A La autoantigen homologue is required for the internal ribosome entry site mediated translation of giardiavirus.
ABSTRACT Translation of Giardiavirus (GLV) mRNA is initiated at an internal ribosome entry site (IRES) in the viral transcript. The IRES localizes to a downstream portion of 5' untranslated region (UTR) and a part of the early downstream coding region of the transcript. Recent studies indicated that the IRES does not require a pre-initiation complex to initiate translation but may directly recruit the small ribosome subunit with the help of a number of trans-activating protein factors. A La autoantigen homologue in the viral host Giardia lamblia, GlLa, was proposed as one of the potential trans-activating factors based on its specific binding to GLV-IRES in vitro. In this study, we further elucidated the functional role of GlLa in GLV-IRES mediated translation in Giardia by knocking down GlLa with antisense morpholino oligo, which resulted in a reduction of GLV-IRES activity by 40%. An over-expression of GlLa in Giardia moderately stimulated GLV-IRES activity by 20%. A yeast inhibitory RNA (IRNA), known to bind mammalian and yeast La autoantigen and inhibit Poliovirus and Hepatitis C virus IRES activities in vitro and in vivo, was also found to bind to GlLa protein in vitro and inhibited GLV-IRES function in vivo. The C-terminal domain of La autoantigen interferes with the dimerization of La and inhibits its function. An over-expression of the C-terminal domain (200-348aa) of GlLa in Giardia showed a dominant-negative effect on GLV-IRES activity, suggesting a potential inhibition of GlLa dimerization. HA tagged GlLa protein was detected mainly in the cytoplasm of Giardia, thus supporting a primary role of GlLa in translation initiation in Giardiavirus.
- SourceAvailable from: ncbi.nlm.nih.gov[Show abstract] [Hide abstract]
ABSTRACT: ncRNAs are key genes in many human diseases including cancer and viral infection, as well as providing critical functions in pathogenic organisms such as fungi, bacteria, viruses, and protists. Until now the identification and characterization of ncRNAs associated with disease has been slow or inaccurate requiring many years of testing to understand complicated RNA and protein gene relationships. High-throughput sequencing now offers the opportunity to characterize miRNAs, siRNAs, small nucleolar RNAs (snoRNAs), and long ncRNAs on a genomic scale, making it faster and easier to clarify how these ncRNAs contribute to the disease state. However, this technology is still relatively new, and ncRNA discovery is not an application of high priority for streamlined bioinformatics. Here we summarize background concepts and practical approaches for ncRNA analysis using high-throughput sequencing, and how it relates to understanding human disease. As a case study, we focus on the parasitic protists Giardia lamblia and Trichomonas vaginalis, where large evolutionary distance has meant difficulties in comparing ncRNAs with those from model eukaryotes. A combination of biological, computational, and sequencing approaches has enabled easier classification of ncRNA classes such as snoRNAs, but has also aided the identification of novel classes. It is hoped that a higher level of understanding of ncRNA expression and interaction may aid in the development of less harsh treatment for protist-based diseases.Frontiers in Genetics 01/2011; 2:96.
A La Autoantigen Homologue Is Required for the Internal
Ribosome Entry Site Mediated Translation of Giardiavirus
Srinivas Garlapati, Ashesh A. Saraiya, Ching C. Wang*
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, United States of America
Translation of Giardiavirus (GLV) mRNA is initiated at an internal ribosome entry site (IRES) in the viral transcript. The IRES
localizes to a downstream portion of 59 untranslated region (UTR) and a part of the early downstream coding region of the
transcript. Recent studies indicated that the IRES does not require a pre-initiation complex to initiate translation but may
directly recruit the small ribosome subunit with the help of a number of trans-activating protein factors. A La autoantigen
homologue in the viral host Giardia lamblia, GlLa, was proposed as one of the potential trans-activating factors based on its
specific binding to GLV-IRES in vitro. In this study, we further elucidated the functional role of GlLa in GLV-IRES mediated
translation in Giardia by knocking down GlLa with antisense morpholino oligo, which resulted in a reduction of GLV-IRES
activity by 40%. An over-expression of GlLa in Giardia moderately stimulated GLV-IRES activity by 20%. A yeast inhibitory
RNA (IRNA), known to bind mammalian and yeast La autoantigen and inhibit Poliovirus and Hepatitis C virus IRES activities
in vitro and in vivo, was also found to bind to GlLa protein in vitro and inhibited GLV-IRES function in vivo. The C-terminal
domain of La autoantigen interferes with the dimerization of La and inhibits its function. An over-expression of the C-
terminal domain (200–348aa) of GlLa in Giardia showed a dominant-negative effect on GLV-IRES activity, suggesting a
potential inhibition of GlLa dimerization. HA tagged GlLa protein was detected mainly in the cytoplasm of Giardia, thus
supporting a primary role of GlLa in translation initiation in Giardiavirus.
Citation: Garlapati S, Saraiya AA, Wang CC (2011) A La Autoantigen Homologue Is Required for the Internal Ribosome Entry Site Mediated Translation of
Giardiavirus. PLoS ONE 6(3): e18263. doi:10.1371/journal.pone.0018263
Editor: Yue Feng, Emory University, United States of America
Received November 18, 2010; Accepted February 28, 2011; Published March 29, 2011
Copyright: ? 2011 Garlapati et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by a National Institutes of Health grant R01 AI-30475. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Recognition of the initiation codon by small ribosomal subunit
is a key step in translation initiation. In eukaryotes, cap-dependent
translation is initiated by the binding of a pre-initiation complex
(the 40S ribosomal subunit combined with eIF1, eIF1A, eIF3 and
eIF2-GTP-tRNA) to the 59 cap of mRNA through an interaction
with eIF4G in the eIF4F complex bound to the cap. This complex
then initiates a downstream scanning along the mRNA for the
initiation codon to begin translation . In an alternative
mechanism, direct binding of a pre-initiation complex (the naked
40S ribosome plus a few protein factors) to the initiation codon is
made possible by highly structured mRNA sequences known as
internal ribosome entry sites (IRESs) [2–4]. IRESs were initially
identified in the 59 untranslated regions (UTRs) of uncapped
messages of picornaviruses . Subsequently, IRESs were also
identified among members of flaviviruses and dicistroviruses .
Recently, numerous capped cellular mRNAs were discovered also
to contain IRESs in their 59-UTRs and shown to utilize IRES
mediated translation initiation when normal cap dependent
translation is severely compromised during conditions of cell
stress, cell cycle, development and diseases .
Most IRESs require only a subset of canonical initiation factors,
whereas others do not require any additional factors to initiate
translation . Some also require a set of non-canonical initiation
factors known as IRES trans-activating factors (ITAFs) [4,6].
Distinctive sets of ITAFs have been indentified with specific types
of viral or cellular IRESs [6,7]. La autoantigen was the first ITAF
identified that stimulated Poliovirus (PV) IRES function both in
vitro and in vivo [8,9]. It was also demonstrated to bind to the
Hepatitis C virus (HCV) IRES near the initiation codon and
stimulate its activity in rabbit reticulocyte lysate . The
functional role of La protein in both viral IRESs was further
confirmed by the inhibitory effects of reducing La protein by
siRNA or by the lack of IRES function in a La dominant negative
mutant, in which a C-terminal domain of La interferes with La
protein dimerization . La also stimulates the IRES activities of
Encephalomyocarditis virus (EMCV) by alleviating the inhibitory
effects of excess polypyrimidine tract binding protein (PTB) in the
cell lysate . For the cellular IRES function, La plays a critical
role in the IRES mediated translation of X-linked inhibitor of
apoptosis  and Bip mRNAs . The La protein thus appears
universally involved in regulating the functions of a variety of
The role of La protein in regulating PV and HCV IRES
function was further elucidated by the identification in Saccharo-
myces cerevisiae of a 60 nt RNA, which has a short hairpin structure
and sequesters La and other RNA binding proteins in yeast cell
. It was referred to as the inhibitory RNA (IRNA) because it
competes with Poliovirus and Hepatitis C virus IRESs for binding
to La protein and inhibits their activity in vitro and in vivo [15–17].
This inherent property of IRNA to bind La protein was attributed
to its specific secondary structure and not due to its primary
PLoS ONE | www.plosone.org1 March 2011 | Volume 6 | Issue 3 | e18263
Giardiavirus (GLV) is a double stranded RNA virus that belongs
to the Totiviridiae family  and specifically infects the vegetative
cells (trophozoites) of the primitive eukaryote Giardia lamblia, which
is a tetraploid . Unlike picornaviruses or flaviviruses, Giardia-
virus does not lyse nor retard the growth of its host G. lamblia
[18,19]. The 6,277 nt Giardiaviral transcript lacks a 59 cap
Figure 1. Effect of a reduced GlLa protein level on GLV-IRES activity. A) Western blot analysis at 48 hrs after the Giardia cells were
transfected with water, control morpholino oligos, or La antisense morpholino oligo. 3XHA-GlLa, expressed under endogenous control, was stained
with the anti-HA antibody whereas anti-a-tubulin antibodies stained tubulins served as loading controls. B) Relative levels of 3XHA-GlLa in transfected
Giardia cells, calculated from densitometric scanning of Western blots from three independent transfection experiments (6 S.D). C) Rluc activities
expressed from 59 cap-dependent and GLV-IRES mediated translations in 48 hrs post-transfected Giardia cells as described in A) (6 S.D). Student t-
test was conducted to calculate P values. P values above 0.05 are considered statistically insignificant, ,0.05 significant, ,0.01 very significant and
,0.005 highly significant. The P values are indicated above each bar for control oligo (compared with water control) and antisense-GlLa oligo (top,
compared with water control; bottom, compared with control oligo).
IRES Mediated Translation in Giardia
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structure and its translation is initiated at an IRES . The IRES
encompasses both a downstream portion of 59UTR and an early
segment of the open reading frame . Several secondary and
tertiary structures of the IRES had been identified. Their potential
roles in IRES function were extensively verified by expressing
transcripts of various GLV- IRES mutants in bicistronic constructs
in Giardia trophozoites [21–23]. The resistance of GLV-IRES
function to the translation initiationinhibitor edeine in vivo indicated
that it does not require recruitment of a pre-initiation complex for
initiating translation . The IRES was also found incapable of
bindingdirectlytothepurified smallribosomalsubunit from Giardia,
suggesting the involvement of certain ITAFs in GLV IRES initiated
translation. Three putative ITAFs have sincebeenidentified by
biochemical and bioinformatics approaches. A member of the
helicase family IBP1 and two homologues of known viral IRES
binding proteins SRp20 and La autoantigen (GlLa) were found to
exhibit specific bindings to GLV IRES RNA in vitro, indicating
potential involvement in the GLV IRES mediated translation .
In the current study, we further investigated the functional role of
GlLa in GLV IRES mediated translation in Giardia.
Knocking down GlLa Inhibits GLV IRES Function
To determine if GlLa plays an essential role in GLV-IRES
mediated translation in Giardia, the endogenous GlLa protein level
was reduced by a custom synthesized antisense morpholino oligo
. The latter was introduced by electroporation into Giardia
trophozoites (see Materials and Methods), in which one of the four
chromosomal copies of the GlLa gene was tagged with a 3XHA
epitope and expressed at the endogenous level [26–28]. Western
analysis of the lysate from the electroporated cells with anti-HA
antibodies indicated that after 48 hrs post-transfection, the GlLa
protein level was reduced by 40% as compared to mock
transfected cells or cells transfected with nonspecific oligos
(Fig. 1A and B). Though this reduced expression was observed
on the tagged GlLa, which constituted only one fourth of the total
GlLa protein, we believe that the data provided an accurate
estimate of reduction of the total GlLa protein.
The translation machinery in Giardia has been found to lack the
mechanism of ribosome scanning . Thus the GLV-IRES
initiated translation of transcripts from uncapped monocistronic
constructs resulted always in the same outcome from that of
dicistronic constructs . The 48-hr knockdown cells from above
were then transfected with an uncapped in vitro transcript from the
monocistronic template pC631Rluc containing a Renilla luciferase
reporter driven by GLV-IRES. The Rluc activity was assayed
5 hrs post-transfection and the outcome showed that the GLV-
IRES mediated translation of Rluc reporter was inhibited by
,40% in the GlLa knockdown cells when compared to the
controls (Fig. 1C). In contrast, cap-dependent translation of the
same reporter gene under the same experimental conditions was
Figure 2. Effect of an over-expressed GlLa protein on GLV-IRES activity. A) Western analysis of GlLa-HA levels in un-induced and
tetracycline-induced Giardia cells 3 hrs after transfection. GlLa-HA was detected by anti-HA antibody. The lower panel shows a-tubulin as the loading
control. B) Relative Rluc expression from 59-cap mediated or GLV-IRES mediated translation initiation in un-induced (black columns) or tetracycline-
induced (grey coulmns) Giardia trophozoites 3 hrs after the previously described transfection. The results were derived from three independent
transfection experiments (6 S.D). P values are indicated above each bar in the graph.
IRES Mediated Translation in Giardia
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not affected by GlLa knockdown, indicating that GlLa does not
play a role in cap-mediated translation in Giardia cells (Fig. 1C).
GlLa Stimulates GLV-IRES Function
HA (hemmaglutinin) tagged GlLa was expressed in Giardia using
a tetracycline inducible Ran promoter system in a plasmid
construct . Western analysis of the lysate from transfected cells
induced with tetracycline for 24 hrs indicated the presence of a
HA tagged GlLa protein, whereas none were detected in the un-
induced cells (Fig. 2A). To determine if the increased level of
tagged GlLa has an effect on GLV-IRES function, the cells over-
expressing HA-tagged GlLa was further transfected with an in vitro
pC631Rluc transcript and assayed for Rluc activity 5–7 hrs post-
transfection. The Rluc activity in Tet-induced cells was 25%
higher than that in the un-induced cells, indicating that a higher
level of GlLa protein has a stimulatory effect on GLV-IRES
mediated translation (Fig. 2B). The cap-dependent translation of
Rluc reporter was not changed in Tet-induced versus un-induced
cells, indicating that GlLa exerts no effect on 59 cap dependent
translation initiation (Fig. 2B).
IRNA binds GlLa in vitro and Inhibits GLV IRES Function in
To further confirm the essential role of GlLa in mediating GLV
IRES function, we used in vitro synthesized yeast IRNA, which has
been shown to inhibit the functions of HCV-IRES and PV-IRES
both in vitro and in vivo by specifically sequestering La protein
[16,17]. In a gel-shift assay, purified recombinant GlLa protein
was shown to bind radiolabeled IRNA and the binding was
competed out by 5 to 20 fold molar excess of unlabeled IRNA but
not by an excess of an unlabeled non-specific RNA of a similar size
(Fig. 3A). We then tested the ability of IRNA to compete with32P-
GLV-IRES for binding to GlLa protein in a gel-shift assay.
Synthetic IRNA was found capable of replacing the radiolabeled
59UTR portion of GLV IRES from binding to recombinant GlLa
protein in a dose dependent manner, whereas a non-specific RNA
of similar size did not exert any detectable effect (Fig. 3B). IRNA
could thus block the binding between GlLa and GLV-IRES in
vitro. To test if the same will happen in vivo, an excessive amount of
IRNA was introduced with the transcript from pC631Rluc into
Giardia cells via transfection and the Rluc activity was assayed after
5 hrs. The results indicated that GLV IRES mediated translation
was reduced by 30% compared to mock transfected or non-
specific RNA transfected cells (Fig. 4). The same experiment
performed on cap-dependent translation of the reporter gene
indicated that IRNA has no detectable effect.
To further confirm that the inhibitory effect of IRNA is due to a
specific sequestering of GlLa protein, the Giardia trophozoites over-
expressing GlLa (Fig. 2A) were transfected with IRNA and the
transcript from pC631Rluc. The inhibitory effect of IRNA on
GLV-IRES-mediated translation of Rluc was abolished in the cells
over-expressing GlLa (Fig. 4), thus reinforcing the conclusion that
GlLa is required for GLV IRES function and that its inhibition by
IRNA is through the interaction of IRNA with GlLa.
An Over-expression of the C-terminal domain of GlLa
Blocks the GlLa Function in Giardia
Human La has been found to function as a dimer. An over-
expression of the C-terminal domain of human La has an
interfering effect on the dimerization and leads to inhibition of
certain viral and cellular IRES mediated translation initiations in
vivo . In order to determine if GlLa also functions as a dimer
and whether the dimerization could be inhibited by its C-terminal
domain in Giardia, a 3XHA tagged GlLa C-terminal domain (aa
200–348) was over-expressed in Giardia using the Tet-inducible
promoter system , and detected as a 21 KDa band in Western
blot analysis after 24 hrs of Tet induction (Fig. 5A). The Tet-
induced and un-induced cells were then transfected with the
transcript from pC631Rluc and assayed for Rluc activity 5 hrs
later. The Rluc activity was reduced by 30% in cells expressing the
GlLa C-terminal domain when compared with the un-induced
cells (Fig. 5B). In contrast, the cap-dependent translation of Rluc
was unaffected by the GlLa C-terminal domain, indicating once
again that GlLa is not involved in a cap-mediated translation
(Fig. 5B). These results indicate that the C-terminal domain of
GlLa has a dominant negative effect on GLV-IRES function,
likely by inhibiting dimerization of GlLa.
GlLa Localizes to the Cytoplasm of Giardia cells
La autoantigen is a nucleo-cytoplasmic protein and is primarily
localized in the nucleus of mammalian and yeast cells . To
determine if GlLa also shares a similar localization pattern in
Figure 3. Yeast IRNA binds to GlLa and competes with the 59 9
UTR portion of GLV-IRES RNA for binding. A) Binding of yeast
IRNA to GlLa (lane 2) was competitively inhibited by 5 to 20 fold excess
of cold yeast IRNA (lanes 3–5) but not by 5 to 20 fold non-specific cold
60 nt RNA (lanes 6–8). B) Binding of radiolabeled 59 UTR of GLV-IRES
RNA (lane 2) was competitively inhibited by 10 to 50-fold excess of cold
yeast IRNA (lanes 3–5), but not by 10 to 50-fold excess of cold 60 nt
non-specific RNA (lanes 6–8).
IRES Mediated Translation in Giardia
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Giardia, one of the four chromosomal copies of GlLa was tagged
with 3XHA was generated using an endogenous tagging method
as described previously [26–28]. When the cells expressing HA-
tagged GlLa were immunostained with anti-HA antibodies and
examined with fluorescence microscopy, the fluorescence was
found primarily localized in the cytoplasm and very little was
found in the nucleus (Fig. 6B), suggesting a primary role of GlLa in
regulating GLV-IRES mediated translation, which takes place in
the cytoplasm. Similar results were obtained when GFP tagged
GlLa was used to observe its localization in the cell (data not
In order to understand the mechanism of translation
initiation at an IRES, it is essential to identify the cellular
trans-acting proteins that bind to it and recruit ribosomes to
initiate the process. Our current study demonstrates that GlLa is
likely an essential auxiliary protein of GLV-IRES mediated
translation. A partial depletion of endogenous GlLa protein
from Giardia led to a corresponding decrease in GLV-IRES
function, whereas an over-expression of GlLa stimulated it.
GlLa binds to yeast IRNA and the latter competitively inhibits
the binding of GlLa to radiolabeled GLV-IRES RNA in vitro. In
Giardia trophozoites, introduction of yeast IRNA inhibited the
function of GLV-IRES, but the inhibition could be reversed by
an over-expression of GILa. All the evidence thus indicates that
GlLa plays an essential role in GLV-IRES mediated translation
Extensive analysis has been done to understand the mechanism
of human La function in IRES mediated translation. It was
demonstrated that the N-terminal half of the protein containing
the La motif, RRM1 and RRM 2, is involved in RNA binding
[30–34], whereas the C-terminal domain is responsible for
dimerization of the La protein . It has been proposed that the
dimer functions as a molecular chaperone by binding the
secondary structures of various IRESs and inducing structural
changes of the IRESs that favor further binding of other initiation
factors and the small ribosome [9,35,36]. A dominant negative
effect was observed in the C-terminal domain of La in preventing
formation of the functional dimer . Our current observation
that an over-expressed C-terminal domain of GlLa has a dominant
negative effect on the GLV-IRES function in Giardia suggests that
GlLa may also first form a dimer via its C-terminal domain and
then act on GLV-IRES .
Although little sequence similarity exists among IRESs,
picornavirus IRESs do appear to share some similarities in their
secondary structures and can be classified into three groups;
enteroviruses and rhinoviruses (Type I IRES elements); cardio-
viruses and apthoviruses (Type II IRES elements); and hepato-
viruses . Free energy minimization modeling suggested that
different types of IRESs could have a common three-dimensional
structure core , providing the structural platform for bindings
of ITAFs to different IRESs [39,40]. GLV-IRES has a highly
complex secondary structure [23,24]. It does not belong to either
Type I or II structure.
Human La is a nuclear protein of 408 amino acid residues
shuttling between the cytoplasm and the nucleus [41,42]. It is
involved in a variety of cellular processes in addition to activating
IRES mediated translation. It protects the 39 ends of nascent pol
III transcripts, ribosomal RNA processing and RNA transport
across the nuclear membrane [43–45]. These features of La are
effectively exploited by some of the viral as well as cellular IRESs.
For poliovirus, the viral protease 3Cprocleaves La at Gln358-
Figure 4. Yeast IRNA inhibits GLV-IRES activity in vivo. Relative Rluc activities in Giardia WB strain trophozoites transfected with Rluc reporter
transcripts driven by 59-cap (A) or GLV-IRES (B) in control (black), presence of the 60 nt non-specific RNA (white) or presence of yeast IRNA (grey). (C)
Relative Rluc activities in Giardia WB cells over-expressing GlLa-HA and transfected with GLV-IRES driven RLuc reporter transcripts in the control
(black), the presence of 60 nt non-specific RNA (white) or the presence of yeast IRNA (grey). The results were derived from three independent
transfection experiments (6 S.D). The P values are indicated above each bar for non-specific RNA (compared with control) and IRNA (top, compared
with control; bottom, compared with non-specific RNA).
IRES Mediated Translation in Giardia
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Gly359 to remove the C-terminal nuclear localization signal of La
and prevents its redistribution to the nucleus . The truncated
La, still retaining the C-terminal dimerization domain, is utilized
for poliovirus IRES mediated translation in the cytoplasm
[11,35,45]. Similarly, UV irradiation causes the redistribution of
La into the cytoplasm where it stimulates IRES mediated
translation of XIAP, a protein involved in preventing apoptosis
. In contrast, our study showed that GlLa localizes primarily in
the cytoplasm of Giardia trophozoite. GlLa is a protein of 348
amino acid residues much smaller than human La and may not
contain a corresponding nuclear targeting signal at the C-terminus
Yeast IRNA has served as an invaluable tool in elucidating the
essential role of human La protein in HCV and poliovirus IRES
mediated translation initiation [15–17]. It was demonstrated that
both sense and antisense sequences of IRNA fold into a similar
secondary structure and exhibit similar binding affinities for La
protein . Thus, La may specifically recognize a secondary
structure of IRNA rather than a specific nucleotide sequence in it.
The same could also apply to the interaction between GlLa and
IRNA. Since IRNA lacks any sequence similarity with GLV-
IRES, the competition between the two in binding to GlLa may
suggest similar secondary structure in the two RNA molecules
recognizing and binding to GlLa. A related question would be
whether Giardia has an IRNA-equivalent in interacting with GlLa
and controlling the function of GLV-IRES. A BLAST of the
Giardia genomic database with IRNA found two homologues. One
(GL50803_6927) has a stretch of 26 bases matching 32 bases in
IRNA (81% identity), whereas the other (GL50803_8865) has 31
bases identical to 44 bases in IRNA (70% identity) (unpublished
work). While there is nothing known about the two homologues,
further study will find out if they have transcripts functioning like
Materials and Methods
Endogenous Tagging of GlLa
Tagging one of the four copies of the endogenous GlLa gene
was carried out  by following the original strategy developed
for Schizosaccharomyces pombe [27,28]. A portion of GlLa gene
lacking the first encoded 30 amino acids was amplified by PCR
and cloned in-frame with a C-terminal 3XHA tag into the
endogenous tagging plasmid pc-3HABSR carrying a blasticidin
(BSR) resistance marker . The resulting construct pc-
GlLa3HABSR was linearized at the Eco47III site located in the
middle of the coding region of GlLa (Figure S1) and electropo-
rated into Giardia WB strain (WB clone C6, ATCC 50803)
trophozoites for homologous recombination between the 39 half of
Figure 5. The dominant negative effect on GLV-IRES activity from over-expressing the C-terminal domain of GlLa. A) Western analysis
with anti-HA antibody of un-induced and tetracycline induced Giardia cells over-expressing the HA-tagged C-terminal domain of GlLa. a-Tubulin was
monitored as the loading control. B) Relative Rluc activities expressed from 59-cap mediated or GLV-IRES mediated translation initiation in un-induced
control (black) and tetracycline-induced (grey) GlLa C-terminal domain over-expression in Giardia trophozoites 3 hrs after transfection. The results
were derived from three independent transfections (6 S.D). P values are indicated above each bar in the graph.
IRES Mediated Translation in Giardia
PLoS ONE | www.plosone.org6March 2011 | Volume 6 | Issue 3 | e18263
the 3X-HA tagged GlLa gene and one of the endogenous GlLa
genes [25,26]. The transfected cells were selected for Blasticidin
resistance (50 mg/mL of blasticidin) and maintained in 100 mg/
mL of blasticidin for further analysis. The expression of the
endogenously integrated 3XHA tagged GlLa was monitored by
Western analysis using mouse anti-HA monoclonal antibody
A Knockdown of GlLa Gene Expression with the
Expression of GlLa gene was post-transcriptionally inhibited
using an anti-sense morpholino oligo essentially as previously
described . A 25mer morpholino-oligonucleotide complemen-
tary to the 9 nts of the 59 UTR and 16 nts of the initial coding
sequence of the GlLa mRNA was custom synthesized (Gene Tools,
LLC). Approximately 30 mL of the 1 mM stock of either the
antisense-GlLa morpholino-oligo or a standard control morpho-
lino-oligo were electroporated into 36106Giardia WB strain
trophozoites expressing endogenously regulated 3XHA-tagged
GlLa in 270 mL of the culture medium to generate a final
morpholino-oligo concentration of 100 mM. The cells were
incubated thereafter for 24, 48 and 72 hrs, respectively, and
analyzed for reduction in the level of 3X-HA-GlLa protein with a
Western analysis using the anti-HA antibody (Sigma).
Gel Shift Assays
The cloning, expression and purification of full-length GlLa-
6XHis fusion protein was described previously . For gel shift
assays, varying amounts of the fusion protein were mixed with
approximately 100 ng of 59-32P-labeled IRNA (see below) in the
binding buffer (20 mM Tris, pH 7.6, 50 mM KCl, 2.5 mM
MgOAc, 0.05% NP40, 1 mM DTT) and incubated at 30uC for
20 min. The RNA-protein complexes were fractionated in a 6%
non-denaturing polyacrylamide gel and visualized by a phoso-
phoimager (Amersham). For competition assays, RNA molecules
with sequence of the multiple cloning site of pGEM-T plasmid was
added as a non-specific competitor. For competition with the
GLV-IRES RNA, the 59UTR portion of GLV-IRES was
synthesized and labeled with
recombinant GlLa protein and competing with unlabeled IRNA
or non-specific RNA.
32P and used for binding to the
In vitro Transcription
The in vitro transcript of pC631Rluc was synthesized using
Megascript T7 transcription kit as described previously . The
59-capped transcript of Renilla luciferase gene with a poly (A) tail
was synthesized from the linearized pRL plasmid using a
MessageMachine T7 transcription kit.
In vitro Synthesis of IRNA
Two complementary oligonucleotide primers containing the
Saccharomyces cerevisiae IRNA sequences  were annealed and
inserted into the ApaI and EcoRI sites of pGEM-T easy vector to
generate the pIRNA plasmid. IRNA transcript was generated
from the linearized pIRNA plamsmid using T7 Megascript
transcription kit and was purified using G-25 spin columns to
remove un-incorporated nucleotides and enzymes. The IRNA
thus synthesized was quantified using NanoDrop 2000 (Thermo-
Transfection of Giardia Trophozoites and the Luciferase
Transient transfection of Giardia WB strain trophozoites was
performed as described previously . The cells were harvested
5 hrs after transfection and the cell lysate was assayed for Rluc
activities using Renilla Luciferase activity kit (Promega).
Over-expression of 3XHA Tagged GlLa and GlLa
C-terminal Domain in Giardia
Genes encoding full-length GlLa or the C-terminal domain
(211–348aa) of GlLa were each amplified from Giardia genomic
DNA with a modification of the stop codon and an incorporation
of a NheI site at the 39-end. The amplified sequence was cloned
into the NcoI and EcoRI sites of pLop2 plasmid . A sequence
encoding 3XHA was inserted at the 39-end of the full-length GlLa
or the GlLa C-terminal domain using the NheI and EcoRI sites.
The two fusion constructs were each moved into a pNLop2-
GitetR plasmid using NheI and SalI sites as described previously
. The final constructs pNlop2-GlLaHA and pNlop2-LaDNHA
were each electroporated into Giardia WB strain trophozoites and
selected with 50 mg/mL of G418. The transfected cells were
treated with 10 mg/mL of tetracycline for 24 hours to induce the
expression of GlLa-3XHA or GlLa C-terminal domain-3XHA,
which was monitored with Western analysis using the anti-HA
antibody (Sigma). Anti-tubulin antibodies (Sigma) were used to
monitor the loading controls.
Giardia WB strain trophozoites expressing HA-tagged full-length
GlLa at the endogenous level were harvested by placing the
culture tubes on ice for 15 min, centrifuging them (2,500 rpm for
10 min.) to pellet the cells, suspending the cells in 1 ml of modified
Figure 6. GlLa-HA localizes in the cytoplasm of Giardia
trophozoites. Immunostaining of A) Wild type WB and B) GlLa-HA
expressing WB Giardia cells with FITC labeled anti-HA antibody and
visualized by Nikon TE2001 microscope.
IRES Mediated Translation in Giardia
PLoS ONE | www.plosone.org7 March 2011 | Volume 6 | Issue 3 | e18263
TYI-S- 33 culture medium, placing the suspension on cover slips
pretreated with 0.1% poly-L-lysine, and incubating at 37uC for
30 min to allow the trophozoites to adhere. The attached cells
were fixed with pre-warmed 4% paraformaldehyde for 30 min at
room temperature and washed three times with PBS. The cells
were then permeabilized with 0.5% Triton X-100 for 15 min at
room temperature, washed three times with PBS, blocked with 5%
BSA in TBS/TNT (20 mM Tris-HCl (pH 7.5), 150 mM NaCl,
0.3% Tween-20, 0.2% NP-40, and 0.05% Triton X-100) for
20 min and washed three times with TBS/TNT. The cells were
then incubated with an Alexa Fluor 488-labeled anti-HA antibody
(1:500 in 0.1% BSA in TBS/TNT) for 60 min at room
temperature and washed three times with TBS/TNT. The cover
slip was then placed facedown on a clean glass slide with 1 drop of
Vectashield mounting media with DAPI and sealed with clear nail
polish. Cells were examined using a Nikon TE2000E motorized
inverted microscope equipped with bright field and epifluores-
cence optics. Images were acquired with the NIS-Elements
Advanced Research software and analyzed with ImageJ.
epitope. GlLa gene lacking the first 100 nts (,30aa) of the ORF
Endogenous tagging of GlLa with a triple HA
is fused in frame at its 39 end with the coding sequence of a triple
HA epitope. The plasmid construct is linearized by Eco 47III at a
unique restriction site located in the GlLa ORF and introduced
into Giardia WB strain trophozoites by electroporation. Homolo-
gous recombination and integration of the linearized vector into
the chromosomal copy of the GlLa gene generates a full length
ORF with a triple HA tag at its 39 end.
We would like to thank Dr. Stephane Gourguechon and Dr. Zacheus
Cande, University of California Berkeley, for providing the plasmid
Conceived and designed the experiments: SG CCW. Performed the
experiments: SG AAS. Analyzed the data: SG CCW. Contributed
reagents/materials/analysis tools: SG AAS CCW. Wrote the manuscript:
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