Micropeptides and use of same for modulating gene expression

Abstract

Process for detecting and identifying micropeptides (miPEPs) encoded by a nucleotide sequence contained in the sequence of the primary transcript of a microRNA and use thereof for modulating gene expression.

1 . Process for detecting and identifying a micropeptide (miPEP) encoded by a nucleotide sequence contained in the sequence of the primary transcript of a microRNA, comprising:
 a) a step of detecting an open reading frame from 12 to 303 nucleotides in length contained in the sequence of the primary transcript of said microRNA, then   b) a step of comparison between:
 the accumulation of said microRNA in a specified eukaryotic cell expressing said microRNA, 
 in the presence of a peptide encoded by a nucleotide sequence that is identical or degenerate relative to that of said open reading frame, said peptide being present in the cell independently of transcription of the primary transcript of said microRNA, and 
 the accumulation of said microRNA in a eukaryotic cell of the same type as the aforesaid specified eukaryotic cell expressing said microRNA, 
 in the absence of said peptide, 
   
       in which a modulation of the accumulation of said microRNA in the presence of said peptide relative to the accumulation of said microRNA in the absence of said peptide indicates the existence of a micropeptide encoded by said open reading frame. 
     
     
         2 . Process for detecting and identifying a miPEP according to  claim 1 , in which the modulation of the accumulation of said microRNA is a decrease or an increase in the accumulation of said microRNA, in particular an increase. 
     
     
         3 . Process for detecting and identifying a miPEP according to  claim 1 , in which the presence of the peptide in the cell results from:
 the introduction of a nucleic acid encoding said peptide into the cell, or   the introduction of said peptide into the cell.   
     
     
         4 . Process for detecting and identifying a miPEP according to  claim 1 , in which said open reading frame in step a) is contained in the 5′ or 3′ portion of said primary transcript of the microRNA, preferably in the 5′ portion. 
     
     
         5 . Process for detecting and identifying a miPEP according to  claim 1 , in which said microRNA is present in a wild-type plant cell or in a wild-type animal cell, and in which said eukaryotic cell used in step b) is a plant cell, preferably of a crucifer, of a leguminous plant or of a plant of the Solanaceae family or is an animal cell, preferably a human cell or  Drosophila  cell. 
     
     
         6 . Process for detecting and identifying a miPEP according to  claim 1 , in which said microRNA is of endogenous origin in said eukaryotic cell used in step b). 
     
     
         7 . Process for detecting and identifying a miPEP according to  claim 1 , in which said microRNA is of exogenous origin in said eukaryotic cell used in step b), said eukaryotic cell containing a vector allowing the expression of said microRNA. 
     
     
         8 . Process for detecting and identifying a miPEP according to  claim 1 , in which the accumulation of said microRNA is determined using quantitative RT-PCR, Northern blot, a DNA or RNA chip. 
     
     
         9 . MiPEP encoded by the nucleotide sequence as obtained by the process according to  claim 1 . 
     
     
         10 . MiPEP from 3 to 100 amino acids, preferably from 4 to 40 amino acids, encoded by n open reading frame from 12 to 303 nucleotids contained in a nucleotide sequence contained in the primary transcript of a microRNA, said miPEP being capable of modulating the accumulation of said microRNA in a eukaryotic cell, said nucleotide sequence being contained in the 5′ or 3′ portion of said primary transcript of a microRNA, preferably in the 5′ portion,
 the modulation of the microRNA by said miPEP being evidenced by observing a variation in the amounts of microRNA quantities between cells in the presence or in the absence of miPEP. 
 
     
     
         11 . MiPEP according to  claim 9 , said miPEP being selected from the group of peptides consisting of:
 SEQ ID NO: 1 to SEQ ID NO: 104 and SEQ ID NO: 355,   SEQ ID NO: 375 to SEQ ID NO: 386, or   SEQ ID NO: 424.   
     
     
         12 . Nucleic acid molecule encoding a miPEP as defined according to  claim 9 , said nucleic acid molecule being selected in particular from the group of nucleic acids consisting of:
 SEQ ID NO: 105 to SEQ ID NO: 208 and SEQ ID NO: 356,   SEQ ID NO: 387 to SEQ ID NO: 399, or   SEQ ID NO: 425.   
     
     
         13 . A method for modulating the expression of a gene in a specified eukaryotic cell, comprising exposing said specified eukaryotic cell at least:
 a miPEP as defined according to  claim 9 ,   a nucleic acid encoding said miPEP, or   a vector containing said nucleic acid,   said specified eukaryotic cell being capable of expressing a microRNA, the primary transcript of which contains at least one nucleotide sequence encoding said at least one miPEP and the accumulation of which is modulated by said at least one miPEP,   the expression of said gene being regulated by said microRNA,   said miPEP in particular being selected from the group of peptides consisting of:   SEQ ID NO: 1 to SEQ ID NO: 104 and SEQ ID NO: 355,   SEQ ID NO: 375 to SEQ ID NO: 386, or   SEQ ID NO: 424,   in particular:   SEQ ID NO: 59, SEQ ID NO: 24, SEQ ID NO: 43, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 14, SEQ ID NO: 63 and SEQ ID NO: 424,   said nucleic acid in particular being selected from the group of nucleic acids consisting of:   SEQ ID NO: 105 to SEQ ID NO: 208 and SEQ ID NO: 356,   SEQ ID NO: 387 to SEQ ID NO: 399, or   SEQ ID NO: 425,   in particular:   SEQ ID NO: 163, SEQ ID NO: 128, SEQ ID NO: 147, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 118, SEQ ID NO: 425 and SEQ ID NO: 167,   said microRNA being selected in particular from the group of nucleic acids consisting of:   SEQ ID NO: 282 to SEQ ID NO: 354, and SEQ ID NO: 358,   SEQ ID NO: 412 to SEQ ID NO: 423, or   SEQ ID NO: 427,   in particular SEQ ID NO: 319, SEQ ID NO: 297, SEQ ID NO: 305, SEQ ID NO: 331, SEQ ID NO: 291, SEQ ID NO: 322 and SEQ ID NO: 427.   
     
     
         14 . Composition comprising at least:
 a miPEP as defined according to  claim 9 ,   a nucleic acid encoding said miPEP, or   a vector containing said nucleic acid.   
       for use as a human or veterinary medicament. 
     
     
         15 . Process for modulating the expression of a gene regulated by a microRNA in a eukaryotic plant or animal cell, in particular a human cell,
 comprising carrying out a step of accumulation of a miPEP in said eukaryotic cell, said miPEP having:
 a size from 3 to 100 amino acids, preferably 4 to 20 amino acids, and 
 a peptide sequence identical to that encoded by a nucleotide sequence contained in the primary transcript of a microRNA regulating the expression of said gene, and 
 being capable of modulating the accumulation of said microRNA, 
   in which the accumulation of said miPEP in said eukaryotic cell induces a modulation of the expression of said gene relative to the expression of said gene without accumulation of said miPEP,   said process not being used for surgical or therapeutic treatment of the human body or animal body, nor for modifying the genetic identity of a human being.   
     
     
         16 . A method for promoting the growth and/or development of plants, in particular for modulating the physiological parameters of a plant, in particular the biomass, foliar surface area, flowering, fruit size, production and/or selection of plant seeds, in particular for controlling the parthenocarpy or monoecism of a plant, or for modifying the physiological parameters of plant seeds, in particular germination, establishment of the root system and resistance to water stress, or for preventing or treating plant diseases,in particular for promoting resistance to infectious diseases, comprising administering to a plant in need there of a composition comprising at least:
 a miPEP as defined according to  claim 9 ,   a nucleic acid encoding said miPEP, or   a vector containing said nucleic acid,   
       as a phytopharmaceutical agent. 
     
     
         17 . A method for production and/or selection of plant seeds, comprising applying a coating or film-coating of said plant seeds a composition comprising at least:
 a miPEP as defined according  claim 9 ,   a nucleic acid encoding said miPEP, or   a vector containing said nucleic acid,   
       as a phytopharmaceutical agent. 
     
     
         18 . A method for eradicating plants or slowing their growth comprising administering to the plants a composition at least:
 a miPEP as defined according to  claim 9 ,   a nucleic acid encoding said miPEP, or   a vector containing said nucleic acid,   
       as a herbicide. 
     
     
         19 . A method for eradicating organisms harmful to plants or liable to be classified as such, in particular as insecticide, arachnicide, molluscicide, or rodenticide, comprising applying to a plant or to a support in contact with the plantcomposition comprising at least:
 a miPEP as defined according to  claim 9 ,   a nucleic acid encoding said miPEP, or   a vector containing said nucleic acid,   
       as a pesticide. 
     
     
         20 . A composition comprising, in combination, a quantity of seeds of a plant and a quantity of a peptide the sequence of which comprises or consists of a sequence identical to that of a miPEP naturally present in said plant, in particular said composition being formulated so as to form a coated seed.