JP2006141305A - RIBONUCLEIC ACID BINDING TO TRANSLATION INITIATION FACTOR eIF4G - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substance utilizable for the investigation of the underlying mechanism, diagnosis and treatment of diseases caused by human translation initiation factor eIF4G or a substance utilizable for the investigation of intracellular process such as translation. <P>SOLUTION: An RNA is obtained by SELEX method and binds to human translation initiation factor eIF4G or an RNA expressed by a base sequence obtained by the substitution, deletion or insertion of one or more bases in the base sequence of the RNA, and the RNA has a bonding activity to eIF4G, its family or their fragments. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a ribonucleic acid that binds to a translation initiation factor eIF4G.

  Protein biosynthesis is the final stage of the expression of genetic information and is an extremely important reaction for the maintenance of life. The translation from gene to protein is performed based on the information shown by mRNA, but the initiation process is composed of several stages, which is a complex reaction involving many sequential translation initiation factors. More than 30 types of eukaryotic translation initiation factors (eIF) are known, including the subunits that make them up and related factors. eIF4G is a translation initiation factor that plays a modulator (scaffold) role, and forms a complex called eIF4F by binding to eIF4E that binds to the cap structure at the 5 ′ end of mRNA and eIF4A that is an RNA helicase. eIF4F binds to the mRNA cap structure, leading to a 43S complex consisting of 40S ribosomal subunits and eIF3. At that time, the combination of eIF4G and eIF3 is important. The ribosome complex led to the cap structure moves through the untranslated region of mRNA to the position of the start codon. When the translation initiation factor dissociates from the 40S ribosomal subunit, the 60S ribosomal subunit binds to the 40S ribosomal subunit and initiates translation. eIF4G needs to dissociate from the 40S ribosomal subunit before the 60S ribosomal subunit binds to the 40S ribosomal subunit, but the details are unknown.

  eIF4G is composed of three domains: an N-terminal domain, a central domain, and a C-terminal domain. The N-terminal domain contains the binding site for eIF4E and PABP. The central domain contains eIF4A and eIF3 binding sites and RNA binding sites. The C-terminal domain contains the binding site for eIF4A and Mnk1.

  There are two known isoforms of eIF4G: eIF4GI and eIF4GII. These amino acids have 46% homology in amino acid sequences and play similar roles in vivo. Further, Paip1 and DAP5 are known as proteins having high homology with eIF4G. Paip1 binds to PABP and activates translation. DAP5, also known as p97 or NAT1, suppresses cap-dependent translation. These highly homologous proteins are called the eIF4G family.

  The virus suppresses host cap-dependent translation in infected cells and initiates translation of the virus itself, in which eIF4G plays a central role. Rotavirus has a cap structure at the 5 'end of the mRNA and a consensus sequence (UGACC) at the 3' end. A protein called NSP3 binds to this 3'-end consensus sequence and eIF4GI, and is involved in the formation of mRNA loop structures. Since NSP3 has a higher affinity for eIF4GI than PABP, eIF4GI binds more to NSP3, and the host translation shifts to viral translation. In addition, foot-and-mouth disease virus, HIV virus, and the like cleave the N-terminal domain containing the binding site of eIF4G with eIF4E with its own protease so that eIF4G cannot interact with host mRNA. The remaining part of eIF4G binds to the IRES of the viral mRNA and viral translation is initiated.

  eIF4G is thought to play an important role in processes other than the translation initiation process. eIF4G is known to bind to a protein called Dcp1, which is important during mRNA degradation. This suggests that eIF4G is involved in both mRNA translation and degradation. EIF4G is also known to interact with eRF3, a translation termination factor, via PABP, and may be involved in both translation initiation and termination. Furthermore, it is suggested that eIF4G binds to the CBC complex that binds to the cap structure of mRNA immediately after splicing, and is involved in mRNA quality control.

  As described above, eIF4G is a very important factor for cell maintenance, but it is known that abnormal expression of this protein is closely related to cell carcinogenesis. Brass et al. Found that eIF4G gene amplification was found in lung cancer cells. Shimogori et al. Also reported that overexpression of eIF4G in NIH3T3 cells causes the cells to become cancerous. In addition, eIF4E and eIF4A are known as translation initiation factors other than eIF4G that are considered to be related to canceration.

In recent years, application of RNA aptamers to pharmaceuticals has attracted attention, and several RNA aptamers have entered the clinical stage. An RNA aptamer is an RNA that specifically binds to a target substance such as a protein, and can be obtained by the SELEX method. The SELEX method is a method of selecting RNA that specifically binds to a target substance from an RNA pool containing RNA having different base sequences of about 10 ¹⁴ . RNA has a structure in which a random sequence of about 40 residues is sandwiched between primer sequences. This RNA pool is associated with the target substance, and only the RNA bound to the target substance is recovered using a filter or the like. The recovered RNA is amplified by RT-PCR and used as a template for the next round. By repeating this operation about 10 times, an RNA aptamer that specifically binds to the target substance can be obtained. When the obtained RNA aptamer has a physiological activity that promotes or inhibits the function of the target substance, the RNA aptamer can be applied to pharmaceuticals and the like.

Japanese Patent Laid-Open No. 2002-300088 Ellington, A.D. and Szostak, J.W. (1990) In vitro selection of RNA molecules that bind specific ligands.Nature, 346, 818-822. Tuerk, C. and Gold, L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science, 249, 505-510. Oguro, A., Ohtsu, T., Svikin, YV, Sonenberg, N. and Nakamura, Y. (2003) RNA aptamers to initiation factor 4A helicase hinder cap-dependent translation by blocking ATP hydrolysis. RNA, 9, 394-407 .

  INDUSTRIAL APPLICABILITY The present invention can be used to elucidate the pathogenesis of diseases caused by the human translation initiation factor eIF4G or a family thereof or a fragment thereof, a substance that can be used for diagnosis and treatment, or a study of intracellular processes such as translation. Aims to provide new materials.

  The inventor prepared an RNA aptamer that specifically binds to eIF4G using the SELEX method, confirmed the binding activity of the obtained RNA aptamer by a filter binding experiment using a nitrocellulose filter, and further obtained RNA The translation inhibitory activity of aptamers was confirmed by experiments using rabbit reticulocyte extract. The present invention has been completed based on the above findings.

  That is, the present invention provides the following (1) to (9).

(1) RNA represented by the nucleotide sequence of SEQ ID NOS: 1-9.

(2) RNA represented by a base sequence in which one or several bases are substituted, deleted or inserted in the base sequence of the RNA described in (1), wherein eIF4G or a family thereof or a fragment thereof RNA having binding activity.

(3) RNA obtained by adding another RNA to the RNA described in (1) or (2), and having binding activity to eIF4G or a family thereof or a fragment thereof.

(4) RNA shortened by cleaving a part of the RNA described in (1) to (3), and having binding activity to eIF4G or a family thereof or a fragment thereof.

(5) An RNA having at least one modified nucleotide introduced in the RNA according to (1) to (4), wherein the RNA has binding activity to eIF4G or a family thereof or a fragment thereof.

(6) The RNA according to (1) to (5), which is labeled.

(7) DNA having a base sequence complementary to the base sequence of RNA described in (1) to (4).

(8) A vector into which the DNA according to (7) is inserted.

(9) A method for detecting or quantifying eIF4G or a family thereof or a fragment thereof using the RNA according to (1) to (5).

  The present invention provides an RNA aptamer capable of binding to eIF4G. The RNA aptamer of the present invention can be used for functional analysis of eIF4G and diagnosis and treatment of diseases such as cancer.

  Hereinafter, the present invention will be described in detail.

  The RNA of the present invention includes the following RNAs (a) to (e).

(a) RNA represented by the nucleotide sequence of SEQ ID NOS: 1-9.

(b) RNA represented by a base sequence in which one or several bases are substituted, deleted or inserted in the base sequence of the RNA described in (a), wherein the RNA is represented by eIF4G or a family thereof or a fragment thereof RNA having binding activity.

(c) RNA obtained by adding another RNA to the RNA described in (a) or (b), and having binding activity to eIF4G or a family thereof or a fragment thereof.

(d) RNA obtained by cleaving a part of the RNA described in (a) to (c) and having a binding activity to eIF4G or a family thereof or a fragment thereof.

(e) RNA having at least one modified nucleotide introduced in the RNA described in (a) to (d), and having binding activity to eIF4G or a family thereof or a fragment thereof.

  The RNA of (a) is RNA actually obtained by the SELEX method by the present inventors. The secondary structure of RNA (a) is as shown in FIGS. Among the RNAs in (a), it has been confirmed that RNAs represented by the nucleotide sequences of SEQ ID NOs: 1 to 3 inhibit translation depending on the cap structure in a translation inhibition experiment using a rabbit reticulocyte extract. Since there is a close relationship between overexpression of eIF4G and cell carcinogenesis, these aptamers that inhibit eIF4G function can be potential candidates for preventive or therapeutic agents for cancer. The RNA represented by the nucleotide sequence of SEQ ID NO: 4 inhibits translation depending on cap structure in translation inhibition experiments using rabbit reticulocyte extract, and also translation dependent on IRES (HCV IRES) of hepatitis C virus. It has been confirmed to inhibit. The RNA represented by SEQ ID NO: 2 binds mainly to the central domain of eIF4G and competes with the binding of eIF4A and eIF3, but the RNA represented by SEQ ID NO: 1 binds mainly to the C-terminal domain of eIF4G. Does not compete with eIF4A binding. In this way, RNA in (a) has different secondary structures and retains different properties. By taking advantage of the characteristics of each, elucidation of the pathogenesis of diseases caused by eIF4G and It can be used to study intracellular processes such as translation.

  The RNA of (b) is a RNA in which a mutation is introduced into the RNA of (a), and is an RNA having a binding activity to eIF4G or a family thereof or a fragment thereof in the same manner as the RNA of (a). Here, “having binding activity” means having higher binding activity than a random sequence RNA pool. The “eIF4G family” refers to a protein (including non-natural proteins) composed of a domain similar to the domain constituting eIF4G, and includes, for example, PAIP1 and DAP5. A “fragment” is a part of eIF4G or a family thereof, and its amino acid sequence can be recognized as a part of eIF4G or a family.

  By introducing a mutation into the RNA of (a) and performing SELEX again, the affinity of the RNA of (a) for eIF4G can be changed. The amount of mutation to be introduced is not particularly limited as long as it does not lose the binding activity, but about 1 to 20 bases is considered appropriate. The affinity between RNA (a) and eIF4G can also be changed by deleting or inserting about 1 to 20 bases.

  The RNA (c) is RNA obtained by adding another RNA to the RNA (a) or (b). For example, by adding about 16 nucleotides of adenylic acid to the 5 ′ end or 3 ′ end, RNA of (a) or (b) can be immobilized on beads of oligo (dT) cellulose (Amersham). it can. In addition, RNA that specifically recognizes two or more molecules by adding RNA with a length of about 30 to 100 residues having affinity for another target molecule to the RNA of (a) or (b) Can be produced. Thus, RNA aptamers suitable for various purposes can be prepared by adding another RNA.

  The RNA (d) is obtained by shortening the RNAs (a) to (c) according to the purpose. For example, the part of the RNA (a) to (c) that does not form the 5'-end and 3'-end base pair may be cleaved partly or entirely while retaining the binding activity and physiological activity. it can. The number of bases to be truncated is not particularly limited, but it is generally considered to be shorter as long as the binding activity does not drop. For example, when RNA aptamers are introduced into cells, generally shorter RNAs are less cytotoxic. In addition, if the length can be shortened to about 50 residues or less, the RNA can be efficiently chemically synthesized.

  The RNA of (e) is obtained by modifying the ribose part, the nucleobase part, the 5 ′ end or the 3 ′ end of the RNAs (a) to (d). Such modifications can enhance RNA stability, binding activity, and physiological activity. For example, fluorination of the ribose 2′-OH of the pyrimidine nucleotides makes it resistant to ribonuclease A and dramatically improves stability.

  The RNAs (a) to (e) above can be labeled with a fluorescent substance, a radioisotope, biotin, digoxigenin, or the like. RNA labeled in this way can be used for detection and quantification of eIF4G. For example, by introducing the RNA labeled with a fluorescent substance into a cell, the behavior of eIF4G in the cell can be observed. In addition, an eIF4G separation column using an RNA aptamer can be prepared by biotinylating the end and immobilizing it.

  In addition to the RNA of the present invention described above, the present invention also includes a DNA having a base sequence complementary to the base sequence of the RNA of the present invention and a vector into which such a DNA has been inserted. Such DNA and vectors can be used for gene therapy of diseases caused by abnormalities in eIF4G.

  Hereinafter, the present invention will be described in more detail with reference to examples.

  First, the outline of the experiment described in this example will be described.

  The RNA aptamer for eIF4G was prepared using the SELEX method. Human eIF4GI consisting of a GST-tagged central domain and C-terminal domain was immobilized on glutathione sepharose for selection. RNA with a random sequence of 40 residues was used as the first pool. After performing 12 round selections, the base sequence of 48 clones was examined. As a result, 18 clones of RNA represented by the base sequence of SEQ ID NO: 2 and 3 clones of RNA represented by SEQ ID NO: 1 were represented by SEQ ID NO: 3. There were 3 clones of RNA that were made, and the others all consisted of different sequences.

  Next, the binding activity of RNA represented by SEQ ID NOs: 1 to 9 to eIF4G was examined by a filter binding experiment using a nitrocellulose filter. As eIF4G, human eIF4GI consisting of a central domain without a GST tag and a C-terminal domain was used. The RNAs represented by SEQ ID NOs: 4 to 9 were randomly selected from 24 clones in which only one clone was present as a result of nucleotide sequence analysis.

  As a result of the experiment, it was found that all nine types of RNA bind to eIF4G more preferentially than the random sequence RNA pool (Figure 1). The dissociation constants of these RNA aptamers were in the range of 0.1-4 μM.

  Next, in order to see the effect of these RNA aptamers on translation, we conducted a translation inhibition experiment using rabbit reticulocyte extract. Bicistronic mRNA containing chloramphenicol acetyltransferase (CAT) and luciferase (LUC) genes was used as a substrate (FIG. 2A). A cap structure was added to the 5 ′ end of CAT mRNA, and HCV IRES was added upstream of LUC mRNA (cap-CAT / HCV / LUC). As a result of the experiment, the RNA represented by the nucleotide sequences of SEQ ID NOs: 1 to 3 inhibited the translation of CAT depending on the cap structure (FIG. 2B). On the other hand, translation of LUC depending on HCV IRES was not inhibited. The RNA represented by the nucleotide sequence of SEQ ID NO: 4 inhibited both CAT and LUC translation. Other RNAs did not inhibit translation.

  Translations that depend on the Cap structure require eIF4G, whereas translations that depend on HCV IRES do not require eIF4G. Therefore, the results of the translation inhibition experiment described above strongly support that the RNAs represented by the nucleotide sequences of SEQ ID NOs: 1 to 3 specifically bind to eIF4G and inhibit translation.

  Next, the RNA binding activity and translation inhibitory activity of the RNA represented by the nucleotide sequence of SEQ ID NO: 1 with 6 bases truncated at the 5 ′ and 3 ′ ends were examined. As a result of the filter binding experiment, it was found that the binding activity of the truncated RNA was almost the same as that of the original RNA. In translation inhibition experiments using rabbit reticulocyte extract, the truncated RNA inhibited CAT translation depending on the cap structure to the same extent as the original RNA.

[Example 1] Purified protein The human eIF4GI consisting of the central domain and C-terminal domain used in the present invention was overexpressed in E. coli as a GST-tagged fusion protein, and Ni-NTA agarose (Qiagen) was used. The crude product was purified, and further purified by an anion exchange column Resource Q (manufactured by Amersham Biosciences).

[Example 2] Acquisition of RNA aptamer
SELEX was performed by modifying the method of Ellington et al. (Ellington and Szostak, Nature 346, 818-822, 1990) and the method of Tuerk et al. (Tuerk and Gold, Science 249, 505-510, 1990). The RNA used in the first round was obtained by transcription of DNA obtained by chemical synthesis with T7 RNA polymerase. A DNA template having a length of 90 residues with a primer sequence on both sides of a 40-residue random sequence was used. The DNA template sequence and primer sequence are shown below.

DNA template: 5'-ctctcatgtcggccgtta-40N-cgtccattgtgtccctatagtgagtcgtatta-3 '(SEQ ID NO: 10)
Primer A: 5'-taatacgactcactatagggacacaatggacg-3 '(SEQ ID NO: 11)
Primer B: 5'-ctctcatgtcggccgtta-3 '(SEQ ID NO: 12)
Primer A contains the T7 RNA polymerase promoter sequence. The RNA pool variation used in the first round was theoretically 10 ¹⁴ .

  The target eIF4G was adsorbed on glutathione sepharose beads (Amersham) and immobilized. The RNA pool was added to this and kept at room temperature for 30 minutes, and then RNA that did not bind to the target eIF4G was washed away with solution A. Here, the solution A is a mixed solution of 80 mM potassium acetate (KOAc), 2.5 mM magnesium acetate (MgOAc), and 20 mM pH 7.6 Tris. The RNA bound to the target eIF4G was collected by adding reduced glutathione, amplified by RT-PCR, transcribed with T7 RNA polymerase, and used in the next round. After 12 rounds, the PCR product was subcloned into pGEM-T Easy vector (Promega) and transformed into E. coli strain NovaBlue (Novagen). After extracting a plasmid from a single colony, the base sequence was determined with a DNA sequencer (ABI PRISM 3100, manufactured by ABI) (including SEQ ID NOs: 1 to 9). The secondary structure of RNA was predicted using the MFOLDROOT program (Zuker, Nucleic Acids Res. 31, 3406-3415, 2003) (FIGS. 3 to 11).

[Example 3] Evaluation of binding activity
Of the RNAs obtained after 12 rounds, the binding activity of 9 types of RNA (SEQ ID NOs: 1 to 9) to eIF4G was evaluated by a filter binding experiment using a nitrocellulose filter. As eIF4G, eIF4GI consisting of a central domain without a GST tag and a C-terminal domain was used. ^The RNA labeled with ³² P and eIF4G were mixed and held at room temperature for 30 minutes, and then the mixture was filtered through a nitrocellulose filter. Since proteins are easily adsorbed to the nitrocellulose filter, RNA bound to eIF4G remains on the filter. The filter was washed with solution A to wash away RNA that did not bind to eIF4G, and then the count of ³² P remaining on the filter was measured with a scintillator (Beckman). The binding rate was expressed as a relative amount of ³² P count of RNA adsorbed on the filter to ³² P count of total RNA added to the system. The binding constant was determined from the relationship of RNA _initial / RNA _trapped = Kd / [eIF4G] _{initial through} experiments with varying eIF4G concentrations.

  The result of the above experiment is shown in FIG. As shown in the figure, all of the RNAs represented by SEQ ID NOs: 1 to 9 bound to eIF4G more preferentially than the random sequence RNA pool.

[Example 4] Translation inhibition experiment A translation inhibition experiment was performed using a rabbit reticulocyte extract (Promega). Bicistronic mRNA containing chloramphenicol acetyltransferase (CAT) and luciferase (LUC) genes was used as a substrate. The 5 ′ end of the CAT gene was capped, and HCV IRES was added upstream of LUC (cap-CAT / HCV / LUC). Capping of mRNA was performed by adding m ⁷ GpppG cap analog (Promega) during transcription with T7 RNA polymerase. T7 Ribo MAX system (Promega) was used for transcription.

  In vitro translation was performed using a mixed solution having the following composition.

  The above mixed solution was kept at 30 ° C. for 30 minutes, and a part thereof was subjected to SDS polyacrylamide gel electrophoresis. The gel was dried and exposed to an imaging plate for about 4 hours, and then analyzed with BAS-2000 (Fuji Film).

  The results of the translation inhibition experiment using the RNA represented by SEQ ID NO: 1 (concentration 0-4 μM) are shown in FIG. 2B. CAT% in the figure indicates the ratio of the CAT translation amount when the RNA aptamer is added to the CAT translation amount when the RNA aptamer is not added (not corrected by the LUC translation amount). As shown in the figure, when 0.5 μM RNA was added, the translation amount of CAT decreased to 30%, but the translation amount of LUC did not decrease.

[Example 5] Preparation of terminal-deficient RNA Using the following primers chemically synthesized with the RNA represented by SEQ ID NO: 1, 6 bases were deleted from the 5 'end and 3' end of the RNA represented by SEQ ID NO: 1. RNA was prepared. Due to transcription by T7 RNA polymerase, one residue of guanosine was added to the 5 ′ end to give a total length of 65 residues.

Primer C: 5'-taatacgactcactatagacaatggacgcacctcc-3 '(SEQ ID NO: 13)
Primer D: 5'-tgtcggccgttagcatttag-3 '(SEQ ID NO: 14)
Experiments similar to Example 3 (evaluation of binding activity) and Example 4 (translation inhibition experiment) were performed using this terminal-deficient RNA. As a result of the experiment, the terminal-deficient RNA showed almost the same binding activity and translation inhibition activity as the original RNA.

The figure which shows the result of the filter coupling | bonding experiment using a nitrocellulose filter. FIG. 2 is a schematic diagram of mRNA used in Example 4 (FIG. 2A) and a diagram showing the results of translation inhibition experiments in Example 4 (FIG. 2B). The secondary structure prediction figure of RNA represented by sequence number 1. The secondary structure prediction figure of RNA represented by sequence number 2. The secondary structure prediction figure of RNA represented by sequence number 3. The secondary structure prediction figure of RNA represented by sequence number 4. The secondary structure prediction figure of RNA represented by sequence number 5. The secondary structure prediction figure of RNA represented by sequence number 6. The secondary structure prediction figure of RNA represented by sequence number 7. The secondary structure prediction figure of RNA represented by sequence number 8. The secondary structure prediction figure of RNA represented by sequence number 9.

Claims (9)

  RNA represented by the nucleotide sequence of SEQ ID NOS: 1-9.   An RNA represented by a base sequence in which one or several bases are substituted, deleted or inserted in the base sequence of RNA according to claim 1, and has binding activity to eIF4G or a family thereof or a fragment thereof. Having RNA.   An RNA obtained by adding another RNA to the RNA according to claim 1 or 2, wherein the RNA has binding activity to eIF4G or a family thereof or a fragment thereof.   4. RNA shortened by cutting part of the RNA according to claim 1, wherein the RNA has binding activity to eIF4G or a family thereof or a fragment thereof.   5. An RNA having at least one modified nucleotide introduced therein, wherein the RNA has binding activity to eIF4G or a family thereof or a fragment thereof.   The RNA according to claims 1 to 5, which is labeled.   DNA which has a base sequence complementary to the base sequence of RNA of Claims 1-4.   A vector into which the DNA according to claim 7 is inserted.   A method for detecting or quantifying eIF4G or a family thereof or a fragment thereof using the RNA according to claim 1.