Regulation of translation of expressed genes

Abstract

The present invention describes mRNA usage improving and/or translation-enhancing nucleic acid sequences, nucleic acid constructs comprising such sequences, and host cells comprising such nucleic acid constructs. The invention further pertains to a method for expressing a protein of interest in a cell or organism using such nucleic acid sequences, as well as their uses for increasing integration of such nucleic acid construct into a genome, for enhancing mRNA usage and/or translation of a recombinantly expressed polypeptide, and for increasing the number of transformants upon transformation of a cell with such nucleic acid construct.

1 . A method for expressing a protein of interest in a cell or organism, said organism not being a human, said method comprising the steps of:
 a) providing a nucleic acid construct comprising:
 i) a first nucleotide sequence that comprises: a TC-rich nucleotide sequence comprising at least 8 consecutive C or T nucleotides, at least 3 A-rich nucleotide sequences comprising at least 5 consecutive A nucleotides, a GT-rich nucleotide sequence comprising at least 10 nucleotides, at least 80% of which are G or T nucleotides, and an intron sequence comprising at least splice sites required for splicing of the intron sequence, and optionally, a GAA repeat nucleotide sequence, 
 ii) a first nucleotide sequence that comprises: a TC-rich nucleotide sequence comprising at least 8 consecutive C or T nucleotides, at least 3 A-rich nucleotide sequences comprising at least 5 consecutive A nucleotides, and a GT-rich nucleotide sequence comprising at least 10 nucleotides, at least 80% of which are G or T nucleotides, said first nucleotide sequence not comprising a GAA repeat nucleotide sequence, or 
 iii) a first nucleotide sequence that comprises: a GAA repeat nucleotide sequence, a TC-rich nucleotide sequence comprising at least 8 consecutive C or T nucleotides, at least 3 A-rich nucleotide sequences comprising at least 5 consecutive A nucleotides, a GT-rich nucleotide sequence comprising at least 10 nucleotides, at least 80% of which are G or T nucleotides, wherein said GAA repeat nucleotide sequence is located 3′ of any one or more of said TC-rich nucleotide sequence, A-rich nucleotide sequences, and/or GT-rich nucleotide sequence, 
 operably linked to a second nucleotide sequence encoding a protein or polypeptide of interest, and further operably linked to a promoter, 
   b) contacting a cell with said nucleic acid construct to obtain a transformed cell, and   c) allowing said transformed cell to express the protein or polypeptide of interest, and optionally recovering said protein or polypeptide.   
     
     
         2 . A method according to  claim 1 , wherein the protein is expressed in an organism, said method further comprising the steps of:
 a) contacting a target cell and/or target tissue of said organism with said nucleic acid construct to obtain a transformed target cell and/or transformed target tissue and/or transformed organism, and   b) optionally, allowing said transformed target cell and/or transformed target tissue to develop into a transformed organism, and   c) allowing said transformed organism to express the protein or polypeptide of interest, and optionally recovering said protein or polypeptide.   
     
     
         3 . A method according to  claim 1 , wherein the cell is selected from the group consisting of an animal, such as mammalian or insect, cell, plant cell, fungal cell, yeast cell, and prokaryotic cell, or wherein the organism is selected from the group consisting of a non-human animal, and a plant. 
     
     
         4 . An isolated nucleotide sequence comprising: a TC-rich nucleotide sequence comprising at least 8 consecutive C or T nucleotides, at least 3 A-rich nucleotide sequences comprising at least 5 consecutive A nucleotides, a GT-rich nucleotide sequence comprising at least 10 nucleotides, at least 80% of which are G or T nucleotides, and an intron sequence comprising at least splice sites required for splicing of the intron sequence, and, optionally, a GAA repeat nucleotide sequence. 
     
     
         5 . An isolated nucleotide sequence according to  claim 4 , wherein a GAA repeat nucleotide sequence is present, and said GAA repeat nucleotide sequence is located 5′ of said TC-rich nucleotide sequence, A-rich nucleotide sequences, GT-rich nucleotide sequence, and intron sequence, or said GAA repeat nucleotide sequence is located 3′ of any one or more of said TC-rich nucleotide sequence, A-rich nucleotide sequences, GT-rich nucleotide sequence, and/or intron sequence. 
     
     
         6 . An isolated nucleotide sequence comprising: a TC-rich nucleotide sequence comprising at least 8 consecutive C or T nucleotides, at least 3 A-rich nucleotide sequences comprising at least 5 consecutive A nucleotides, and a GT-rich nucleotide sequence comprising at least 10 nucleotides, at least 80% of which are G or T nucleotides, said nucleotide sequence not comprising a GAA repeat nucleotide sequence. 
     
     
         7 . An isolated nucleotide sequence comprising: a GAA repeat nucleotide sequence, a TC-rich nucleotide sequence comprising at least 8 consecutive C or T nucleotides, at least 3 A-rich nucleotide sequences comprising at least 5 consecutive A nucleotides, a GT-rich nucleotide sequence comprising at least 10 nucleotides, at least 80% of which are G or T nucleotides, wherein said GAA repeat nucleotide sequence is located 3′ of any one or more of said TC-rich nucleotide sequence, A-rich nucleotide sequences, and/or GT-rich nucleotide sequence. 
     
     
         8 . A nucleotide sequence according to  claim 4 , wherein the nucleotide sequence further comprises a CAA repeat nucleotide sequence. 
     
     
         9 . A nucleotide sequence according to  claim 4 , wherein the nucleotide sequence further comprises a TATA nucleotide sequence. 
     
     
         10 . A nucleotide sequence according to  claim 8 , wherein the CAA repeat nucleotide sequence replaces a GAA repeat nucleotide sequence. 
     
     
         11 . A nucleic acid construct comprising an isolated nucleic acid sequence according to  claim 4 , operably linked to a second nucleotide sequence encoding a protein or polypeptide of interest, and further operably linked to a promoter. 
     
     
         12 . A host cell comprising a nucleic acid sequence according to  claim 4  or a nucleic acid construct according to  claim 11 . 
     
     
         13 . A host cell according to  claim 12 , being selected from the group consisting of an animal, such as mammalian or insect, cell, plant cell, fungal cell, yeast cell, and prokaryotic cell. 
     
     
         14 . A non-human transformed organism transformed with a nucleic acid sequence according  claim 4 , or a nucleic acid construct according to  claim 11 . 
     
     
         15 . An organism according to  claim 14 , said organism being selected from a non-human animal and a plant.