CA2227508A1 - 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 - Google Patents

Abstract

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 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.

Description

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 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, 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.

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 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 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 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 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 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.

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.

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 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.

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 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.

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.

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 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 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 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 (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 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.

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 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%

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 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 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.

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 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 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 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 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.

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 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 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 post-transfection using luciferase lysis buffer (Promega) for the assay of luciferase. Luciferase activity was measured using a BIOORBITT""
bioluminometer.

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.

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.

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).

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 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).

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.

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 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 expression level of PAIP-1 and decrease translation and perhaps other cellular functions.

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

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.

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.

Claims (8)

1. An isolated PABP-interacting protein (PAIP) exhibiting homology to eIF4G and interacting with eIF4A.

2. The isolated PAIP of claim 1 having an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:
(a) amino acids from about 1 to about 480 of Figure 1;
(b) amino acids from about 2 to about 480 of Figure 1;
(c) amino acids from about 415 to about 480 of Figure 1; and (d) the amino acid sequence of an epitope-bearing portion of any one of the polypeptides of (a), (b), (c), or (d).

3. An isolated nucleic molecule comprising a polynucleotide sequence at least 95% identical to a sequence selected from the group consisting of:
(a) a nucleotide sequence encoding a PAIP polypeptide comprising amino acids from about 1 to about 480 in Figure 1;
(b) a nucleotide sequence encoding a PAIP polypeptide comprising amino acids from about 2 to about 480 in Figure 1;
(c) a nucleotide sequence encoding a PAIP polypeptide comprising amino acids from about 415 to about 480 in Figure 1; and (d) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), or (d).

4. A recombinant vector comprising said isolated nucleic acid molecule of claim 3.

5. A method of making a recombinant host cell comprising introducing the recombinant vector of claim 4 into a host cell.

6. A recombinant host cell produced by the method of claim 5.

7. A recombinant method for producing PAIP
polypeptide, comprising culturing said host cell of claim 6 under conditions such that said polypeptide is expressed and recovering said PAIP polypeptide.

8. 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, and/or PAIP encoding nucleic acid molecule andlor PAIP-activity modulator together with a pharmaceutically acceptable carrier.