Translational control system using rna-protein interaction motif

Abstract

A translational control method using an RNA-protein interaction motif is provided. The method comprises a step of introducing an mRNA having: a 5′UTR regulation structure comprising: (1) a cap structure at the 5′ terminus, (2) a spacer positioned on the 3′ side of the cap structure, and (3) one or more RNA motifs positioned on the 3′ side of the spacer, which comprises an RNA-protein interaction motif-derived nucleotide sequence or a variant thereof; and a nucleotide sequence encoding a target protein gene on the 3′ side of the 5′UTR regulation structure, into a cell in the presence of a protein specifically binding to the RNA motifs, wherein a translational level is decreased as the number of bases of the spacer decreases, and the translational level is decreased as the number of the RNA motifs increases.

1 . A translational control method using an RNA-protein interaction motif, comprising a step of introducing an mRNA having: a 5′UTR regulation structure comprising:
 (1) a cap structure at the 5′ terminus, 
 (2) a spacer positioned on the 3′ side of the cap structure, and 
 (3) one or more RNA motifs positioned on the 3′ side of the spacer, which comprises an RNA-protein interaction motif-derived nucleotide sequence or a variant thereof; and a nucleotide sequence encoding a target protein gene on the 3′ side of the 5′UTR regulation structure, into a cell in the presence of a protein specifically binding to the RNA motifs, 
 wherein a translational level is decreased as the number of bases of the spacer decreases, and the translational level is decreased as the number of the RNA motifs increases. 
 
     
     
         2 . The method according to  claim 1 , wherein the number of bases of the spacer is 0 to 350. 
     
     
         3 . The method according to  claim 1 , wherein the number of the RNA motifs is 1 to 4. 
     
     
         4 . The method according to  claim 1 , wherein each of the RNA motifs is a binding sequence selected from the group consisting of a binding sequence of L7Ae protein, a binding sequence of MS2 phage coat protein and a binding sequence of  Bacillus stearothermophilus  ribosomal protein S15. 
     
     
         5 . An mRNA translational level decreasing method, comprising the step of providing, on the 5′ side of a nucleotide sequence encoding a target protein gene, a 5′UTR regulation structure comprising:
 (1) a cap structure at the 5′ terminus; 
 (2) a spacer positioned on the 3′ side of the cap structure; and 
 (3) one or more RNA motifs positioned on the 3′ side of the spacer, which comprises an RNA-protein interaction motif-derived nucleotide sequence or a variant thereof, 
 wherein translational level is decreased as the number of bases of the spacer decreases, and the translational level is decreased as the number of the RNA motifs increases. 
 
     
     
         6 . The method according to  claim 5 , wherein the number of bases of the spacer is 0 to 350. 
     
     
         7 . The method according to  claim 5 , wherein the number of the RNA motifs is 1 to 4. 
     
     
         8 . The method according to  claim 5 , wherein each of the RNA motifs is a binding sequence selected from the group consisting of a binding sequence of L7Ae protein, a binding sequence of MS2 phage coat protein and a binding sequence of  Bacillus stearothermophilus  ribosomal protein S15. 
     
     
         9 . An mRNA comprising:
 a 5′UTR regulation structure comprising
 (1) a cap structure at the 5′ terminus, 
 (2) a spacer positioned on the 3′ side of the cap structure, and 
 (3) one or more RNA motifs positioned on the 3′ side of the spacer, which comprises an RNA-protein interaction motif-derived nucleotide sequence or a variant thereof; and 
   a nucleotide sequence encoding a target protein gene on the 3′ side of the 5′UTR regulation structure,   wherein translational level of the target protein is decreased in the presence of a protein specifically binding to the RNA motifs.   
     
     
         10 . The mRNA according to  claim 9 , wherein the number of bases of the spacer is 0 to 350. 
     
     
         11 . The mRNA according to  claim 10 , wherein the number of bases of the spacer is 51 to 350. 
     
     
         12 . The mRNA according to  claim 9 , wherein the number of the RNA motifs is 1 to 4. 
     
     
         13 . The mRNA according to  claim 12 , wherein the number of the RNA motifs is 2 to 4. 
     
     
         14 . The mRNA according to  claim 9 , wherein each of the RNA motifs is a binding sequence selected from the group consisting of a binding sequence of L7Ae protein, a binding sequence of MS2 phage coat protein and a binding sequence of  Bacillus stearothermophilus  ribosomal protein S15. 
     
     
         15 . A translational control system comprising the mRNA according to  claim 9  and a protein specifically binding to the RNA motifs. 
     
     
         16 . A vector comprising a nucleic acid sequence encoding the mRNA according to  claim 9 . 
     
     
         17 . A method for selecting an exogenous mRNA that translates a protein at a freely selected level in a cell, comprising the steps of:
 (1) introducing the mRNA according to  claim 9  into a cell that expresses a protein specifically binding to a corresponding RNA motif; and   (2) measuring a translational level of the protein to identify the mRNA providing a desired translational level.   
     
     
         18 . A method for regulating expression levels of target proteins which proteins are independently at different levels from a plurality of different mRNAs encoding different target protein genes, comprising the steps of:
 introducing a first mRNA, which has a cap structure at the 5′ terminus, comprises a nucleotide sequence encoding a first target protein gene, and has, on the 3′ side of the cap structure and the 5′ side of an initiation codon, a first regulation structure comprising a spacer and one or more first RNA motifs of an RNA-protein interaction motif-derived nucleotide sequence or a variant thereof, into a cell in the presence of a protein specifically binding to the first RNA motifs; and   introducing a second mRNA, which has a cap structure at the 5′ terminus, comprises a nucleotide sequence encoding a second target protein gene, and has, on the 3′ side of the cap structure and the 5′ side of an initiation codon, a second regulation structure comprising a spacer, and one or more second RNA motifs of an RNA-protein interaction motif-derived nucleotide sequence or a variant thereof, into the cell in the presence of a protein specifically binding to the second RNA motifs,   wherein the first regulation structure and the second regulation structure are different from each other in the number of bases of the spacer and/or the number of the RNA motifs, and   the first RNA motif and the second RNA motif are the same as each other, or the first RNA motif and the second RNA motif are variants specifically binding to the same protein but having different dissociation constants for the same protein.   
     
     
         19 . The method according to  claim 18 , wherein the number of bases of the spacer is 0 to 350. 
     
     
         20 . The method according to  claim 18 , wherein the number of the RNA motifs is 1 to 4. 
     
     
         21 . The method according to  claim 18 , wherein each of the RNA motifs is a binding sequence selected from the group consisting of a binding sequence of L7Ae protein, a binding sequence of MS2 phage coat protein and a binding sequence of  Bacillus stearothermophilus  ribosomal protein S15.