Micropeptides and use thereof 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 15 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 as obtained by the process according to  claim 1 . 
     
     
         10 . MiPEP from 4 to 100 amino acids, preferably from 4 to 40 amino acids, encoded by 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. 
     
     
         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, SEQ ID NO: 375 to 386 and SEQ ID NO: 355. 
     
     
         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, SEQ ID NO 387 to SEQ ID NO: 399 and SEQ ID NO: 356. 
     
     
         13 . A method for modulating the expression of a gene in a specified eukaryotic cell, comprising inserting into said specified eukaryotic cell at least one of an miPEP as defined in  claim 10 , 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 104, SEQ ID NO: 375 to SEQ ID NO: 386 and SEQ ID NO: 355, in particular SEQ ID NO: 59, SEQ ID NO: 43, and SEQ ID NO: 77, said nucleic acid in particular being selected from the group of nucleic acids consisting of SEQ ID NO: 105 to 208, SEQ ID NO: 387 to SEQ ID NO: 399 and SEQ ID NO: 356, in particular SEQ ID NO: 163, SEQ ID NO: 147, 181, said microRNA being selected in particular from the group of nucleic acids consisting of SEQ ID NO: 282 to 354, SEQ ID NO: 412 to 423 and SEQ ID NO 358, in particular SEQ ID NO: 319, SEQ ID NO: 305 and SEQ ID NO: 331.   
     
     
         14 . Composition comprising at least one:
 miPEP as defined according to  claim 10 ,   nucleic acid encoding said miPEP, or   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 4 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 (i) 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 (ii) for preventing or treating plant diseases, in particular for promoting resistance to infectious diseases, comprising administering to a plant a composition comprising at least one of an miPEP as defined according to  claim 10 , a nucleic acid encoding said miPEP, or a vector containing said nucleic acid, as a phytopharmaceutical agent.   
     
     
         17 . A method for eradicating plants or slowing their growth, comprising administering to said plants a composition comprising at least one of an miPEP as defined according to  claim 10 , a nucleic acid encoding said miPEP, or a vector containing said nucleic acid, as an herbicide. 
     
     
         18 . 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 a composition comprising at least one of an miPEP as defined according to  claim 10 , a nucleic acid encoding said miPEP, as a pesticide, to a plant or to a support in contact with the plant.