Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TITLE OF THE INVENTION
PAIP, A NOVEL HUMAN PROTEIN WHICH BRIDGES
THE 3' AND 5' END OF mRNA, AND USE THEREOF AS A TARGET
FOR TRANSLATIONAL MODULATION TO TREAT HUMAN DISEASE
FIELD OF THE INVENTION
The present invention relates to the process of protein
synthesis in eukaryotic cells. More particularly, the present invention
relates to translation and more specifically to the initiation of translation
in such cells. More specifically, the present invention relates to proteins
involved in the initiation of translation in animal cells, to the modulation
of
translation thereby and to the coupling of the 3' and 5' ends mRNA by the
proteins of the present invention. The present invention also relates to
isolated nucleic acid molecules encoding a human protein, a poly(A)
binding Protein-Interacting Protein (PAIP), as well as vectors, host cells
harboring same. In addition, the present invention relates to screening
assays for identifying modulators of PAIP activity.
BACKGROUND OF THE INVENTION
The initiation of translation in eukaryotes involves
binding of the small ribosomal subunit to the mRNA via recognition of the
5' cap structure (m'GpppX, where X is an nucleotide) by the cap binding
complex eIF4F (Merrick et al., 1996, in Translational Control., Eds
Hershey et al. 31-69, Cold Spring Harbor, New York: Cold Spring Harbor
Laboratory Press). eIF4F is a three subunit complex composed of eIF4E,
the cap binding protein (Sonenberg et al., 1979, Proc. Natl. Acad. Sci.
USA 76:4345-4349); eIF4A, an ATP-dependent RNA helicase (Rozen
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et al., 1990, Mol. Cell. Biol. 10:1134-1144); and eIF4G, which interacts
with both eIF4A and eIF4E, and enhances the interaction of eIF4E with
the cap (Haghighat et al., 1997, J. Biol. Chem. 272:21677-21680). eIF4G
has also been suggested to have RNA-binding properties (coyer et al.,
1993, Mol. Cell. Biol. 13:4860-4874).
The mRNA 3' polyadenylate (A) tail and the associated
poly(A) binding protein (PABP) also regulate translation initiation (Munroe
et al., 1990, Mol. Cell. Biol. 10:3441-3455), most likely through an
interaction with the 5' end of the mRNA (Jacobson, 1996, in Translational
Control., supra, 451-480; Sachs et al., 1997, Cell 89:831-838). In yeast,
PABP interacts directly with eIF4G (Tarun et al., 1996, EMBO J.
15:7168-7177; Tarun et al., 1997, Proc. Natl. Acad. Sci. USA
94:9046-9051 ), and a similar interaction was observed in plants (Le et al.,
1997, J. Biol. Chem. 272:16247-16255). No such interaction has been
reported in mammalian cells.
There thus remain a need to assess whether the
interaction between PABP and eIF4G observed in yeast and in plants is
common to all eukaryotes and more particularly to mammalian cells.
While it had been initially envisaged that specific control
of translation inhibition by specific RNA-protein interactions would be
generally limited to interactions at or near the 5' end of mRNA, it has now
become clear that the 3' end of mRNAs plays a significant role in
translation control. Moreover, recent evidence support an important role
for the 3' end of mRNA in the control of initiation of translation (Standart
et al., 1944, Biochimie 76:867-879). It is interesting to note that the
messages which have been shown to demonstrate a cross-talk between
the 3' and 5' end of mRNAs are from generally highly regulated genes,
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which are often involved in maturation, embryogenesis and early
development (Standart et al., 1994, supra). In addition, several mRNAs
encoding cytokines and oncoproteins have been shown to contain
UA-rich (UAR) sequences which can strongly affect their translational
efficiency (Kruys et al., 1994, Biochimie 76:862-866). Non-limiting
examples of such mRNAs include c-fos, c-myc, IFN-~3, GM-CSF, TNF,
IFNs, IL-1, IL-5. IL-10 and IL-13 (Kruys et al., 1994, supra).
Based on the complexity of the interactions operating at
the 5' and 3' end of mRNAs and on the importance of the messages
displaying or suggesting a role for the 3' end thereof on translation
control, the dissection and elucidation of the mechanism which enables
such a bridging between the 3' and 5' end of mRNAs should provide
significant information on gene expression and cellular homeostasis in
general. Furthermore, such a dissection and elucidation could provide
means to relieve (or promote depending on the situation) the translational
repression operating at the 3' end of messages involved in diseases or
conditions. Non-limiting examples of such diseases include cancer and
disease or conditions linked to virus infections or disease or conditions
which display a inflammatory response (Caput et al., 1986, Proc. Natl.
Acad. Sci. 83:1670-1674; Han et al., 1990, European Cytokine Network
1: 71-75; Kruys et al., 1992, Proc. Natl. Acad. Sci. 89:673-677; Han et al.,
1991, J. Immunol. 146:1843-1848). Since a number of viruses target
amongst other things the 3'-5' coupling of the host mRNAs, they can
inhibit the translation thereof. The identification of a bridging agent
between the 3' and 5' end of mRNA might thus permit a relief of the viral
induced translation inhibition.
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There thus remains a need to elucidate how the
coupling of the 3' and 5' ends of mammalian mRNAs is effected and to
understand how this coupling affects gene expression and more
particularly translation initiation andlor mRNA stability.
The diversity of functions which are affected by an
interaction between the 5' and 3' end of mRNAs is not limited to
cytoplasmic functions, such as for example, localization, stability,
decapping, and translation (Decker et al., 1995, Curr. Opinion Cell. Biol.
7:386-392). Indeed, the yeast pPABP has been reported to be localized
to the nucleus (Sachs et al., 1992, Cell 70:461-973; and ibid., 1997,
supra). Thus, the binding of the 5' and 3' ends of mRNA might also impact
fundamental functions in the nucleus. One such example of nucleus
function is nucleo-cytoplasmic transport. Such role in nucleo-cytoplasmic
transport is supported by the nuclear localization of eIF4E in the nucleus
of mammalian cells (Lejbkowicz et al., 1992, Proc. Natl. Acad. Sci.
89:9612-9616).
The present invention seeks to meet these and other
needs.
The present description refers to a number of
documents, the content of which is herein incorporated by reference.
SUMMARY OF THE INVENTION
The invention concerns a novel animal translation factor
termed PABP-Interacting Protein, and more particularly to mammalian
PAIP and especially to human PAIP-1 that exhibits homology to the
central portion of eIF4G, and interacts with eIF4A. More particularly, the
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present invention provides isolated polypeptides having the amino acid
sequences shown in Figure 1 and Figure 6.
The present invention further relates to isolated nucleic
acid molecules comprising polynucleotides which encode a PAIP
polypeptide, and more particularly to isolated nucleic acid molecules
encoding the PAIP polypeptide having the amino acid sequence shown
in Figure 1 and Figure 2.
The invention in addition relates to recombinant vectors
harboring the isolated nucleic acid molecules of the present invention.
More particularly, the invention relates to expression vectors which
express the PAIP polypeptides of the present invention. The present
invention further relates to host cells containing such recombinant vectors
or expression vectors, to methods of making such host cells, and to
methods of making such vectors.
Further, the present invention provides screening assays
and methods for identifying modulators of PAIP activity. More particularly,
the present invention relates to assays and methods for screening and
identifying compounds which can enhance or inhibit the biological activity
of PAIP. In one particular embodiment of the present invention, the
screening assay for identifying modulators of PAIP activity comprises
contacting cells or extracts containing PAIP and a candidate compound,
assaying a cellular response or biological function of PAIP, wherein the
potential modulating compound is selected when the cellular response or
PAIP biological activity in the presence of the candidate compound is
measurably different than in the absence thereof and whereby an
increase in cellular response or PAIP biological activity over the control
without compound indicates that the compound is an agonist while a
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decrease in cellular response or PAIP biological activity indicates that the
compound is an antagonist.
In addition, the present invention relates to methods for
treating an animal (such as a human) in need of a modulation of PAIP
level and/or activity, which comprises administration thereto of a
composition comprising a therapeutically effective amount of PAIP
polypeptide, and for PAIP nucleic acid molecule encoding same, andlor
modulators of PAIP activity.
The invention further relates to the use of polypeptides
of the present invention, andlor modulators of PAIP activity in in vitro
translation systems, and to methods of modulating translation in cells or
extracts thereof comprising an addition of the polypeptides, andlor nucleic
acid molecules, andlor modulators of PAIP activity of the present
invention.
In accordance with the present invention, there is
therefore provided, an isolated PABP-interacting protein (PAIP) exhibiting
homology to eIF4G and interacting with eIF4A.
In accordance with the present invention, there is also
provided, an isolated nucleic acid molecule comprising a polynucleotide
sequence encoding PAIP.
In accordance with another aspect of the present
invention, there is provided, an isolated nucleic acid molecule comprising
a polynucleotide sequence which hybridizes under stringent conditions to
a polynucleotide sequence encoding PAIP-1 or to a sequence which is
complementary thereto.
In accordance with yet another aspect of the present
invention, there is provided a method of constructing a recombinant
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vector which comprises inserting an isolated nucleic acid molecule
encoding PAIP into a vector.
In accordance with a further aspect of the present
invention, there is provided a method of making a recombinant cell
comprising introducing thereinto a recombinant vector harboring a nucleic
acid sequence encoding PAIP.
In accordance with an additional aspect of the present
invention, there is provided an antibody which recognizes specifically a
PAIP polypeptide or derivative thereof.
In accordance with yet an additional aspect of the
present invention, there is provided a method for treating an animal in
need of modulation of PAIP level andlor activity, comprising administering
thereinto a therapeutically effective amount of a PAIP polypeptide, andlor
PAIP encoding nucleic acid molecule andlor PAIP-activity modulator
together with a pharmaceutically acceptable carrier.
In accordance with a further additional aspect of the
present invention, there is provided a method of increasing the
translational efficiency in a cell by increasing the ratio of PAIP to PABP,
comprising introducing in the cell, an effective amount of PAIP
polypeptide or PAIP-encoding nucleic acid molecule.
In accordance with yet a further additional aspect of the
present invention, there is provided a method to uncouple the interaction
between the 3' and 5' ends of an mRNAby targeting the bridging factor
PAIP.
The PAIP polypeptides and nucleic acid molecules of
the instant invention have been isolated from human. Nevertheless, it will
be clear to the person of ordinary skill that the present invention should
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not be so limited. Indeed, using the teachings of the present invention
and those of the art, homologues of PAIP can be identified and isolated
from other animal species. Non-limiting examples thereof include monkey,
mouse, rat, rabbit, and frog.
BRIEF DESCRIPTION OF THE DRAWINGS
Having thus generally described the invention, reference
will now be made to the accompanying drawings, showing by way of
illustration a preferred embodiment thereof, and in which:
Figure 1 a-b show the nucleotide and deduced amino
acid sequence of human PABP-Interacting Protein, PAIP-1.
Figure 2 shows a schematic representation of PAIP-1
and an alignment illustrating the homology with eIF4G. The eIF4E binding
site (Mader et al., 1995, Mol. Cell. Biol. 15:4990-4997) is underlined. Two
recently characterized eIF4A binding sites within eIF4G (Imataka et al.,
1997, Mol. Cell. Biol. 17:6940-6947) are indicated. Alignment of the
amino acid sequences of PAIP-1 and eIF4G was carried out using PIMA
multi-sequence alignment (Baylor College of Medicine Search Launcher).
Identical amino acid residues are boxed and conservative changes are
shaded. Amino acid numbers are shown on the left.
Figure 3 a-d show the interaction of PAIP-1 and PABP.
a) shows that PAIP-1 binds to poly(A) and FLAG monoclonal Antibody
(mAb)- coupled Sepharose in the presence of PABP. Coomassie blue
staining of input His-PAIP-1 (lane 1), FLAG-PABP (lane 2), proteins
bound to poly(A)-Sepharose (lanes 3-5), and to FLAG mAb-Sepharose
(lanes 6-8); b) shows that PAIP-1 and PABP antisera do not cross-react.
Purified His-PAIP-1 and His-PABP were immunoprecipitated with both
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PAIP-1 and PABP-specific antibodies followed by Western blotting with
anti-Xpress antibody (Invitrogen); c) Co-immunoprecipitation of PABP
and eIF4A with PAIP-1 is shown. HeLa S10 extracts were incubated with
affinity purified GST and PAIP-1 specific antibodies or with a PABP mAb,
and Western blotted with different antibodies against the proteins
indicated to the right; and d) shows the mapping of PABP binding site on
PAIP-1. Coomassie blue staining (top panel) of purified GST (lane 2) and
GST-PAIP-1 fusion proteins (lanes 3-9). FLAG-PABP protein (lane 1,
bottom panel) was pre-incubated with GST and GST-PAIP-1 fusion
proteins, precipitated with glutathione-Sepharose beads and detected by
Western blotting.
Figure 4 a-c show that PAIP-1 overexpression enhances
translation. COS-7 cells were transiently co-transfected with a luciferase
reporter cDNA and either pcDNA3 alone, or the indicated amounts of
pcDNA3-PAIP-1 (wt) or pcDNA3-PAIP-1 (~Ct) plasmids; a) Luciferase
activity levels are shown. The mean value for 6 assays in three
independent transfections is shown with the standard deviation as error
bars; b) shows a representative RNase protection assay. Antisense actin
and luciferase probes (lanes 1 and 2) were tested against tRNA (lane 3),
and RNA from mock (lane 4) or transfected cells (lanes 5-7), as described
below. The positions of the full length probes and protected fragments are
indicated on the right; and c) shows that Wild-type PAIP-1 and PAIP-1
(OCt) proteins are expressed to similar levels in COS-7 cells. Protein
extracts from transfected cells were subjected to SDS-10% PAGE
followed by Western blotting with PAIP-1 antisera. Molecular mass
markers are shown on the left in kDa.
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Figure 5 a-b show a model for the bridging of 5' and 3'
mRNA ends in eukaryotes; a) shows the association of PAIP-1 with eIF4A
with PABP linking the mRNA termini in animal cells; b) shows a direct
interaction between PABP and eIF4G in yeast and plant cells (adapted
5 from Sachs et al., 1997, Trends Biochem. Sci. 22:189-192). The position
of the cap structure, initiator methionine codon (AUG), and poly(A) tail are
indicated in boxes. eIF4E is shown binding the cap structure. eIF4G
enhances binding of eIF4E to the cap via interaction with the mRNA
(Haghighat et al., 1997, supra). eIF4A recycles through the cap binding
10 complex and is thought to act together with eIF4B in mRNA unwinding
(Merrick et al., 1996, supra; Rozen et al., 1990, supra).
Figure 6 shows an alignment of the amino acid
sequence of PAIP-1 with that of human eIF4Gl (4G1) and human el4Gll
(4G11). The alignment was carried out as mentioned previously. Once
again,ldentical amino acid residues are boxed and conservative changes
are shaded. Amino acid numbers are shown on the left.
Figure 7 shows a further alignment of the amino acid
sequence of PAIP-1 with that of human eIF4Gl (4G1), human el4Gll
(4G11)) p97, and yeast eIF4G (TIF 4631 and TIF4632). The alignment was
carried out as mentioned for Figure 2. Once again, identical amino acid
residues are boxed and conservative changes are shaded. Amino acid
numbers are shown on the left.
Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
non-restrictive description of preferred embodiments with reference to the
accompanying drawing which is exemplary and should not be interpreted
as limiting the scope of the present invention.
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DESCRIPTION OF THE PREFERRED EMBODIMENT
A polypeptide which interacts with PABP, exhibits
homology to the central portion of eIF4G and interacts with eIF4A has
thus been identified in animal cells and termed PAIP. The nucleic acid
and amino acid sequences of a human PAIP (PAIP-1 ) is described herein
below and the functional role of this factor assessed.
Nucleotide sequences are presented herein by single
strand, in the 5' to 3' direction, from left to right, using the one letter
nucleotide symbols as commonly used in the art and in accordance with
the recommendations of the IUPAC-IUB Biochemical Nomenclature
Commission.
Unless defined otherwise, the scientific and
technological terms and nomenclature used herein have the same
meaning as commonly understood by a person of ordinary skill to which
this invention pertains. Generally, the procedures for cell cultures,
infection, molecular biology methods and the like are common methods
used in the art. Such standard techniques can be found in reference
manuals such as for example Sambrook et al. (1989, Molecular Cloning
- A Laboratory Manual, Cold Spring Harbor Laboratories) and Ausubel et
al. (1994, Current Protocols in Molecular Biology, Wiley, New York).
The present description refers to a number of routinely
used recombinant DNA (rDNA) technology terms. Nevertheless,
definitions of selected examples of such rDNA terms are provided for
clarity and consistency.
As used herein, "nucleic acid molecule", refers to a
polymer of nucleotides. Non-limiting examples thereof include DNA (i.e.
genomic DNA, cDNA) and RNA molecules (i.e. mRNA). The nucleic acid
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molecule can be obtained by cloning techniques or synthesized. DNA can
be double-stranded or single stranded (coding strand or non-coding
strand [antisense]).
The term "isolated nucleic acid molecule" refers to a
nucleic acid molecule purified from its natural environment. Non-limiting
examples of an isolated nucleic acid molecule is a DNA sequence
inserted into a vector, and a partially purified polynucleotide sequence in
solution.
The term "recombinant DNA" as known in the art refers
to a DNA molecule resulting from the joining of DNA segments. This is
often referred to as genetic engineering.
The term "DNA segment", is used herein, to refer to a
DNA molecule comprising a linear stretch or sequence of nucleotides.
This sequence when read in accordance with the genetic code, can
encode a linear stretch or sequence of amino acids which can be referred
to as a polypeptide, protein, protein fragment and the like.
The terminology "amplification pair" refers herein to a
pair of oligonucleotides (oligos) of the present invention, which are
selected to be used together in amplifying a selected nucleic acid
sequence by one of a number of types of amplification processes,
preferably a polymerise chain reaction. Other types of amplification
processes include ligase chain reaction, strand displacement
amplification, or nucleic acid sequence-based amplification, as explained
in greater detail below. As commonly known in the art, the oligos are
designed to bind to a complementary sequence under selected
conditions.
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The nucleic acid (i.e. DNA or RNA) for practicing the
present invention may be obtained according to well known methods.
As used herein, the term "physiologically relevant" is
meant to describe interactions which can modulate transcription of a gene
in its natural setting.
Oligonucleotide probes or primers of the present
invention may be of any suitable length, depending on the particular
assay format and the particular needs and targeted genomes employed.
In general, the oligonucleotide probes or primers are at least 12
nucleotides in length, preferably between 15 and 24 molecules, and they
may be adapted to be especially suited to a chosen nucleic acid
amplification system. As commonly known in the art, the oligonucleotide
probes and primers can be designed by taking into consideration the
melting point of hydrizidation thereof with its targeted sequence (see
below and in Sambrook et al., 1989, Molecular Cloning - A Laboratory
Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1989, in Current
Protocols in Molecular Biology, John Wiley & Sons Inc., N.Y.).
The terms "DNA oligonucleotide", or "DNA molecule" or
"DNA sequence" refer to a molecule comprised of the
deoxyribonucleotides adenine (A), guanine (G), thymine (T) andlor
cytosine (C). Oligonucleotide or DNA can be found in linear DNA
molecules or fragments, viruses, plasmids, vectors, chromosomes or
synthetically derived DNA.
"Nucleic acid hybridization" refers generally to the
hybridization of two single-stranded nucleic acid molecules having
complementary base sequences, which under appropriate conditions will
form a thermodynamically favored double-stranded structure. Examples
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of hybridization conditions can be found in the two laboratory manuals
referred above (Sambrook et al., 1989, supra and Ausubel et al., 1989,
supra) and are commonly known in the art. In the case of a hybridization
to a nitrocellulose filter, as for example in the well known Southern
blotting procedure, a nitrocellulose filter can be incubated overnight at
65°C with a labeled probe in a solution containing high salt ( 5 x SSC
or
5 x SSPE), 5 x Denhardt's solution, 1 % SDS, and 100 pg/ml denatured
carried DNA ( i.e. salmon sperm DNA). The non-specifically binding
probe can then be washed off the filter by several washes in 0.2 x
SSCI0.1 % SDS at a temperature which is selected in view of the desired
stringency: room temperature (low stringency), 42°C (moderate
stringency) or 65°C (high stringency). The selected temperature is
based
on the melting temperature (Tm) of the DNA hybrid. Of course, RNA-DNA
hybrids can also be formed and detected. In such cases, the conditions
of hybridization and washing can be adapted according to well known
methods by the person of ordinary skill. Stringent conditions will be
preferably used (Sambrook et al.,1989, supra). As well known in the art
other stringent hybridization conditions can be used (i.e. 42°C in the
presence of 50% of formamide).
Probes of the invention can be utilized with naturally
occurring sugar-phosphate backbones as well as modified backbones
including phosphorothioates, dithionates, alkyl phosphonates and
a-nucleotides and the like. Modified sugar-phosphate backbones are
generally taught by Miller, 1988 (Ann. Reports Med. Chem. 23:295) and
Moran et al., 1987 (Nucl. Acids Res., 14:5019). Probes of the invention
can be constructed of either ribonucleic acid (RNA) or deoxyribonucleic
acid (DNA), and preferably of DNA.
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The types of detection methods in which probes can be
used include Southern blots (DNA detection), dot or slot blots (DNA,
RNA), and Northern blots (RNA detection). Although less prepared,
labeled proteins could also be used to detect a particular nucleic acid
5 sequence to which it binds. Other detection methods include kits
containing probes on a dipstick setup and the like.
Although the present invention is not specifically
dependent on the use of a label for the detection of a particular nucleic
acid sequence, such a label might be beneficial, by increasing the
10 sensitivity of the detection. Furthermore, it enables automation. Probes
can be labeled according to numerous well known methods (Sambrook
et al., 1989, supra). Non-limiting examples of labels include 3H, '4C, 3zP,
and 35S. Non-limiting examples of detectable markers include ligands,
fluorophores, chemiluminescent agents, enzymes, and antibodies. Other
15 detectable markers for use with probes, which can enable an increase in
sensitivity of the method of the invention, include biotin and
radionucleotides. It will become evident to the person of ordinary skill that
the choice of a particular label dictates the manner in which it is bound to
the probe.
As commonly known, radioactive nucleotides can be
incorporated into probes of the invention by several methods. Non-limiting
examples thereof include kinasing the 5' ends of the probes using gamma
szP ATP and polynucleotide kinase, using the Klenow fragment of Pol I of
E, coli in the presence of radioactive dNTP (i.e. uniformly labeled DNA
probe using random oligonucleotide primers in low-melt gels), using the
SP6IT7 system to transcribe a DNA segment in the presence of one or
more radioactive NTP, and the like.
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As used herein, "oligonucleotides" or "oligos" define a
molecule having two or more nucleotides (ribo or deoxyribonucleotides).
The size of the oligo will be dictated by the particular situation and
ultimately on the particular use thereof and adapted accordingly by the
person of ordinary skill. An oligonucleotide can be synthetised chemically
or derived by cloning according to well known methods.
As used herein, a "primer" defines an oligonucleotide
which is capable of annealing to a target sequence, thereby creating a
double stranded region which can serve as an initiation point for DNA
synthesis under suitable conditions.
Amplification of a selected, or target, nucleic acid
sequence may be carried out by a number of suitable methods. See
generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25. Numerous
amplification techniques have been described and can be readily adapted
to suit particular needs of a person of ordinary skill. Non-limiting examples
of amplification techniques include polymerase chain reaction (PCR),
ligase chain reaction (LCR), strand displacement amplification (SDA),
transcription-based amplification, the Q[3 replicase system and NASBA
(Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et
al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods Mol.
Biol., 28:253-260; and Sambrook et al., 1989, supra). Preferably,
amplification will be carried out using PCR. ,
Polymerase chain reaction (PCR) is carried out in
accordance with known techniques. See, e.g., U.S. Pat. Nos. 4,683,195;
4,683,202; 4,800,159; and 4,965,188 (the disclosures of all three U.S.
Patent are incorporated herein by reference). In general, PCR involves,
a treatment of a nucleic acid sample (e.g., in the presence of a heat
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stable DNA polymerase) under hybridizing conditions, with one
oligonucleotide primer for each strand of the specific sequence to be
detected. An extension product of each primer which is synthesized is
complementary to each of the two nucleic acid strands, with the primers
sufficiently complementary to each strand of the specific sequence to
hybridize therewith. The extension product synthesized from each primer
can also serve as a template for further synthesis of extension products
using the same primers. Following a sufficient number of rounds of
synthesis of extension products, the sample is analysed to assess
whether the sequence or sequences to be detected are present.
Detection of the amplified sequence may be carried out by visualization
following EtBr staining of the DNA following gel electrophores, or using
a detectable label in accordance with known techniques, and the like. For
a review on PCR techniques (see PCR Protocols, A Guide to Methods
and Amplifications, Michael et al. Eds, Acad. Press, 1990).
Ligase chain reaction (LCR) is carried out in accordance
with known techniques (Weiss, 1991, Science 254:1292). Adaptation of
the protocol to meet the desired needs can be carried out by a person of
ordinary skill. Strand displacement amplification (SDA) is also carried out
in accordance with known techniques or adaptations thereof to meet the
particular needs (Walker et al., 1992, Proc. Natl. Acad. Sci. USA
89:392-396; and ibid., 1992, Nucleic Acids Res. 20:1691-1696.
As used herein, the term "gene" is well known in the art
and relates to a nucleic acid sequence defining a single protein or
polypeptide. A "structural gene" defines a DNA sequence which is
transcribed into RNA and translated into a protein having a specific amino
acid sequence thereby giving rise the a specific polypeptide or protein. It
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will readily recognized by the person of ordinary skill, that the nucleic acid
sequence of the present invention can be incorporated into anyone of
numerous established kit formats which are well known in the art.
A "heterologous" (i.e. a heterologous gene) region of a
DNA molecule is a subsegment segment of DNA within a larger segment
that is not found in association therewith in nature. The term
"heterologous" can be similarly used to define two polypeptidic segments
not joined together in nature. Non-limiting examples of heterologous
genes include reporter genes such as luciferase, chloramphenicol acetyl
transferase, ~i-galactosidase, and the like which can be juxtaposed or
joined to heterologous control regions or to heterologous polypeptides.
The term "vector" is commonly known in the art and
defines a plasmid DNA, phage DNA, viral DNA and the like, which can
serve as a DNA vehicle into which DNA of the present invention can be
cloned. Numerous types of vectars exist and are well known in the art.
The term "expression" defines the process by which a
structural gene is transcribed into mRNA (transcription), the mRNA is then
being translated (translation) inta one polypeptide (or protein) or more.
The terminology "expression vector" defines a vector or
vehicle as described above but designed to enable the expression of an
inserted sequence following transformation into a host. The cloned gene
(inserted sequence) is usually placed under the control of control element
sequences such as promoter sequences. The placing of a cloned gene
under such control sequences is often refered to as being operably linked
to control elements or sequences.
Operably linked sequences may also include two
segments that are transcribed onto the same RNA transcript. Thus, two
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sequences, such as a promoter and a "reporter sequence" are operably
linked if transcription commencing in the promoter will produce an RNA
transcript of the reporter sequence. In order to be "operably linked" it is
not necessary that two sequences be immediately adjacent to one
another.
Expression control sequences will vary depending on
whether the vector is designed to express the operably linked gene in a
prokaryotic or eukaryotic host or both (shuttle vectors) and can
additionally contain transcriptional elements such as enhancer elements,
termination sequences, tissue-specificity elements, andlor translational
initiation and termination sites. Typically, expression vectors are
prokaryote specific or eukaryote specific although shuttle vectors are also
widely available.
Prokaryotic expression are useful for the preparation of
large quantities of the protein encoded by the DNA sequence of interest.
This protein can be purified according to standard protocols that take
advantage of the intrinsic properties thereof, such as size and charge (i.e.
SDS gel electrophoresis, gel filtration, centrifugation, ion exchange
chromatography...). In addition, the protein of interest can be purified via
affinity chromatography using polyclonal or monoclonal antibodies. The
purified protein can be used for therapeutic applications.
The DNA construct can be a vector comprising a
promoter that is operably linked to an oligonucleotide sequence of the
present invention, which is in turn, operably linked to a heterologous
gene, such as the gene for the luciferase reporter molecule. "Promoter"
refers to a DNA regulatory region capable of binding directly or indirectly
to RNA polymerase in a cell and initiating transcription of a downstream
CA 02227508 1998-04-O1
(3' direction) coding sequence. For purposes of the present invention, the
promoter is bound at its 3' terminus by the transcription initiation site and
extends upstream (5' direction) to include the minimum number of bases
or elements necessary to initiate transcription at levels detectable above
5 background. Within the promoter will be found a transcription initiation
site
(conveniently defined by mapping with S1 nuclease), as well as protein
binding domains (consensus sequences) responsible for the binding of
RNA polymerase. Eukaryotic promoters will often, but not always, contain
"TATA" boxes and "CCAT" boxes. Prokaryotic promoters contain
10 Shine-Dalgarno sequences in addition to the -10 and -35 consensus
sequences.
As used herein, the designation "functional derivative"
denotes, in the context of a functional derivative of a sequence whether
an nucleic acid or amino acid sequence, a molecule that retains a
15 biological activity (either function or structural) that is substantially
similar
to that of the original sequence. This functional derivative or equivalent
may be a natural derivatives or may be prepared synthetically. Such
derivatives include amino acid sequences having substitutions, deletions,
or additions of one or more amino acids, provided that the biological
20 activity of the protein is conserved. The same applies to derivatives of
nucleic acid sequences which can have substitutions, deletions, or
additions of one or more nucleotides, provided that the biological activity
of the sequence is generally maintained. When relating to a protein
sequence, the substituting amino acid as chemico-physical properties
which are similar to that of the substituted amino acid. The similar
chemico-physical properties include, similarities in charge, bulkiness,
hydrophobicity, hydrophylicity and the like. The term "functional
CA 02227508 1998-04-O1
21
derivatives" is intended to include "functional fragments", "functional
segments", "functional variants", "functional analogs" or "functional
chemical derivatives" of the subject matter of the present invention.
"Fragments" of the nucleic acid molecules according to the present
invention refer to such molecules having at least 12 nt, more particularly
at least 18 nt, and even more preferably at least 24 nt which have utility
as diagnostic probes and/or primers. It will become apparent to the
person of ordinary skill that larger fragments of 100 nt, 1000 nt, 2000 nt
and more also find utility in accordance with the present invention.
The term "at least 24 nt" is meant to refer to 24
contiguous nt of a chosen sequence such as shown for example in
Figure 1.
The term "functional variant" refers herein to a protein
or nucleic acid molecule which is substantially similar in structure and
biological activity to the protein or nucleic acid of the present invention.
The functional derivatives of the present invention can
be synthesized chemically or produced through recombinant DNA
technology, all these methods are well known in the art.
The term "molecule" is used herein in a broad sense and
is intended to include natural molecules, synthetic molecules, and mixture
of natural and synthetic molecules. The term "molecule" is also meant to
cover a mixture of more than one molecule such as for example pools or
libraries of molecules. Non-limiting examples of molecules include
chemicals, biological macromolecules, cell extracts and the like. The term
"compound" is used herein interchangeably with molecule and is similarly
defined.
CA 02227508 1998-04-O1
22
Nucleic acid fragments in accordance with the present
invention include epitope-encoding portions of the polypeptides of the
invention. Such portions can be identified by the person of ordinary skill
using the nucleic acid sequences of the present invention in accordance
with well known methods. Such epitopes are useful in raising antibodies
that are specific to the polypeptides of the present invention. The
invention also provides nucleic acid molecules which comprise
polynucleotide sequences capable of hybridizing under stringent
conditions to the polynucleotide sequences of the present invention or to
portions thereof.
The term hybridizing to a "portion of a polynucleotide
sequence" refers to a polynucleotide which hybridizes to at least 12 nt,
more preferably at least 18 nt, even more preferably at least 24 nt and
especially to about 50 nt of a polynucleotide sequence of the present
invention.
The present invention further provides isolated nucleic
acid molecules comprising a polynucleotide sequences which is at least
95% identical, and preferably from 96% to 99% identical to the
polynucleic acid sequence encoding the full length PAIP polypeptide or
fragments andlor derivatives thereof. Methods to compare sequences
and determine their homologylidentity are well known in the art.
As used herein, "chemical derivatives" is meant to cover
additional chemical moieties not normally part of the subject matter of the
invention. Such moieties could affect the physico-chemical characteristic
of the derivative (i.e. solubility, absorption, half life and the like,
decrease
of toxicity). Such moieties are examplified in Remington's Pharmaceutical
CA 02227508 1998-04-O1
23
Sciences (1980). Methods of coupling these chemical-physical moieties
to a polypeptide are well known in the art.
The term "allele" defines an alternative form of a gene
which occupies a given locus on a chromosome.
As commonly known, a "mutation" is a detectable
change in the genetic material which can be transmitted to a daughter
cell. As well known, a mutation can be, for example, a detectable change
in one or more deoxyribonucleotide. For example, nucleotides can be
added, deleted, substituted for, inverted, or transposed to a new position.
Spontaneous mutations and experimentally induced mutations exist. The
result of a mutations of nucleic acid molecule is a mutant nucleic acid
molecule. A mutant polypeptide can be encoded from this mutant nucleic
acid molecule.
As used herein, the term "purified" refers to a molecule
having been separated from a cellular component. Thus, for example, a
"purified protein" has been purified to a level not found in nature. A
"substantially pure" molecule is a molecule that is lacking in all other
cellular components.
The term "isolated polypeptide" refers to a polypeptide
removed from its natural environment. Non-limiting examples of isolated
polypeptides include a polypeptide produced recombinantly in a host cell
and partially or substantially purified polypeptides from such host cells.
The polypeptides of the present invention comprise polypeptides encoded
by the nucleic acid molecules of the present invention, as shown for
example in Figure 1. The present invention also provides polypeptides
comprising amino acids sequences which are at least 95% homologous,
preferably from 96-99% homologous, even more preferably at least 95%
CA 02227508 1998-04-O1
24
identical and especially preferably from 96% to 99% identical to the full
length PAIP polypeptide sequence or fragments or derivatives thereof.
As used herein, the terms "molecule", "compound" or
"ligand" are used interchangeably and broadly to refer to natural,
synthetic or semi-synthetic molecules or compounds. The term "molecule"
therefore denotes for examples chemicals, macromolecules, cell or tissue
extracts (from plants or animals} and the like. Non-limiting examples of
molecules include nucleic acid molecules, peptides, antibodies,
carbohydrates and pharmaceutical agents. The agents can be selected
and screened by a variety of means including random screening, rational
selection and by rational design using for example protein or ligand
modelling methods such as computer modelling. The terms "rationally
selected" or "rationally designed" are meant to define compounds which
have been chosen based on the configuration of the interaction domains
of the present invention. As will be understood by the person of ordinary
skill, macromolecules having non-naturally occurring modifications are
also within the scope of the term "molecule". For example,
peptidomimetics, well known in the pharmaceutical industry and generally
referred to as peptide analogs can be generated by modelling as
mentioned above. Similarly, in a preferred embodiment, the polypeptides
of the present invention are modified to enhance their stability. It should
be understood that in most cases this modification should not alter the
biological activity of the interaction domain. The molecules identified in
accordance with the teachings of the present invention have a therapeutic
value is diseases or conditions in which the physiology or homeastasis of
the cell andlor tissue is compromised by a defect in in modulating gene
expression andlor translation. Alternatively, the molecules identified in
CA 02227508 1998-04-O1
accordance with the teachings of the present invention find utility in the
development of more efficient cell lines or cell extracts for translating
mRNAs.
As used herein, agonists and antagonists of translation
5 activity also include potentiators of known compounds with such agonist
or antagonist properties. In one embodiment, agonists can be detected
by contacting the indicator cell with a compound or mixture or library of
molecules, for a fixed period of time, and then determining the effect of
the compound on the cell.
10 The level of gene expression of the reporter gene (e.g.
the level of luciferase, or (3-gal, produced) within the treated cells can be
compared to that of the reporter gene in the absence of the molecule(s).
The difference between the levels of gene expression indicates whether
the molecules) of interest agonizes the aforementioned interaction. The
15 magnitude of the level of reporter gene product expressed (treated vs.
untreated cells) provides a relative indication of the strength of that
molecules) as an agonist. The same type of approach can also be used
in the presence of an antagonist(s).
Alternatively, an indicator cell in accordance with the
20 present invention can be used to identify antagonists. For example, the
test molecule or molecules are incubated with the host cell in conjunction
with one or more agonists held at a fixed concentration. An indication and
relative strength of the antagonistic properties of the molecules) can be
provided by comparing the level of gene expression in the indicator cell
25 in the presence of the agonist, in the absence of test molecules vs in the
presence thereof. Of course, the antagonistic effect of a molecule can
also be determined in the absence of agonist, simply by comparing the
CA 02227508 1998-04-O1
26
level of expression of the reporter gene product in the presence and
absence of the test molecule(s).
It shall be understood that the "in vivo" experimental
model can also be used to carry out an "in vitro" assay. For example,
cellular extracts from the indicator cells can be prepared and used in one
of the aforementioned "in vitro" tests (such as binding assays or in vitro
translations).
As used herein the recitation "indicator cells" refers to
cells that express PAIP and PABP andlor eIF4A andlor eIF3 interacting,
and wherein an interaction between these domains is coupled to an
identifiable or selectable phenotype or characteristic such that it provides
an assessment of the interaction between these domains. Such indicator
cells can be used in the screening assays of the present invention. In
certain embodiments, the indicator cells have been engineered so as to
express a chosen derivative, fragment, homolog, or mutant of PAIP (i.e.
the PABP, eIF4A interacting domains). The cells can be yeast cells or
higher eukaryotic cells such as mammalian cells (WO 96/41169). In one
particular embodiment, the indicator cell is a yeast cell harboring vectors
enabling the use of the two hybrid system technology, as well known in
the art (Ausubel et al., 1994, supra) and can be used to test a compound
or a library thereof. In one embodiment, a reporter gene encoding a
selectable marker or an assayable protein can be operably linked to a
control element such that expression of the selectable marker or
assayable protein is dependent on the interaction of the a PAIP domains
with its binding partner (i.e. PABP, eIF4A). Such an indicator cell could be
used to rapidly screen at high-throughput a vast array of test molecules.
In a particular embodiment, the reporter gene is luciferase or ~i-Gal.
CA 02227508 1998-04-O1
27
As exemplified herein below in one embodiment, at least
one of PAIP domain may be provided as a fusion protein. The design of
constructs therefor and the expression and production of fusion proteins
are exemplified herein and are well known in the art (Sambrook et al.,
1989, supra; and Ausubel et al., 1994, supra). In a particular embodiment,
both the PABP interaction domain of PAIP and PABP are part of fusion
proteins. For example, in a particular embodiment, the fusions are a
LexA-PAIP fusion (DNA-binding domain - PAIP; bait) and a B42-PABP
fusion (transactivator domain - PABP; prey). In still a particularly preferred
embodiment, the LexA-PAIP and B42-PABP fusion proteins are
expressed in a yeast cell also harboring a reporter gene operably linked
to a LexA operator andlor LexA responsive element.
Non-limiting examples of such fusion proteins include a
hemaglutinin fusions and Gluthione-S-transferase (GST) fusions, HIS
fusions, FLAG fusions, and Maltose binding protein (MBP) fusions. In
certain embodiments, it might be beneficial to introduce a protease
cleavage site between the two polypeptide sequences which have been
fused. Such protease cleavage sites between two heterologously fused
polypeptides are well known in the art.
In certain embodiments, it might also be beneficial to
fuse the interaction domains of the present invention to signal peptide
sequences enabling a secretion of the fusion protein from the host cell.
Signal peptides from diverse organisms are well known in the art.
Bacterial OmpA and yeast Suc2 are two non-limiting examples of proteins
containing signal sequences. In certain embodiments, it might also be
beneficial to introduce a linker (commonly known) between the interaction
domain and the heterologous polypeptide portion. Such fusion protein find
CA 02227508 1998-04-O1
28
utility in the assays of the present invention as well as for purification
purposes, detection purposes and the like.
For certainty, the sequences and polypeptides useful to
practice the invention include without being limited thereto mutants,
homologs, subtypes, alleles and the like. It shall be understood that
generally, the sequences of the present invention should encode a
functional (albeit defective) interaction domain. It will be clear to the
person of ordinary skill that whether an interaction domain of the present
invention, variant, derivative, or fragment thereof retains its function in
binding to its partner can be readily determined by using the teachings
and assays of the present invention and the general teachings of the art.
As exemplified herein below, the interaction domains of
the present invention can be modified, for example by in vitro
mutagenesis, to dissect the structure-function relationship thereof and
permit a better design and identification of modulating compounds.
However, some derivative or analogs having lost their biological function
of interacting with their respective interaction partner may still find
utility,
for example for raising antibodies. Such analogs or derivatives could be
used for example to raise antibodies to the interaction domains of the
present invention. These antibodies could be used for detection or
purification purposes. In addition, these antibodies could also act as
competitive or non-competitive inhibitor and be found to be modulators of
PAIP activity.
A host cell or indicator cell has been "transfected" by
exogenous or heterologous DNA (e.g. a DNA construct) when such DNA
has been introduced inside the cell. The transfecting DNA may or may not
be integrated (covalently linked) into chromosomal DNA making up the
CA 02227508 1998-04-O1
29
genome of the cell. In prokaryotes, yeast, and mammalian cells for
example, the transfecting DNA may be maintained on an episomal
element such as a plasmid. With respect to eukaryotic cells, a stably
transfected cell is one in which the transfecting DNA has become
integrated into a chromosome so that it is inherited by daughter cells
through chromosome replication. This stability is demonstrated by the
ability of the eukaryotic cell to establish cell lines or clones comprised of
a population of daughter cells containing the transfecting DNA.
Transfection methods are well known in the art (Sambrook et al., 1989,
supra; Ausubel et al., 1994, supra). The use of an animal cells is
preferred as indicator cells since in lower eukaryotes the PABP directly
interacts with eIF4G (without an apparent need for the bridging factor
PAIP. It will be understood that extracts from animal cells or mammalian
cells for example could be used in certain embodiments, to compensate
for the lack of certain factors in lower eukaryotic indicator cells.
In general, techniques for preparing antibodies
(including monoclonal antibodies and hybridomas) and for detecting
antigens using antibodies are well known in the art (Campbell, 1984, In
"Monoclonal Antibody Technology: Laboratory Techniques in
Biochemistry and Molecular Biology", Elsevier Science Publisher,
Amsterdam, The Netherlands) and in Harlow et al., 1988 (in: Antibody -
A Laboratory Manual, CSH Laboratories). The present invention also
provides polyclonal, monoclonal antibodies, or humanized versions
thereof, chimeric antibodies and the like which inhibit or neutralize their
respective interaction domains and/or are specific thereto.
The present invention also provides antisense nucleic
acid molecules which can be used for example to decrease or abrogate
CA 02227508 1998-04-O1
the expression of PAIP. An antisense nucleic acid molecule according to
the present invention refers to a molecule capable of forming a stable
duplex or triplex with a portion of its targeted nucleic acid sequence (DNA
or RNA). The use of antisense nucleic acid molecules and the design and
5 modification of such molecules is well known in the art as described for
example in WO 96132966, WO 96111266, WO 94/15646, WO 93/08845,
and USP 5,593,974. Antisense nucleic acid molecules according to the
present invention can be derived from the nucleic acid sequences and
modified in accordance to well known methods. For example, some
10 antisense molecules can be designed to be more resistant to degradation
to increase their affinity to their targeted sequence, to affect their
transport to chosen cell types or cell compartments, andlor to enhance
their lipid solubility by using nucleotide analogs andlor substituting chosen
chemical fragments thereof, as commonly known in the art.
15 From the specification and appended claims, the term
therapeutic agent should be taken in a broad sense so as to also include
a combination of at least two such therapeutic agents. Further, the DNA
segments or proteins according to the present invention can be
introduced into individuals in a number of ways. For example,
20 erythropoietic cells can be isolated from the afflicted individual,
transformed with a DNA construct according to the invention and
reintroduced to the afflicted individual in a number of ways, including
intravenous injection. Alternatively, the DNA construct can be
administered directly to the afflicted individual, for example, by injection
25 in the bone marrow. The DNA construct can also be delivered through a
vehicle such as a liposome, or nanoerythrosome which can be designed
CA 02227508 1998-04-O1
31
to be targeted to a specific cell type, and engineered to be administered
through different routes.
For administration to humans, the prescribing medical
professional will ultimately determine the appropriate form and dosage for
a given patient, and this can be expected to vary according to the chosen
therapeutic regimen (i.e DNA construct, protein, cells), the response and
condition of the patient as well as the severity of the disease.
Composition within the scope of the present invention
should contain the active agent (i.e. fusion protein, nucleic acid, and
molecule) in an amount effective to achieve an inhibitory effect on HIV
and related viruses while avoiding adverse side effects. Typically, the
nucleic acids in accordance with the present invention can be
administered to mammals (i.e. humans) in doses ranging from 0.005 to
1 mg per kg of body weight per day of the mammal which is treated.
Pharmaceutically acceptable preparations and salts of the active agent
are within the scope of the present invention and are well known in the art
(Remington's Pharmaceutical Science, 16th Ed., Mack Ed.). For the
administration of polypeptides, antagonists, agonists and the like, the
amount administered should be chosen so as to avoid adverse side
effects. The dosage will be adapted by the clinician in accordance with
conventional factors such as the extent of the disease and different
parameters from the patient. Typically, 0.001 to 50 mglkg/day will be
administered to the mammal.
The present invention is illustrated in further detail by the
following non-limiting examples.
CA 02227508 1998-04-O1
32
EXAMPLE 1
Material and Methods
Plasmids
A 1.7 kb clone obtained by Far-Western screening with
32P-FLAG-HMK-PABP of a human placental cDNA library, was digested
with Sall and subcloned into the Sall site of KS vector (Stratagene)
forming the KS-S plasmid. A larger clone (2.8 kb) containing the complete
coding region for PAIP-1, was obtained by re-screening the library and
was designated KS-S8. A 115 nucleotide truncation of the 5' UTR (S8D5')
was generated by digestion of KS-S8 with NarIIXbaI followed by ligation
to SK (Stratagene) digested with AccIIXbaI.
A KpnIIXbaI fragment from SK-S8D5' was cloned in
pcDNA3 vector (Invitrogen) for expression of PAIP-1 in mammalian cells.
A C-terminal deletion construct [PAIP-1 (OCt )] was generated by
digestion of SK-S8D5' with KpnIIPvull (which truncates the coding region
at amino acid 415) and ligated to pcDNA3 previously digested with
KpnI/EcoRV.
For generation of a glutathione-S-transferase
(GST)-PAIP-1 fusion protein, the Sall fragment from the KS-S plasmid
(encoding amino acids 4-480) was ligated to pGexHMK (Blanar et al.,
1992, Science 256:1014-1018). GST-PAIP-1 (135-415) was generated
by ligation of the AseIIPvull from KS-S to pGex3X (Pharmacia).
GST-PAIP-1 (162-346) was obtained by ligation of an Xmnl fragment
from KS-S to pGexHMK. GST-PAIP-1 (186-325) and (186-415) fusions
were generated by ligation of BsaIIHincll and BsaIIPvull fragments to
pGex3X. GST-PAIP-1 (326-480) and (415-480) were generated by
CA 02227508 1998-04-O1
33
ligation of HincIIICIaI and PvuIIICIaI fragments from KS-S8 to pGexHMK
and pGex3X.
Generation of recombinant baculoviruses
To generate a FLAG-HMK (Blanar et al., 1992, supra)
fusion of PABP, an Ncol-BamHl fragment of pHu73 encoding human
PABP (Grange et al., 1987, Nucleic Acids Res. 15:4771-4778) was
subcloned into the EcoRl site of the baculovirus transfer vector
pVL1392-FLAGHMK (Haghighat et al., 1996, J. Virol. 70:8444-8450)
(Pharmingen). Recombinant baculovirus was generated with the
BaculoGoIdT"" expression system (Pharmingen). FLAG-PABP was
immunopurified using a commercial anti-FLAG mAb column (Kodak).
PAIP-1 and PABP were expressed in Sf9 cells with six histidine and
Xpress epitope N-terminal tags (fused to the Sall fragment from clone S,
and the NcoIIBamHI fragment from pHu73 (Grange et al., 1987, supra),
respectively), using the pBIueBacHis2 baculovirus vector as
recommended by the manufacturer (Invitrogen) and purified on Ni2+ -NTA
resin (QIAGEN).
Screening of a human expression cDNA library
A human placenta hgt11 expression cDNA library (kindly
provided by M. Park, McGill University) was plated (1 x 106 phages), and
transferred to IPTG-soaked nitrocellulose filters (Amersham). The
Far-Western probe was generated by incubation of purified
FLAG-HMK-PABP (2 mg) with heart muscle kinase (10 u, Sigma) in the
presence of [y32P]-ATP (50 mCi) as described (Blanar et al., 1992, supra).
Isolation of RNA and RNase Protection Assay
Total RNA was isolated from COS-7 cells using TrizoIT""
as suggested by the manufacturer (Gibco-BRL). RNA (5 mg) was
CA 02227508 1998-04-O1
34
hybridized overnight to 32P-labeled anti-sense RNA probes (1x105 cpm)
specific for actin (Ambion) and luciferase (bonze et al., 1995, Nucleic
Acids Res. 23:861-868), followed by digestion with RNaseA/T1 (Ambion)
and electrophoresis on a denaturing urea-acrylamide gel.
Purification of GST fusion proteins.
BL21 bacteria were transformed with GST-PAIP-1 fusion
constructs, and the proteins were purified on glutathione-Sepharose resin
(Pharmacia) as per the manufacturer's instructions.
Western blotting
The following antibodies were used: PAIP-1 antiserum
(raised against the GST-PAIP-1 fusion protein, rabbit #1702) was used
at a 1:1200 dilution; monoclonal antibodies against the FLAG-epitope
(Kodak), the Xpress epitope (Invitrogen), and human PABP (mAb 10E10;
Gorlach et al., 1994, Exp. Gell Res. 211:400-407) were used at 1:1000
dilution; eIF4G polyclonal antiserum (raised against the N-terminus of
eIF4G, rabbit #1620) was used at a dilution of 1:1000; hybridoma
supernatant of a monoclonal antibody against eIF4A (kindly provided by
H. Traschel) was used at a 1:10 dilution. For immunoprecipitation, PAIP-1
antiserum was affinity purified into PAIP-1 and GST-specific antibodies
by passing over GST and GST-PAIP-1 coupled resins, generated with
AminoLink Plus Coupling Gel (Pierce).
Transient transfections
COS-7 cells were grown in 60 mm dishes (to ~50%
confluence) and transfected with 0.5 mg pcDNA3-Luc and a total of 5 mg
of the pcDNA3 plasmids (as indicated in Fig. 4A; a total of 5 mg was
maintained in all cases by supplementing with pcDNA3 vector) using
Lipofectamine (15 ml, Gibco-BRL). Cells were harvested 48 h
CA 02227508 1998-04-O1
post-transfection using luciferase lysis buffer (Promega) for the assay of
luciferase. Luciferase activity was measured using a BIOORBITT""
bioluminometer.
5 EXAMPLE 2
Identification of proteins which bind to PABP
To identify proteins that interact with mammalian PABP,
recombinant methods carried out with human PABP. Human PABP was
thus used in a Far-Western assay (Blanar et al., 1992, supra), a
10 recombinant baculovirus was generated expressing an N-terminal
FLAG-HMK fused to PABP. The FLAG-epitope allowed for affinity
purification of the ~80 kDa FLAG-PABP fusion protein from insect cells,
that was then labeled with 32P using heart muscle kinase, and used to
probe membranes containing purified translation initiation factors. No
15 interaction was detected with known initiation factors, including eIF4G
(data not shown). However, a small number of polypeptides in a HeLa
extract, including a 70 kDa polypeptide, interacted with FLAG-PABP in
Far-Western assays (data not shown). Consequently, a human placental
cDNA expression library was screened using the Far-Western technique,
20 and a partial cDNA was identified which formed an in-frame fusion with
b-galactosidase. Using a DNA probe from this clone, several additional
cDNAs were identified. The largest cDNA (~ 2.8 kb) encoded a 480 amino
acid protein (termed PABP-Interacting Protein-1, PAIP-1 ). The nucleic
acid sequence of this PAIP-1 cDNA of 2.768 by and its predicted amino
25 acid sequence are shown in Fig. 1.
Thus, in human, a novel polypeptide appears to be a
candidate polypeptide that acts to bridge the 3' and 5' ends of mRNAs.
CA 02227508 1998-04-O1
36
EXAMPLE 3
Human PAIP-1 shows significant homology with eIF4G
The predicted amino acid sequence of PAIP-1 was
aligned with human eIF4G and as shown in Figure 2 is shown to display
significant homology (25% identity and 39% similarity) with the central
portion of human eIF4G (Yon et al., 1992, J. Biol. Chem.
267:23226-23231 ) (amino acids 420 and 890). The central portion of
eIF4G contains one of two recently characterized eIF4A binding regions
(Imataka et al., 1997, Mol. Cell. Biol. 17:6940-6947) and also binds an
additional initiation factor, eIF3 (Imataka et al., 1997, supra; Lamphear
et al., 1995, J. Biol. Chem. 270:21975-21983). In addition, PAIP-1 bears
significant homology to the eIF4G-related protein p97 (Imataka et al.,
1997, EMBO J. 16:817-825), also called DAP-5 and NAT-1 (Levy-Strumpf
et al., 1997, Mol. Cell. Biol. 17:1615-1625; Yamanaka et al., 1997, Genes
Dev. 11:321-333). The residues within eIF4G that are required for
eIF4E-binding (Mader et al., 1995, Mol. Cell. Biol. 15:4990-4997) are
underlined, and are not present in PAIP-1. Computer searches using
human PAIP-1 sequences also revealed significant homologylidentity with
expressed sequence tags (ESTs) from mouse, rat, and frog. It therefore
appears that PAIP has been conserved throughout evolution. PAIP is
therefore thought to be a key player in gene expression and especially in
translational control. The functional complementation described in
Example 7, support this contention.
CA 02227508 1998-04-O1
37
EXAMPLE 4
PAIP-1 and PABP directlyr interact in vitro
To demonstrate a direct interaction of PABP and PAIP-1
in vitro, histidine-tagged (His-) PAIP-1 was incubated with
poly(A)-Sepharose or FLAG monoclonal antibody (mAb)-coupled
Sepharose, in the absence or presence of FLAG-PABP. Bound proteins
were resolved by SDS-PAGE and detected by Coomassie blue staining
(Fig. 2a). His-PAIP-1 migrated as a ~70 kDa protein (lane 1; indicated by
a dot), larger than its predicted molecular mass of 54 kDa, most likely due
to its proline-rich N-terminus. Purified FLAG-PABP (lane 2; indicated by
a dot) bound efficiently to both poly(A)- and FLAG mAb-coupled
Sepharose (lanes 4 and 5, 7 and 8; >70% binding). In contrast, PAIP-1
did not bind by itself to the poly(A)- and FLAG mAb-coupled Sepharose
(lanes 3 and 6), but was recovered with the resins in the presence of
PABP (lanes 5 and 8; a minor protein species of ~75 kDa that co-purified
with His-PAIP-1 [lane 1] was recovered with poly(A)- [lanes 3 and 5], but
not with FLAG mAb- Sepharose [lanes 6 and 8]). The amount of PAIP-1
recovered with PABP on the poly(A)- Sepharose was ~20% (lane 5) and
~60% of the input with the FLAG mAb-coupled Sepharose (lane 8).
These results indicate an efficient interaction between purified PAIP-1
and PABP in vitro. This interaction is not dependent on poly(A) RNA or
most probably any other RNA, since microccocal nuclease treatment had
no effect on binding in Far-Western and co-precipitation assays (data not
shown).
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38
EXAMPLE 5
PAIP-1 and PABP interact in vivo
To test for association of native PAIP-1 and PABP in
vivo, co-immunoprecipitation assays were carried out using a HeLa S10
extract. The PAIP-1 and PABP antibodies were tested for cross-reactivity
by immunoprecipitating recombinant His-PAIP-1 and His-PABP proteins
followed by Western blotting with an antibody directed against the Xpress
epitope that is present at the N-terminus of both proteins (see Example
1). His-PAIP-1, but not PABP, was recovered with PAIP-1 antisera
(Fig. 3b, lanes 1 and 2), while the opposite result was observed with the
PABP antiserum (lanes 3 and 4); this demonstrates that there is no
cross-reactivity between PABP and PAIP-1 antisera. Next, a HeLa S10
extract was incubated with either affinity purified GST, PAIP-1 or PABP-
specific antibodies. Following precipitation with Protein-G Sepharose, the
bound proteins were subjected to Western blotting with antibodies
directed against the proteins indicated below, and the amount of protein
precipitated was compared to the load (Fig. 3c, lane 1; 1I5 of the input
was analyzed). PABP was detected in both the PAIP-1 and PABP
immunoprecipitates (top panel, compare lanes 3 and 4 and 1; 12% and
40% recovery, respectively), but not in the GST-immunoprecipitate (lane
2). PAIP-1 (lane 1, second panel from top) was recovered efficiently with
PAIP-1 antisera (lane 3, 36% of input; the 50 kDa band in lanes 2 and 3
is the antibody heavy chain), and was also co-immunoprecipitated with
the PABP antibody C 4% of input (lane 4) subtracting a small amount of
PAIP-1 that was immunoprecipitated with antibody to GST (lane 2)].
Thus, PAIP-1 and PABP form a complex in HeLa extracts. Since PAIP-1
contains a region with significant homology to one of two eIF4A binding
CA 02227508 1998-04-O1
39
domains in eIF4G (Imataka et al., 1997, supra), we also tested for
co-immunoprecipitation of eIF4A with the PAIP-1 antiserum. Indeed,
eIF4A was recovered in the PAIP-1 immunoprecipitate (lane 3, second
panel from bottom, 10% of input) and to a lesser extent in the PABP
immunoprecipitate (lane 4, 3%), suggesting a ternary complex between
PAIP-1, PABP and eIF4A. Co-immunoprecipitation of eIF4G was also
examined (bottom panel). However, eIF4G was only detected in the input
(lane 1 ) and not in any of the immunoprecipitates (lanes 2-4). The
association of PAIP-1 with PABP and eIF4A, and the lack of interaction
of PABP with eIF4G, were further confirmed using the two-hybrid assay
in yeast (data not shown).
EXAMPLE 6
Identification of the PABP interaction site within PAIP-1
To identify the PABP interaction site within PAIP-1,
various portions of PAIP-1 were fused to GST and the proteins were
expressed in E. coli . The purified proteins (Fig. 3d, top panel) were
pre-incubated with FLAG-PABP, precipitated with glutathione-Sepharose,
and subjected to Western blotting with an antibody against PABP (bottom
panel). PABP was detected in the input material (lane 1 ) and in the
reactions containing fusion proteins retaining the C-terminus of PAIP-1
(lanes 7-9). This portion of PAIP-1 (amino acids 415 to 480) is very rich
in acidic amino acids, and is not conserved in eIF4G (Figs. 2 and 6).
These results demonstrate that the C-terminus of PAIP-1, spanning
amino acids 415 to 480, is sufficient for interaction with PABP.
CA 02227508 1998-04-O1
EXAMPLE 7
PAIP-1 enhances the translation activiy in vivo
To assess the possible role of PAIP-1 in translation,
COS-7 cells were co-transfected with vectors expressing either wild-type
5 PAIP-1 or a C-terminal deletion mutant lacking the PABP-binding site,
PAIP-1 (~Ct), and a firefly luciferase reporter plasmid (Fig. 4). With
increasing amounts of the wild-type PAIP-1 plasmid, a dose-dependent
increase of up to 2.8 fold in luciferase activity was observed compared to
that for the vector alone (Fig. 4a). Transfection with the PAIP-1 (~Ct)
10 plasmid caused no stimulation, suggesting that the C-terminus of PAI P-1
is required for this effect. Since this portion of PAIP-1 confers the
association with PABP, this would suggest that stimulation requires
interaction with PABP. To exclude the possibility that the effects seen
reflect differences in mRNA concentrations, RNase protection assays
15 were performed on RNA extracted from parallel transfections using
anti-sense actin and luciferase probes (Fig. 4b, lanes 1 and 2). As
expected, no protected fragments were observed for the tRNA control
(lane 3), and only actin mRNA was detected in mock-transfected cells
(lane 4, bottom panel). The luciferaselactin mRNA ratios were 1.2 t 0.1
20 for wild-type, and 0.8 t 0.1 for PAIP-1 (~Ct), relative to vector (set at 1
)
for three experiments (lanes 5-7). These results suggest that there may
be small differences in luciferase mRNA levels, but not of the magnitude
to explain the increase in luciferase activity. Therefore, the
overexpression of PAIP-1 likely enhances translation primarily. Western
25 blotting with PAIP-1 antisera indicated that wild-type and PAIP-1 (~Ct)
proteins were expressed to similar levels (Fig. 4c). The overexpressed
wild-type PAIP-1 co-migrated with a protein from COS cells that was
CA 02227508 1998-04-O1
41
detected with the PAIP-1 antisera (compare lane 1 with lanes 2-4) and
likely corresponds to the monkey homologue of PAIP-1. Since PAIP-1 is
present at ~6 fold lower levels than PABP (data not shown), the
overexpression of PAIP-1 would likely result in more PAIP-11PABP
complex and potentiate contacts with the 5' end of the mRNA via
interaction with eIF4A. Thus, it appears that human PAIP-1 can
functionally replace its monkey homologue to increase translation
efficiency in green monkey cells (Cos cells). The monkey PAIP (or other
mammalian or animal PAIP) could thus be isolated and characterized
using the method of the present invention or by using high or low
stringency hybridization. For example, the PABP interacting domain of
PAIP-1 spanning amino acids 415 to 480 could be used in hybridization
experiments to identify animal homologues. Of course, the person of
ordinary skill will be able to adapt the hybridization conditions to take into
account the phylogenic relationship between the probe (or primer) and
the homolog to be identified. The observed increase in translation
following expression of human PAIP-1 in COS cells strongly suggest that
such increase in translation is predicted to occur whenever the ratio of the
level of PAIP-11PABP protein expression in a given cell is lower than one.
The present invention therefore provides a method to increase the
translation activity of numerous types of cells.
It will also be understood that in certain situations the
translation efficiency of cells (or extracts) could be decreased by
changing the level of PAIP-11PABP. Non-limiting examples of such
approaches include anti-PAIP antibodies (such as PAIP-1 antibodies) or
PAIP antisense (such as PAIP-1 antisense) that could lower the
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42
expression level of PAIP-1 and decrease translation and perhaps other
cellular functions.
EXAMPLE 8
Tentative model of the role of PAIP-1 in translation control
Of what significance is the interaction of PAIP-1 with
both PABP and eIF4A? The eIF4A subunit of eIF4F is required for
translation of all mRNAs, as dominant-negative mutants of eIF4A repress
both cap-dependent and cap-independent translation (Pause et al., 1994,
EMBO J. 13:1205-1215). It has been proposed that the 'loading' of eIF4A
onto the 5' UTR, is followed by the unwinding of mRNA secondary
structure, thus resulting in enhanced ribosome binding (Merrick et al.,
1996, supra). The interaction between PAIP-1 and eIF4A could allow for
bridging to occur between the PAIP-11PABP complex on the poly(A) tail
and the 5' UTR-bound eIF4A (see model, Fig. 5a). The interaction
between the mRNA 5' and 3' ends may thus serve to select only intact
mRNAs (i.e. containing both a cap and poly(A) tail) as templates for the
translational machinery, and may protect the mRNA from degradation.
Also, the proximity of mRNA ends may promote the re-initiation of
terminating ribosomes on the same mRNA, thus enhancing translation
rates. An earlier study on translation in rabbit reticulocyte lysates
indicated that re-initiating ribosomes are less sensitive to inhibition by cap
analogues than those undergoing primary initiation events (Asselbergs
et al., 1978, Eur. J. Biochem. 88:483-488). This is consistent with a
re-initiating ribosome utilizing an alternative, cap-independent route to the
mRNA, such as that provided by the putative link formed between the
mRNA termini (Fig. 5). It is possible that PAIP-1 links PABP to the 40S
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ribosome as predicted in yeast (Tarun et al., 1996, supra; Tarun et al.,
1995, Genes Dev. 9:2997-3007) through an interaction with eIF3,
although we could not detect such an interaction.
Recent studies in yeast and plants indicate that the
interaction between Pab1 p and eIF4G provide a bridge between the cap
and poly(A) tail (Tarun et al., 1996, supra; Tarun et al., 1997, supra; Le
et al., 1997, supra) (Fig. 5b). However, the lack of interaction between
human PABP and eIF4G (Fig. 3c) suggests that this model (Sachs et al.,
1997, supra; Tarun et al., 1996, supra; Hentze et al., 1997, Science
275:500-501 ) does not extend to mammalian cells. We have recently
identified a novel human functional homologue of eIF4G, as well as a
longer form of the original eIF4G (Gradi et al., 1998, Mol. Cell. Biol.
18:334-342), but neither of these proteins showed an interaction with
PABP (data not shown). The bridging of the mRNA termini by an adaptor
protein like PAIP-1 in animal cells may allow for additional levels of
regulation not required in yeast. For instance, PAIP-1 is upregulated
following T cell activation, and also interacts with the T cell-inducible
iPABP (Yang et al., 1995, Mol. Cell. Biol. 15:6770-6776). This is
consistent with PAIP-1 being a positive effector of translation. Thus, the
presence of PAIP-1 in animal cells may reflect an evolutionary advantage
for higher eukaryotes to link PABP to eIF4A function without affecting the
cap binding process directly.
The present invention therefore provides the
identification of a positive effector of translation in animal cells. As well,
the present invention identifies a target (PAIP) for modulating gene
expression and translation in animal cells.
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44
Based on the virtually ubiquitous nature of the cap
structure and poly(A) tail of mRNAs and their diversified role in cellular
metabolism and homeostasis, PAIP could prove to be a key element in
other fundamental cellular activities (i.e. nucleo-cytoplasmic transport).
Although the present invention has been described
hereinabove by way of preferred embodiments thereof, it can be modified,
without departing from the spirit and nature of the subject invention as
defined in the appended claims.
