Protection against environmental toxicity through manipulation of the processing of messenger RNA precursors

Abstract

This invention describes the identification of pre-messenger RNA processing as a novel target of environmental stress caused by example of lithium and sodium toxicity. Overexpression of different types of proteins (or protein fragments) from different organisms but all involved in pre-mRNA processing, protects yeast from salt stress, which indicates that any stimulation of this process, independently of its mechanism, may counteract the toxic effects of mineral salts. A similar phenotype of tolerance to NaCl and to LiCl has been observed by overexpression of these types of proteins in transgenic Arabidopsis plants, demonstrating the generality of this protective effect in eukaryotic cells and organisms.

1 . A method to enhance stress tolerance in cells and organisms comprising the manipulation of the process of processing messenger RNA precursors. 
     
     
         2 . A method to enhance salt toxicity in cells and organisms comprising the manipulation of the process of processing messenger RNA precursors. 
     
     
         3 . A method according to  claim 1  or  2  wherein said manipulation comprises the genetic or biochemical manipulation of a molecule involved in or interfering with the process of processing messenger RNA precursors. 
     
     
         4 . A method according to  claim 1  wherein said stress tolerance comprises tolerance against osmotic, drought, cold or freezing stress. 
     
     
         5 . A method according to  claim 1  comprising the genetic or biochemical manipulation of a protein possessing a domain with a high content in Arg-Ser, Arg-Glu and Arg-Asp dipeptides (RS domain). 
     
     
         6 . A method according to  claim 1  comprising the genetic or biochemical manipulation of a protein of an RNA binding protein. 
     
     
         7 . A method according to  claim 1  comprising the genetic or biochemical manipulation of a component of the U1-snRNP or the U2-snRNP complex. 
     
     
         8 . A method according to  claim 1  comprising the genetic or biochemical manipulation of a transcription factor. 
     
     
         9 . A method according to  claim 1  comprising the genetic or biochemical manipulation of a nuclear movement protein. 
     
     
         10 . An isolated nucleic acid molecule encoding a protein or an immunologically active and/or functional fragment thereof selected from the group consisting of:
 (a) a nucleic acid molecule comprising a DNA sequence as set forth in any of SEQ ID NOs: 1, 2, 5, 7, 9, 11, 13, 15, 17 or 19 or the complement thereof,   (b) a nucleic acid molecule comprising an RNA sequence corresponding to any of SEQ ID NOs 1, 2, 5, 7, 9, 11, 13, 15, 17 or 19 as in (a) or the complement thereof,   (c) a nucleic acid molecule specifically hybridizing to the nucleotide sequence as defined in (a) or (b),   (d) a nucleic acid encoding a protein with an amino acid sequence which is at least 95% identical to the polypeptide set forth in any of SEQ ID NOs: 3, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21,   (e) a nucleic acid molecule encoding a protein comprising the amino acid sequence as given in any of SEQ ID NOs: 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 21 or 22,   (f) a nucleic acid molecule which is degenerated as a result of the genetic code to a nucleotide sequence of a nucleic acid molecule as set forth in any of SEQ ID NOs: 1, 2, 5, 7, 9, 11, 13, 15, 17 or 19 or as defined (a) to (e),   (g) a nucleic acid molecule which is diverging due to the differences in codon usage between the organisms to a nucleotide sequence encoding a protein as set forth in any of SEQ ID NOs 3, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21 or as defined in (a) to (e),   (h) a nucleic acid molecule which is diverging due to the differences in alleles encoding a protein as set forth in any of SEQ ID NOs: 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 21 or as defined in (a) to (e),   (i) a nucleic acid molecule encoding an immunologically active and/or functional fragment of a protein encoded by a DNA sequence as set forth in any of SEQ ID NOs: 1, 2, 5, 7, 9, 11, 13, 15, 17 or 19 or as defined in (a) to (e),   (j) a nucleic acid molecule encoding a protein as set forth in SEQ ID NOs: 3, 4, 6, 8, 10, 12, 14, 16, 18, 20 or 21 or as defined in (a) to (i) characterized in that said sequence is DNA, cDNA, genomic DNA or synthetic DNA.   
     
     
         11 . A nucleic acid molecule of at least 15 nucleotides in length specifically hybridizing with a nucleic acid molecule of  claim 10 . 
     
     
         12 . A nucleic acid molecule of at least 15 nucleotides in length specifically amplifying a nucleic acid molecule of  claim 10 . 
     
     
         13 . A vector comprising a nucleic acid molecule according to  claim 10 . 
     
     
         14 . A vector according to  claim 13  which is an expression vector wherein the nucleic acid is operably linked to one or more control sequences allowing the expression of said sequence in prokaryotic and/or eukaryotic host cells. 
     
     
         15 . A host cell containing a nucleic acid molecule according to  claim 10  or a vector according to  claim 13  or  14 . 
     
     
         16 . A host cell comprising the vector of  claim 14  wherein the host cell is a bacterial, insect, fungal, yeast, plant or animal cell. 
     
     
         17 . An anti-sense molecule targeted to at least one nucleic acid molecule selected from the group consisting of nucleic acid molecules having sequences as set forth in SEQ ID NOs: 7, 9, 11, 13, 15, 17 or 19. 
     
     
         18 . The method of  claim 1  wherein the cells express an anti-sense molecule targeted to at least one nucleic acid molecule selected from the group consisting of nucleic acid molecules having sequences as set forth in SEQ ID NOs: 7, 9, 11, 13, 15, 17, or 19. 
     
     
         19 . A polypeptide encodable by at least one of the nucleic acid molecules defined in  claim 10 , or a homologue thereof or a derivative thereof, or an immunologically active and/or functional fragment thereof. 
     
     
         20 . The polypeptide of  claim 19  comprising an amino acid sequence as given in any of SEQ ID Nos 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 21 or 22 or a homologue thereof or a derivative thereof, or an immunologically active and/or functional fragment thereof. 
     
     
         21 . A method of producing a polypeptide according to  claim 19  or  20  comprising culturing a host cell of  claim 16  under conditions allowing expression of the polypeptide and recovering the produced polypeptide from the culture. 
     
     
         22 . A method for the production of transgenic plant, plant cell or plant tissue comprising the introduction of a nucleic acid molecule according to  claim 10  in an expressible format or a vector in said plant, plant cell or plant tissue. 
     
     
         23 . A method for the production of altered plants, plant cells or plant tissues comprising the introduction of a polypeptide of  claim 19  or  20  directly into a cell, a tissue or an organ of said plant. 
     
     
         24 . A method for effecting the expression of a polypeptide of  claim 19  or  20  comprising the introduction of a nucleic acid molecule of  claim 10  operably linked to one or more control sequences stably into the genome of a plant cell. 
     
     
         25 . The method of  claim 22  further comprising regenerating a plant from said plant cell. 
     
     
         26 . The transgenic plant cell obtainable by a method of  claim 25  wherein said nucleic acid is stably integrated into the genome of said plant cell. 
     
     
         27 . Transgenic plants tolerant to salt stress as a result of the expression of at least one of the nucleic acid molecules of  claim 10  or an anti-sense molecule of  claim 17 . 
     
     
         28 . Transgenic plants which as a result of the expression of at least one of the nucleic acid molecules of  claim 10  or an anti-sense molecule of  claim 17  show an alteration of their phenotype. 
     
     
         29 . A harvestable part of a plant of  claim 27 . 
     
     
         30 . The harvestable part of a plant of  claim 29  selected from the group consisting of seeds, leaves, fruits, stem cultures, rhizomes, roots, tubers and bulbs. 
     
     
         31 . Transgenic progeny derived from any of the plants or plant parts of  claim 27 . 
     
     
         32 . A method of enhancing stress tolerance in (a) plant(s) comprising expression of at least one of the nucleic acid molecules of  claim 10  or an anti-sense molecule of  claim 17  in cells, tissues or parts of said plant(s). 
     
     
         33 . A method for altering stress tolerance in (a) plant(s) comprising expression of at least one of the nucleic acid molecules of  claim 10  or at least one of the polypeptides of  claim 19  or  20  or a anti-sense molecule of  claim 17  in cells, tissues or parts of said plant(s). 
     
     
         34 . A method of  claim 32  wherein said stress is selected from osmotic stress, salt stress, drought stress, freezing stress, and cold stress. 
     
     
         35 . New strains of yeast or other unicellular eukaryotes more tolerant to salt stress as a result of the expression of any of the nucleic acid molecules of  claim 10 . 
     
     
         36 . An in vitro cell culture system comprising animal, plant or host cells tolerant to salt, obtained as a result of the expression of at least one of the nucleic acid molecules of  claim 10 , or an antisense molecule of  claim 17 . 
     
     
         37 . An antibody specifically recognizing a polypeptide of  claim 19  or  20  or a specific epitope thereof. 
     
     
         38 . A diagnostic composition comprising at least a nucleic acid molecule of  claims 10  to  12 , or a polypeptide of  claim 19  or  20 . 
     
     
         39 . A method of  claim 1  for increasing yield.