KR101021402B1 - Method for control of nonsense-mediated mRNA decay using gene PNRC2 - Google Patents

Abstract

The present invention provides a gene PNRC2 that can control nonsense-mediated mRNA disruption (hereinafter referred to as 'NMD') and an artificially controlled NMD using the same. Provide a method. Through the molecular biological understanding of NMD and artificial NMD efficiency control technology according to the present invention, it is possible to develop therapeutic agents or inhibitors of NMD-related genetic diseases and cancers.

NMD, PNRC2, Upf1, Dcp1a

Description

Method for control of NMD using gene PNCR2 {Method for control of nonsense-mediated mRNA decay using gene PNRC2}

The present invention relates to a gene PNRC2 (Human proline-rich nuclear receptor coregulatory protein 2) capable of controlling nonsense-mediated mRNA decay (hereinafter referred to as 'NMD') and artificial NMD regulation using the same. It is about a method. In more detail, the novel gene PNRC2 related to NMD binding to Upf1 and Dcpla and NMD regulation using the same It is about a method.

Every cell has many gene expression control mechanisms to express the necessary genetic information in the right place at the right time. Gene expression control mechanisms are essential mechanisms involved in transcription, splicing, and translation processes in human cells, and are responsible for expressing accurate genetic information in cells.

Among many gene expression control mechanisms, nonsense-mediated mRNA Decay (NMD) is well known as a representative mechanism involved in the quality control of mRNA. The NMD mechanism is a mechanism for recognizing this mRNA and finally removing it in cells when abnormal protein termination codons (hereinafter, referred to as 'PTC') are generated on the mRNA.

If the mRNA containing PTC is not recognized by the NMD mechanism, the mRNA expresses an abnormal protein shorter than its normal length, and the resulting short protein may have a dominant-negative effect. If it can inhibit the function of normal protein can inhibit the function of the cell. In other words, the NMD mechanism may be regarded as a kind of quality assurance mechanism that recognizes and removes abnormal short protein expression on mRNA in advance. If problems occur in the proper regulation of gene expression, abnormally expressed genes can directly affect the development of numerous cancers and genetic diseases.

Since NMD has been identified as a cause of numerous cancers and hereditary diseases, development of anti-cancer drugs and genetic disease treatments aimed at controlling NMD efficiency has been actively conducted. If successful control of NMD efficiency is achieved by controlling proteins closely related to NMD in various ways, it may be advantageous to artificially inhibit or activate the efficiency of NMD. Therefore, the molecular biological understanding of NMD and artificial NMD efficiency control technology will be able to develop therapeutic agents or inhibitors of NMD-related genetic diseases and cancers.

It is therefore an object of the present invention to provide target genes and proteins that can regulate the efficiency of NMD.

Another object of the present invention is to provide a method for controlling the efficiency of NMD by using target genes and proteins that can control the efficiency of NMD.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

The present inventors identified PNRC2 (Human proline-rich nuclear receptor coregulatory protein 2) gene which binds Upf1, which is an important factor in NMD, and Dcp1a, which is a decapping enzyme. In addition, the present inventors confirmed that the PNRC2 gene acts as a bridge between Upf1 and Dcpla, and when the PNRC2 protein expression is inhibited, the efficiency of NMD is lowered. Through this, it was confirmed that PNRC2 plays an important role in NMD by acting as a bridge between NMD machinery and Dcpla and completed the present invention.

According to one aspect of the invention, the present invention provides an isolated gene PNRC2 having a nucleotide sequence of SEQ ID NO: 1 involved in NMD and an isolated protein PNRC2 having an amino acid sequence of SEQ ID NO: 2 involved in NMD.

According to another aspect of the present invention, the present invention provides an NMD inhibitor comprising DNA for inhibiting the expression of the isolated gene PNRC2 having the nucleotide sequence of SEQ ID NO: 1.

According to another aspect of the present invention, the present invention provides a method for inhibiting NMD by inhibiting the expression of the protein PNRC2 having the amino acid sequence of SEQ ID NO: 2.

According to another aspect of the invention, the invention provides an NMD activator comprising an isolated gene PNRC2 having the nucleotide sequence of SEQ ID NO: 1.

According to another aspect of the present invention, the present invention provides a method of activating NMD by inducing the expression of protein PNRC2 having the amino acid sequence of SEQ ID NO: 2.

According to another aspect of the present invention, there is provided an isolated mRNA monitoring polyprotein complex comprising protein PNRC2 and protein Upf1 having the amino acid sequence of SEQ ID NO: 2.

According to another aspect of the invention, there is provided an agent which inhibits or modulates binding of Upf1 or Dcpla to protein PNRC2 having the amino acid sequence of SEQ ID NO: 2 or binding of protein PNRC2 to Upf1 or Dcpla.

Hereinafter, the present invention will be described in more detail.

The present invention provides a gene comprising an isolated gene PNRC2 having a nucleotide sequence of SEQ ID NO: 1 involved in NMD, an isolated protein PNRC2 having an amino acid sequence of SEQ ID NO: 2 and a protein PNRC2 having an amino acid sequence of SEQ ID NO: 2 and a protein Upf1. To provide a multiprotein complex.

The present inventors identified PNRC2 (Human proline-rich nuclear receptor coregulatory protein 2) gene which binds Upf1, which is an important factor in NMD, and Dcp1a, which is a decapping enzyme.

Cloning the PNRC2 gene and performing PNRC2 immunoprecipitation (hereinafter, 'IP') and in vitro binding assays showed that PNRC2 binds to Upf1 and Dcpla (Fig. 1a to 1e). In addition, various experiments confirmed that Upf1-PNRC2-Dcp1a was present in the same complex (FIGS. 2A to 2C).

The present invention provides a method for inhibiting NMD by inhibiting the expression of NMD inhibitor including DNA that inhibits the expression of the isolated gene PNRC2 having the nucleotide sequence of SEQ ID NO: 1 and the protein PNRC2 having the amino acid sequence of SEQ ID NO: 2 .

Various methods can be used to inhibit the expression of the isolated gene PNRC2 having the nucleotide sequence of SEQ ID NO: 1. For example, small interfering double-stranded RNA represented by the following SEQ ID NO: 3 or SEQ ID NO: 4 (small interfering double-stranded RNA, referred to as "siRNA") can be specifically inhibited PNRC2 expression.

5'-r (UUGGAAUUCUAGCUUAUCA) d (TT) -3 '(SEQ ID NO: 3)

5'- r (AGUUGGAAUUCUAGCUUAU) d (TT) -3 '(SEQ ID NO: 4)

Inhibiting the expression of PNRC2 according to the present invention, it was confirmed that the efficiency of NMD, which is a lower stage of pioneer round of translation, is inhibited (FIGS. 3A to 3B).

In addition, as a result of confirming the intracellular location of PNRC2 according to the present invention through confocal laser scanning (confocal microscopy), it was found that it exists in the P-body (FIGS. 3C to 3E).

The present invention provides a method for activating NMD by inducing the expression of the NMD activator comprising the isolated gene PNRC2 having the nucleotide sequence of SEQ ID NO: 1 and the protein PNRC2 having the amino acid sequence of SEQ ID NO: 2.

PNRC2 according to the present invention can be artificially linked to activate NMD, and it has been shown to regulate NMD efficiency through interaction with Upf1 (FIGS. 4A to 4D). In addition, it was confirmed that the binding force between PNRC2 and Dcp1a is enhanced by hyperphosphorylation of Upf1 (FIGS. 5A to 5C).

As mentioned above, the present invention provides a novel gene PNRC2 that directly acts on pioneer round of translation, and thus plays an important role in NMD, and NMD inhibitors and methods of inhibiting it. . The present invention can be very useful for developing therapeutic agents or inhibitors of NMD-related genetic diseases and cancers through molecular biological understanding of NMD and artificial NMD efficiency control technology.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Experiment method and material

One. Immunoprecipitation  ( immunoprecipitation , IP )

After overexpressing the desired DNA in Cos7 cells, the expressed protein is precipitated using an antibody. Later, the western blot can be used to find proteins that settle together.

2. RT - PCR

Small genes are inhibited in HeLa cells using small interfering double-helix RNA (siRNA). After 2 days, the reporter used to measure NMD is overexpressed in the cell. After 1 day, RNA is obtained and converted to cDNA using reverse transcriptase. Amplify by PCR using specific primers for the gene of interest. At this time, the DNA amplified by the radiation ³² P. This material is run on a gel to obtain RT-PCR results.

3. Dyeing ( immunostaining )

HeLa cells are grown on cover slip. After overexpressing the desired DNA, two days later, staining is performed. First, it is fixed with paraformaldehyde, and then the cell membrane is pierced with Triton X-100. We stain the protein we want using a primary antibody (Antibody) and a fluorescent secondary antibody (Antibody). The nucleus is then stained with a reagent called DAPI. The resulting material is observed through confocal microscopy.

Example  1. According to the present invention New  gene PNRC2  Sympathy

The inventors have identified a human proline-rich nuclear receptor coregulatory protein 2 (PNRC2) gene (Refs. 2, 3, 4) that binds Upf1 and Dcpla. This was cloned and represented herein as SEQ ID NO: 1.

Hereinafter, the following facts were newly identified through various molecular biological approaches.

Example  2. PNRC2  Wow Upf1 Specific binding of

Yeast two-hybrid screening revealed that PNRC2 and Upf1 bind (FIG. 1A). 1B shows domains of proteins that bind Upf1 and specific sites that bind them. In order to confirm the binding between PNRC2 and Upf1 in different ways, after immunoexpression of FLAG-Upf1 and immunoprecipitation using FLAG antibody (IP), it was observed that the binding to Myc-PNRC2 (Fig. 1c). Conversely, when IP was performed with Myc-PNRC2, FLAG-Upf1 and Dcpla were combined (FIG. 1D). In addition, it was confirmed that PNRC2 directly binds to Upf1 through an in vitro binding assay (FIG. 1E). Therefore, the experiments confirmed that PNRC2 binds to Upf1.

Example  3. PNRC2  Wow Dcpla Specific binding of

Recently, a paper was published that combined PNRC2 with Dcp1a through yeast two-hybrid screening (Ref. 5). Several experiments were performed to confirm the specific binding of these two factors. First, when mammalian two hybrid assays were performed, binding of PNRC2 and Dcp1a could be confirmed in vivo (FIG. 2A). FLAG-Dcpla IP showed that PNRC2 and Upf1 formed a complex regardless of RNA (FIG. 2B). Finally, in vitro binding assays confirmed that PNRC2 binds directly to Dcpla (FIG. 2C). Therefore, the experiments confirmed that PNRC2 binds to Dcpla. These results indicate that Upf1-PNRC2-Dcp1a is present in the same complex.

Example  4. According to the present invention PNRC2 On by NMD Efficiency control and Intracellular  Check location

In order to determine the effect of PNRC2 in NMD, a sub-step of Pioneer round of translation, the expression of PNRC2 in cells was inhibited with siRNA (FIG. 3a), followed by NMD using quantitative RT-PCR techniques. The efficiency was measured. As a result, it could be seen that NMD efficiency is inhibited (FIG. 3B). This experiment confirms that PNRC2 affects NMD efficiency. And confocal laser scanning microscope (confocal microscopy) it can be seen that the PNRC2 present in the P-body according to the present invention (Figs. 3c to 3e).

Example  5. According to the present invention PNRC2 Artificial connection of tethering )through NMD Regulation of

In order to reconfirm whether PNRC2 according to the present invention is related to NMD, it was observed whether normal mRNA breaks when artificial tethering of PNRC2 to mRNA having a normal termination codon occurs.

First, by only connecting the PNRC2 to the back of the normal stop codon (tethering) it can be observed that the normal mRNA is broken and NMD occurs, while the NMD efficiency is reduced in the mRNA that eliminated the stop codon (Fig. 4a) ). When experimenting with other factors related to NMD Upf1, Upf2 it was seen that the NMD occurs with a similar efficiency (Fig. 4b). When the expression of PNRC2 protein was inhibited by siRNA, NMD efficiency by Upf1 and Upf2 was also lowered (FIG. 4C). When siRNA was used to inhibit the expression of Upf1 and Upf2 proteins, the NMD efficiency of PNRC2 was observed. When the upf1 expression was inhibited, NMD efficiency was lowered even though PNRC2 was tethered. However, when the upf2 expression was inhibited, the NMD efficiency remained the same (FIG. 4D). Taken together, we can conclude that PNRC2 binds to Upf1 and is a necessary factor for MND.

Example  6. Upf1 of Overphosphorylation  According PNRC2 Wow Dcp1a Confirm increase of binding with

When IP with FLAG-Upf1 and FLAG-Upf1 in the superphosphorylated form, Upf1 in the superphosphorylated form showed more specific binding with Myc-PNRC2 and Myc-Dcpla (FIG. 5A). Confocal laser microscopy of FLAG-Upf1 and FLAG-Upf1 in hyperphosphorylated form confirmed the location of cells in the p-body (FIG). 5b and 5c). Therefore, it was found that hyperphosphorylation of Upf1 further increased the binding of PNRC2 and Dcp1a, thus placing it in the P-body.

Example  7. PNRC2 When inhibiting the expression of Hyperphosphorylation  Form Upf1 Change of position and Upf1 and Dcpla  Impact on bonding between

When the expression of PNRC2 protein was inhibited through siRNA, the location of the hyperphosphorylated form of FLAG-Upf1 was not observed in the p-body through confocal microscopy (FIG. 6A and FIG. 6). 6b). Dcpla did not change the position in the cell even when inhibited the expression of PNRC2 protein (Figs. 6c and 6d). When IP was performed with FLAG-Dcpla while siRNA inhibited the expression of PNRC2 protein, the binding of the superphosphorylated Myc-Upf1 was significantly reduced (FIG. 6E). Therefore, it was found that when the expression of PNRC2 was inhibited, Upf1 in the superphosphorylated form was not located in the p-body and inhibited the binding between Upf1 and Dcpla.

Through the above experiments, it was confirmed that PNRC2 has high functionality as a target protein in the development of NMD regulation technology as an intracellular factor that regulates the efficiency of NMD. In addition, PNRC2 binds to Upf1 and Dcpla to bridge NMD and decapping machinery by acting as a bridge between them. It was possible to determine how NMD sends a decapping signal, which has not been identified so far, to remove mRNA with abnormal stop codons. As a result, NMD-related anticancer and hereditary diseases can be developed by inhibiting or activating the efficiency of NMD by PNRC2.

Although specific portions of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

[references]

Annu Rev Biochem / Chang, Y. F., Imam, J. S. & Wilkinson, M. F / 76, 51-74 (2007)

Nucleic Acids Res / Zhou and Chen / 29, 3939-3948 (2001)

3. Mol. Endocrinol / Zhou et al / 14, 986-998 (2000)

Nucleic Acids Res / Zhou et al / 34, 5974-5986 (2006)

5. Mol. Cell Proteomics / Albers et al / 4,205-213 (2005)

Figure 1a is a diagram showing the specific binding between PNRC2 and Upf1 according to the present invention as a result of performing yeast two-hybrid screening (yeast two-hybrid screening).

1B shows domains of proteins that bind to Upf1 and specific sites that bind to them.

FIG. 1C is a diagram showing that after overexpressing FLAG-Upf1, immunoprecipitation (IP) using FLAG antibody binds to Myc-PNRC2.

FIG. 1D is a view showing that FLAG-Upf1 and Dcpla are combined when IP is performed with Myc-PNRC2.

1E is a diagram showing that PNRC2 directly binds to Upf1 according to the present invention through an in vitro binding assay.

2A is a diagram showing the results of confirming the binding of PNRC2 and Dcp1a in vivo when the mammalian two-hybrid assay was performed.

2B shows that PNRC2 and Upf1 form a complex regardless of RNA via FLAG-Dcpla IP.

FIG. 2C shows that PNRC2 binds directly to Dcpla through an in vitro binding assay.

Figure 3a is a diagram showing the results confirmed that the inhibition of the expression of PNRC2 in cells with siRNA.

Figure 3b is a diagram showing that the efficiency of NMD was specifically inhibited as a result of inhibiting the expression of PNRC2 in the cells with siRNA.

3C to 3E show confocal microscopy of PNRC2 present in the P-body according to the present invention.

Figure 4a is a diagram showing that the normal mRNA is broken by only connecting (teethering) the PNRC2 in the back of the normal stop codon according to the present invention, NMD efficiency is lowered in the mRNA that eliminates the stop codon.

Figure 4b is a diagram showing that the NMD occurs with similar efficiency when experimenting with other factors related to NMD Upf1, Upf2 together.

Figure 4c is a diagram showing that when the expression of the PNRC2 protein using siRNA, NMD efficiency by Upf1, Upf2 is lowered.

4D shows that when NNR efficiency by PNRC2 was observed after inhibiting the expression of Upf1 and Upf2 proteins using siRNA, NMD efficiency was lowered even though PNRC2 was tethered when Upf1 was inhibited. In the case of inhibiting the expression of Upf2, the NMD efficiency remains the same.

Figure 5a is a diagram showing a more specific binding of the hyperphosphorylated Upf1 and Myc-PNRC2, Myc-Dcpla as a result of IP to FLAG-Upf1 and FLAG-Upf1 in the superphosphorylated form.

5B and 5C show the location of the FLAG-Upf1 and the superphosphorylated form of FLAG-Upf1 in the cell through confocal laser microscopy, where the superphosphorylated form of FLAG-Upf1 is located in the p-body. Figure showing that.

6A and 6B show the results of observation through confocal microscopy that the position of the superphosphorylated form of FLAG-Upf1 does not exist in the p-body when the expression of PNRC2 protein is inhibited through siRNA. Figure showing.

6C and 6D show that there is no change in the position of Dcpla in cells even when the expression of PNRC2 protein is inhibited.

FIG. 6E is a diagram showing that the binding of the hyperphosphorylated Myc-Upf1 is reduced when IP is performed with FLAG-Dcpla while siRNA inhibits the expression of PNRC2 protein.

7 is a schematic diagram showing the relationship between NMD control by PNRC2 according to the present invention.

<110> Korea University Industrial and Academic Collaboration Foundation <120> Method for control of nonsense-mediated mRNA decay using gene          PNRC2 <130> P11-080417-01 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 420 <212> DNA <213> Homo sapiens <400> 1 atgggtggtg gagagaggta taacattcca gcccctcaat ctagaaatgt tagtaagaac 60 caacaacagc ttaacagaca gaagaccaag gaacagaatt cccagatgaa gattgttcat 120 aagaaaaaag aaagaggaca tggttataac tcatcagcag ctgcctggca ggccatgcaa 180 aatgggggga agaacaaaaa ttttccaaat aatcaaagtt ggaattctag cttatcaggt 240 cccaggttac tttttaaatc tcaagctaat cagaactatg ctggtgccaa atttagtgag 300 ccgccatcac caagtgttct tcccaaacca ccaagccact gggtccctgt ttcctttaat 360 ccttcagata aggaaataat gacatttcaa cttaaaacct tacttaaagt acaggtataa 420                                                                          420 <210> 2 <211> 139 <212> PRT <213> Homo sapiens <400> 2 Met Gly Gly Gly Glu Arg Tyr Asn Ile Pro Ala Pro Gln Ser Arg Asn   1 5 10 15 Val Ser Lys Asn Gln Gln Gln Leu Asn Arg Gln Lys Thr Lys Glu Gln              20 25 30 Asn Ser Gln Met Lys Ile Val His Lys Lys Lys Glu Arg Gly His Gly          35 40 45 Tyr Asn Ser Ser Ala Ala Ala Trp Gln Ala Met Gln Asn Gly Lys      50 55 60 Asn Lys Asn Phe Pro Asn Asn Gln Ser Trp Asn Ser Ser Leu Ser Gly  65 70 75 80 Pro Arg Leu Leu Phe Lys Ser Gln Ala Asn Gln Asn Tyr Ala Gly Ala                  85 90 95 Lys Phe Ser Glu Pro Pro Ser Pro Ser Val Leu Pro Lys Pro Pro Ser             100 105 110 His Trp Val Pro Val Ser Phe Asn Pro Ser Asp Lys Glu Ile Met Thr         115 120 125 Phe Gln Leu Lys Thr Leu Leu Lys Val Gln Val     130 135 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> Small interfering double-stranded RNA <220> <223> small interfering double-stranded RNA 5'-          r (UUGGAAUUCUAGCUUAUCA) d (TT) -3 ' <400> 3 uuggaauucu agcuuaucat t 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> Small interfering double-stranded RNA <220> <223> small interfering double-stranded RNA 5'-          r (AGUUGGAAUUCUAGCUUAU) d (TT) -3 ' <400> 4 aguuggaauu cuagcuuaut t 21  

Claims (11)

delete delete In non-human animals, expression of protein PNRC2 having the amino acid sequence of SEQ ID NO: 2 is inhibited by small interfering double-stranded RNA (siRNA) sequences represented by SEQ ID NO: 3 or SEQ ID NO: How to inhibit. 4. The method of claim 3, wherein the protein is involved in pioneer round of translation by acting as a bridge between Upf1 and Dcpla. delete delete delete delete delete delete delete

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