- and C-terminal deletions. The portions of
this predicted helix could also exist in a stable conformation and can
mediate protein-protein interactions. Further experiments are in
progress to distinguish between the possibilities, which would shed
more light on the actual mechanism of the La protein-mediated stimulation of IRES-mediated translation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside
and grown for another 4 h. The cells were pelleted and resuspended in 200 µl of lysis buffer (50 mM
NaH2PO4, 300 mM NaCl, 10 mM imidazole). The extract was made by sonication. The
above crude extracts were mixed with 0.25 volume of Ni-NTA-agarose
slurry (Qiagen) and kept for rocking at 4 °C for 2 h. The
lysate was loaded onto a column, and the flow-through was discarded.
The column was washed with 5 ml of wash buffer (50 mM
NaH2PO4, 300 mM NaCl, 40 mM imidazole). The bound protein was eluted with 500 µl
of elution buffer containing 500 mM imidazole. The eluted
proteins were dialyzed at 4 °C for 4-6 h in 500 ml of dialysis
buffer (50 mM Tris, pH 7.4, 100 mM KCl, 7 mM
-mercaptoethanol, 20% glycerol), aliquoted, and
stored in a
70 °C freezer.
-32P]uridine
triphosphate (PerkinElmer Life Sciences). The pcDHCV-383 and
pcDHCV-341 were linearized with EcoRI, gel eluted, and
transcribed in vitro to generate the 32P-labeled
RNA. The transcription reaction was carried out under standard
conditions (Promega protocol) using 2.5 µg of linear template DNA at
37 °C for 1 h 30 min. After alcohol precipitation, the RNA was
resuspended in 25 µl of nuclease-free water. 1 µl of the
radiolabeled RNA sample was spotted onto DE81 filter paper, washed with
phosphate buffer, and dried, and the incorporated radioactivity was
measured using liquid scintillation counter.
-32P]HCV 18-383
RNA or [
-32P]HCV 1-341 RNA was incubated with the
proteins at 30 °C for 15 min in RNA binding buffer (containing 5 mM HEPES, pH 7.6, 25 mM KCl, 2 mM
MgCl2, 3.8% glycerol, 2 mM dithiothreitol, and
0.1 mM EDTA), and loaded onto nitrocellulose filters
equilibrated with 2 ml of RNA binding buffer. The filters were then
washed twice with 2 ml of binding buffer and dried, and the counts
retained were measured in liquid scintillation counter. The graph was
plotted with protein concentration (nM) on x
axis and the percentage of bound RNA as the percentage of counts
retained, on the y axis. The relative affinity constants were calculated as the protein concentrations at which 50% RNA was bound.
plates were later spotted as different
dilutions onto plates containing 10 mM 3-aminotriazole.
-Galactosidase Assay--
The yeast colonies were
inoculated into 5 ml of selective medium and grown overnight. The cells
were harvested and suspended in 100 µl of Z buffer (60 mM
Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, 1 mM MgSO4, pH 7.0), 1 mM
phenylmethylsulfonyl fluoride, and 1% SDS. Cells were disrupted by
repeated cycles of vortexing and cooling in presence of glass beads.
0.8 ml of Z buffer, 1 mM phenylmethylsulfonyl fluoride, and
1% SDS was added, and 100 µl was set aside for protein estimation.
To the remaining, 0.2 ml of 4 mg/ml ONPG in 200 mM phosphate buffer, pH 7.0, was added and incubated at room temperature until yellow color developed. The reaction was stopped by adding 0.5 ml
of 1 M Na2CO3, and
A420 was measured.
atoms in the template structures resulted in the identification of the structurally conserved regions (SCRs) and structurally variable regions. The mean of the topologically equivalent C
atoms weighted by the square of the sequence identity (42) between the RRM in the La protein and each one of the template structures define the framework for the family of RRM proteins of known
structure. The sequence of an RRM of La was then aligned with the known
structures and the framework. The regions of RRM of the La protein
equivalent to the framework region were modeled from the SCRs of the
template structures. To model a given SCR of RRM-La, the equivalent SCR
in a homologue of known structure with the best local sequence identity
with RRM-La is superimposed on the framework. The variable regions are
modeled by identifying a suitable segment from a known structure in the
data bank. A search is made for a segment having the desired number of
residues and the proper end-to-end distances across the three
"anchor" C
at the either side of the putative loop such that
the loop can be fitted joining the contiguous conserved regions. A
template matching approach (45) to rank the candidate loops was also used. The best ranking loop with no short contacts with the rest of the
protein is fitted using the ring-closure procedure of F. Eisenmenger.2 Side chains are
modeled either by extrapolating from the equivalent positions in the
basis structure where appropriate or by using rules derived from the
analysis of known protein structures (46).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase). Interaction between the bait RNA and the
prey protein results in the reconstitution of
transcriptional activation of the reporter genes (His and
LacZ), changing the phenotype of the yeast cells. Positive
interactions are detected by selection on His
plates and
assaying the
-galactosidase reporter gene activity. For this
purpose, RNA hybrid vector was generated by cloning the HCV IRES
(18-383 nt) into the pRH5' vector (Invitrogen), which produced HCV
IRES as a fusion to MS2 RNA. The protein hybrid vectors were generated
by cloning the full-length La1-408 as well as the deletions, La1-208
(La-N) and La209-408 (La-C) into pYESTrp2 vector, which produces the
above proteins as a fusion to B42 activation domain (Fig.
1A). The pYESTrp2 protein
hybrid vectors were co-transformed with the pRH5'-HCV RNA hybrid vector
into the host S. cerevisiae (L40uraMS2). The colonies
showing histidine (His)-positive phenotype were selected on
His-negative plates. Different dilutions of the transformants were then
assayed for the ability to grow on His-negative plates containing 10 mM 3-amino-1,2,4-triazole (3-AT) for further screening of
specific interaction (Fig. 1B). The transformants were also
assayed for
-galactosidase activity (
-gal), the relative
-galactosidase activities resulting from the interactions are shown
in Fig. 1C. The host alone was taken as background. When pYESTrp2 La1-408 was co-transformed with pRH5'-HCV construct, the
growth of the transformants on His
-3-AT plates and almost
6-fold increase in
-galactosidase activity over the background
indicated that La protein does interact with the HCV IRES in the
in vivo conditions also. To confirm that this interaction
was because of the bridge formed by the MS2-HCV hybrid RNA and the
B42-La protein, several controls were used. MS2-HCV with IRP did not
show considerable growth at higher dilutions on His
-3-AT
plates and there was no significant increase in
-gal activity. As
expected, the positive controls, MS2-IRE and the B42-IRP hybrid showed
strong interaction (Fig. 1C). MS2 alone with B42-La did not
show significant
-gal activity (data not shown).
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Fig. 1.
Yeast three-hybrid system analysis.
A, schematic representation of the full-length La protein
showing the relative positions of the three putative RRMs. The protein
hybrids and RNA hybrid generated in pYESTrp2 and pRH5' vectors,
respectively, are shown. B, three-hybrid yeast strain,
S. cerevisiae L40uraMS2, was transformed with plasmids
carrying different B42 activation domain fusions and MS2RNA fusions.
The transformants were selected on Trp/Ura/His medium and
subsequently tested for the expression of reporter gene activity. The
positive three-hybrid interactions were further confirmed by growth on
the above medium supplemented with 10 mM 3-aminotriazole.
C, the relative
-galactosidase activity of each
transformant is plotted as vertical bars. The
mean and the standard deviations from the three independent experiments
are shown. The cotransformants in each case are indicated
below the bars. IRE-IRP interaction represents
the positive control.
,
10 mM 3-AT) and modest increase (4-5-fold) in
-galactosidase activity over the negative controls, indicating
significant interaction with the HCV IRES RNA (Fig. 1). Additionally,
we have also investigated the La protein binding with the HCV-RNA
in vitro by UV-cross-linking assays using the full-length
and the deletions La1-208 (La-N) and La209-408 (La-C). Results showed
that, in addition to the full-length protein, both the N- and
C-terminal halves of La protein are able to interact with the HCV IRES
in vitro (data not shown).
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Fig. 2.
The effect of exogenous addition of purified
full-length and truncated La proteins on the HCV and poliovirus
IRES-mediated translation. A, 1 µg of capped HCV
bicistronic RNA was translated in RRL and supplemented with increasing
concentrations of either purified His-tagged full-length La1-408 (25, 50, and 75 ng) or La1-208 (12.5, 25, and 37.5 ng) or La209-408 (12.5, 25, and 37.5 ng) or equimolar amounts of La1-208 and La209-408
proteins together (6.25, 12.5, or 18.75 ng each) as indicated. Both the
reporter genes', Renilla luciferase and firefly
luciferase, activities were measured, and the relative luciferase units
were plotted. The open bars represent the firefly
luciferase activity, and the shaded bars
represent the Renilla luciferase activity.
B, a similar experiment was performed with 1 µg of capped
CAT poliovirus Luc bicistronic RNA supplemented with full-length
La1-408 (250, 500, and 750 ng), La1-208 (125, 250, and 375 ng), or
La209-408 (125, 250, and 375 ng) proteins. Open
bars represent firefly luciferase activity, and
shaded bars represent the CAT activity.
-strands, and the other is located around
position 85, which links a
-strand with the C-terminal helix. A
large majority of the interactions between the protein and RNA involve
the polar atoms in the main chain of the polypeptide chain. Thus,
although the RNA interacting regions are predominantly located in the
loops, these are well conserved within the RRM proteins of known
structure (Fig. 4A). It is well known that the length and
conformation of the "equivalent" loops in homologous proteins vary
enormously (42). However, it can be seen in Fig. 3 that, although the
RNA binding regions are located in the loops, no insertion or deletion
is present in the RNA-interacting loops. Although there are many
residues conserved in the
-
loop, the sequence at the
-
loop is conserved as QYAKTDSD. These conservative features are probably
the result of the need-based requirement of RNA binding.
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Fig. 3.
Structure-based sequence alignment.
Sequences of RRM1, -2, and -3 of La protein were aligned along with the
sequences of known structures of RRM proteins. The structural
environments of the residues in the known structures are encoded in the
representation: uppercase, solvent-inaccessible;
lowercase, solvent-accessible; italics, positive
, one of the Ramachandran angles; bold, side-chain
hydrogen bonded to the main-chain amide; underline,
side-chain hydrogen bonded to the main-chain carbonyl. The
numbers within parentheses represent the first
residue of the given protein in a block. Conserved
-helical and
-strand regions are also indicated. Figure was produced using JOY
(60).
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Fig. 4.
Computational analysis of RNA recognition
motifs. A, superposition of four known structures of
RRM-containing proteins bound to RNA. The C trace of the proteins
are shown in different colors after the optimal match of the
C
positions. The consensus GCAC base sequence regions of the bound
RNA in these structures are also shown. High similarity in the region
of RNA bound to the protein can be seen. B and C,
three-dimensional models of RRM2 bound to the RNA with base sequence
GCAC (panel B) and RRM3 (panel
C). Helical and
-strand regions are highlighted in
different colors. Slightly different conformations in the
region of RNA binding of RRM2 and RRM3 can be noticed.
Panels A-C were produced using SETOR (61).
-
loop (around position 50 of 1aud), there is an insertion
involving a Phe residue in RRM1. The loop conformation here is markedly
different from the conserved loop conformation seen in the known
structures. Most importantly, in the C-terminal
-
region of RRM1,
there are three prolyl residues in a stretch of six residues. Clearly,
this stretch is likely to adopt a polyproline conformation, which is
not appropriate in the place of a
-strand as lack of amide group in
three places will preclude proximity of another
-strand to form a
-sheet structure. Obviously, a Pro-rich region cannot be
accommodated in the helix as well that follows the
-strand in
question. If the Pro-rich region forms a loop linking the
-strand
and the following helix, then the polyproline conformation of the loop
is very different from the bent loop conformation seen in the
"equivalent" loop of the homologous proteins of known structure.
Further, the main chain amide is unavailable, in three positions, for
interacting with RNA. Thus, first of all it is not clear whether RRM1
would form the canonical RRM fold. Even if the fold of RRM1 is similar
to that of known RRM folds, the putative RNA-interacting regions are
likely to adopt conformations very different from those in the RRM
proteins of known structure. Thus, the RRM1 of La, if it is capable of binding to RNA, is likely to bind in a novel mode. A three-dimensional structure-based explanation provided here is consistent with the proposals of Sobel and Wolin (56) and Ohndorf et al.
(57).
-
loop, which should alter the conformation of the
loop to some extent. Another putative RNA binding loop (
-
) has no
deletion or insertion involved, and the nature of residues are such
that the backbone conformation of this loop in RRM3 is likely to be
similar to that of the equivalent region in the homologous structures.
Thus, all of the backbone region involved in RNA binding is preserved
except for a deletion in the
-
loop. This may not prevent the RNA
binding, but may accommodate RNA with sequence motifs different from GCAC.
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Fig. 5.
Filter-binding assay to determine the
relative binding affinities of the RRMs. A,
[32P]HCV IRES RNA (18-383 nt) was bound to increasing
concentrations of either full-length or truncated La proteins
corresponding to different RRMs (La1-100 (RRM1), La101-208 (RRM2),
and La209-300 (RRM3)) as indicated on the x axis. The
amount of bound RNA was determined by binding to nitrocellulose filter.
The percentage of bound RNA was plotted against the protein
concentration (nM). B, the linear region of the
curves in panel A were plotted and the apparent
Kd was calculated as the protein concentration at
which 50% of RNA was retained. C, filter-binding assays
were performed as described above using [32P]HCV 5'-UTR
RNA (1-341) and increasing concentrations of either full-length and
deletion La proteins (La101-208 (RRM2) and La209-300 (RRM3)) as
indicated. The percentage of bound RNA was plotted against the protein
concentration (nM). D, the linear regions of the
curves in panel C were plotted and the apparent
Kd was calculated as the protein concentration at
which 50% of RNA was retained.
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Fig. 6.
Primer extension inhibition (toe-printing)
analysis. A, HCV IRES RNA (18-383 nt) was incubated with
increasing amounts (200, 400, and 600 ng) of La full-length protein
(lanes 6-8), as indicated above the
lanes, and analyzed by primer extension. Lane
5 shows the no protein control. The cDNA products
terminated at the sites indicated on the right. The sequence
corresponding to initiator AUG and GCAC is indicated on the
left of the panel. B, similarly HCV IRES RNA
(18-383 nt) was incubated in absence of any protein (lane
5) or presence of increasing concentrations of (200, 400, and 600 ng) of either La100-208 (RRM2) protein (lanes
6-8) or La209-308 (RRM3) protein (lanes
9-11). The major toe-prints are indicated on the
right and the GCAC motif near AUG is marked on the
left of the panel. Lanes 1-4 in each
panel show the DNA sequencing ladder corresponding to the HCV 18-383
RNA obtained by using the same end-labeled primer.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity over
the control, although the signals were not as high as expected for
optimal length of RNA bait. Additionally, to avoid signals from
nonspecific interaction, we have investigated the growth pattern of the
transformed yeast in presence of 10 mM 3-AT and included
various negative controls. In addition, MS2-HCV (18-383) RNA hybrid
did not show any interaction with another RNA-binding protein, IRP,
indicating high specificity of the assay.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Akio Nomoto and Dr. Tsukiyama-Kohara for the HCV 1b-encoding plasmid pCV, and Dr. Jack Keene for the pET-La construct. We gratefully acknowledge Dr. Nahum Sonenberg for the poliovirus bicistronic construct and Dr. Richard M. Elliot and Dr. Aleem Siddiqui for the HCV bicistronic constructs. We are also grateful to Dr. M. S. Shaila for helpful discussion and critical comments on the manuscript.
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FOOTNOTES |
---|
* This work was supported in part by a grant from the Department of Science and Technology, India (to S. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a predoctoral fellowship from the Council of Scientific and Industrial Research, India.
Supported by a Wellcome Trust grant.
** International Senior Fellow of the Wellcome Trust.
To whom correspondence should be addressed. Tel.:
91-80-394-2886; Fax: 91-80-360-2697; E-mail:
sdas@mcbl.iisc.ernet.in.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M210287200
2 F. Eisenmenger, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
HCV, hepatitis C
virus;
UTR, untranslated region;
IRES, internal ribosome entry site;
La, human La antigen;
RRM, RNA recognition motif;
nt, nucleotide(s);
eIF, eukaryotic initiation factor;
HIV, human immunodeficiency virus;
TAR, trans-activation response element;
RRL, rabbit
reticulocyte lysate;
CAT, chloramphenicol acetyltransferase;
SCR, structurally conserved region;
-gal,
-galactosidase, 3-AT,
3-amino-1,2,4-triazole;
Ni-NTA, nickel-nitrilotriacetic acid;
TE, Tris-EDTA.
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REFERENCES |
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1. | Choo, Q.-L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W., and Houghton, M. (1989) Science 244, 359-362[Medline] [Order article via Infotrieve] |
2. | Houghton, M., Weiner, A., Han, J., Kuo, G., and Choo, Q.-L. (1991) Hepatology 14, 381-388[Medline] [Order article via Infotrieve] |
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