Method of purifying RNA binding protein-RNA complexes

Abstract

The present invention provides methods for purifying RNA molecules interacting with an RNA binding protein (RBP), and the use of such methods to analyze a gene expression profile of a cell. The invention also provides sequences of RNA molecules that mediate binding to an RBP, proteins encoded by the sequences, a method of identifying the sequences, and the use of the sequences in a screen to identify bioactive molecules. The invention also provides RNA motifs found among the sequences and compounds that bind the RNA motifs. In addition, the invention provides methods of treating diseases associated with a function of an RNA binding protein.

1 . A method for purifying an RNA molecule interacting with an RNA binding protein of interest in a biological sample, comprising the steps of: 
 a contacting said biological sample with an agent that creates a covalent bond between said RNA molecule and said RNA binding protein of interest, thereby generating a covalently bound RNA binding protein-RNA complex containing said RNA molecule;    b. cleaving said RNA molecule by contacting said RNA binding protein-RNA complex with an agent capable of cleaving a bond thereof, thereby generating a fragment of said RNA molecule, wherein said fragment is at least 22 nucleotide bases in length;    c. selecting said RNA binding protein-RNA complex with a molecule that specifically interacts with a component of said RNA binding protein-RNA complex; and    d. purifying said RNA binding protein-RNA complex under stringent conditions,    thereby purifying an RNA molecule interacting with an RNA binding protein of interest    
     
     
         2 - 6 . (canceled)  
     
     
         7 . The method of  claim 1 , wherein step (b) comprises utilization of a nuclease.  
     
     
         8 - 11 . (canceled)  
     
     
         12 . The method of  claim 1 , further comprising labeling said RNA molecule.  
     
     
         13 . The method of  claim 1 , wherein step (d) comprises a utilization of an agent capable of disrupting an intermolecular interaction.  
     
     
         14 - 24 . (canceled)  
     
     
         25 . A method of identifying an RNA molecule interacting with an RNA binding protein of interest, comprising purifying said RNA molecule by the method of  claim 1 , and identifying said RNA molecule, thereby identifying an RNA molecule interacting with an RNA binding protein of interest.  
     
     
         26 . The method of  claim 25 , further comprising ligating nucleotide linkers to said RNA molecule.  
     
     
         27 . The method of  claim 26 , wherein said nucleotide linkers are directionally oriented.  
     
     
         28 . The method of  claim 1 , further comprising amplifying said RNA molecule.  
     
     
         29 . A method for identifying a candidate RNA motif that may mediate binding to an RNA binding protein of interest, comprising the steps of: 
 a. purifying a plurality of RNA molecules interacting with said RNA binding protein of interest by the method of  claim 1:     b. obtaining sequences from a subset of said plurality of RNA molecules; and    c. detecting a presence of said candidate RNA motif in two of said sequences,    thereby identifying a candidate RNA motif that may mediate binding to an RNA binding protein of interest.    
     
     
         30 - 35 . (canceled)  
     
     
         36 . A method for purifying an RNA molecule interacting with an RNA binding protein of interest in a biological sample, comprising the steps of: 
 a. contacting said biological sample with an agent that creates a covalent bond between said RNA molecule and said RNA binding protein of interest, thereby generating a covalently bound RNA binding protein-RNA complex containing said RNA molecule;    b. cleaving said RNA molecule with an agent capable of cleaving a bond thereof, thereby generating a fragment of said RNA molecule, wherein said fragment is at least 22 nucleotide bases in length;    c. selecting said RNA binding protein-RNA complex with a molecule that specifically interacts with a component of said RNA binding protein-RNA complex; and    d. purifying said RNA binding protein-RNA complex, wherein said purifying step comprises a denaturing ionic detergent,    thereby purifying an RNA molecule interacting with an RNA binding protein of interest    
     
     
         37 . The method of  claim 36 , wherein step (c) is performed before step (b).  
     
     
         38 - 41 . (canceled)  
     
     
         42 . The method of  claim 36 , wherein step (b) comprises utilization of a nuclease.  
     
     
         43 - 46 . (canceled)  
     
     
         47 . The method of  claim 36 , wherein step (c) comprises immunoprecipitation of said RNA binding protein-RNA complex.  
     
     
         48 . The method of  claim 36 , further comprising labeling said RNA molecule.  
     
     
         49 - 50 . (canceled)  
     
     
         51 . The method of  claim 36 , wherein said denaturing ionic detergent is sodium dodecyl sulfate or a derivative thereof.  
     
     
         52 . The method of  claim 36 , wherein step (d) comprises heating said RNA binding protein-RNA complex in a buffer.  
     
     
         53 . The method of  claim 36 , wherein step (d) comprises a chromatographic method.  
     
     
         54 . The method of  claim 53 , wherein said chromatographic method comprises electrophoresis.  
     
     
         55 . The method of  claim 53 , wherein said chromatographic method comprises an agent capable of disrupting an intermolecular interaction.  
     
     
         56 . The method of  claim 53 , wherein said chromatographic method comprises a pH-buffering agent.  
     
     
         57 . The method of  claim 36 , wherein said step (d) comprises transferring said RNA binding protein-RNA complex to a substrate.  
     
     
         58 . The method of  claim 57 , wherein said substrate comprises nitrocellulose.  
     
     
         59 - 69 . (canceled)  
     
     
         70 . A method for purifying an RNA molecule interacting with an RNA binding protein of interest in a biological sample, comprising the steps of: 
 a. contacting said biological sample with an agent that creates a covalent bond between said RNA molecule and said RNA binding protein of interest, thereby generating a covalently bound RNA binding protein-RNA complex containing said RNA molecule;    b. cleaving said RNA molecule with an agent capable of cleaving a bond thereof, thereby generating a fragment of said RNA molecule, wherein said fragment is at least 22 nucleotide bases in length;    c. selecting said RNA binding protein-RNA complex with a molecule that specifically interacts with a component of said RNA binding protein-RNA complex; and    d. purifying said RNA binding protein-RNA complex, wherein said purifying comprises a chromatographic method,    thereby purifying an RNA molecule interacting with an RNA binding protein of interest.    
     
     
         71 . The method of  claim 70 , wherein step (c) is performed before step (b).  
     
     
         72 - 75 . (canceled)  
     
     
         76 . The method of claim  64 , wherein step (b) comprises utilization of a nuclease.  
     
     
         77 - 81 . (canceled)  
     
     
         82 . The method of  claim 70 , further comprising labeling said RNA molecule.  
     
     
         83 . The method of  claim 70 , wherein step (d) comprises a utilization of an agent capable of disrupting an intermolecular interaction.  
     
     
         84 . The method of  claim 83 , wherein said agent is a detergent.  
     
     
         85 . The method of  claim 84 , wherein said detergent is an ionic detergent.  
     
     
         86 . The method of  claim 85 , wherein said ionic detergent is sodium dodecyl sulfate or a derivative thereof  
     
     
         87 . The method of  claim 70 , wherein step (d) comprises heating said RNA binding protein-RNA complex in a buffer.  
     
     
         88 . The method of  claim 70 , wherein said chromatographic method comprises electrophoresis.  
     
     
         89 . The method of  claim 70 , wherein said chromatographic method comprises an agent capable of disrupting an intermolecular interaction.  
     
     
         90 . The method of  claim 70 , wherein said chromatographic method comprises a pH-buffering agent.  
     
     
         91 . The method of  claim 70 , wherein said step (d) comprises transferring said RNA binding protein-RNA complex to a substrate.  
     
     
         92 . The method of  claim 91 , wherein said substrate comprises nitrocellulose.  
     
     
         93 - 96 . (canceled)  
     
     
         97 . A method for identifying a candidate RNA motif that may mediate binding to an RNA binding protein of interest, comprising the steps of: 
 a. purifying a plurality of RNA molecules interacting with said RNA binding protein of interest by the method of  claim 70;     b. obtaining sequences from a subset of said plurality of RNA molecules; and    c. detecting a presence of said candidate RNA motif in two of said sequences,    thereby identifying a candidate RNA motif that may mediate binding to an RNA binding protein of interest.    
     
     
         98 - 103 . (canceled)  
     
     
         104 . An RNA motif of an RNA molecule having a sequence selected from the group consisting of SEQ ID No 1-449, wherein said RNA motif interacts with an RNA binding protein.  
     
     
         105 . The RNA motif of  claim 104 , wherein said RNA binding protein is Nova-1, a Nova-2, or a combination thereof.  
     
     
         106 - 109 . (canceled)  
     
     
         110 . A method for purifying an RNA binding protein present in an RNA binding protein-RNA complex containing a known component, comprising the steps of: 
 a. contacting said RNA binding protein-RNA complex with an agent that creates a covalent bond between two components of said RNA binding protein-RNA complex;    b. cleaving an RNA molecule of said RNA binding protein-RNA complex by contacting said RNA binding protein-RNA complex with an agent capable of cleaving a bond of said RNA molecule, thereby generating a fragment of said RNA molecule, wherein said fragment is at least 22 nucleotide bases in length;    c. selecting said RNA binding protein-RNA complex with a molecule that specifically interacts with said known component;    d. purifying said RNA binding protein-RNA complex under stringent conditions; and    e. removing said RNA binding protein from said RNA binding protein-RNA complex,    thereby purifying an RNA binding protein present in an RNA binding protein-RNA complex containing a known component.    
     
     
         111 . A method for identifying an RNA binding protein present in an RNA binding protein-RNA complex containing a known component, comprising the steps of: 
 a. contacting said RNA binding protein-RNA complex with an agent that creates a covalent bond between two components of said RNA binding protein-RNA complex;    b. cleaving an RNA molecule of said RNA binding protein-RNA complex by contacting said RNA binding protein-RNA complex with an agent capable of cleaving a bond of said RNA molecules, thereby generating a fragment of said RNA molecule, wherein said fragment is at least 22 nucleotide bases in length;    c selecting said RNA binding protein-RNA complex with a molecule that specifically interacts with said known component;    d. purifying said RNA binding protein-RNA complex under stringent conditions; and    e. identifying said RNA binding protein from said RNA binding protein-RNA complex,    thereby identifying an RNA binding protein present in an RNA binding protein-RNA complex containing a known component.    
     
     
         112 . A method of assessing a level of association of an RNA transcript of interest with an RNA binding protein of interest, comprising the steps of: 
 a contacting an RNA binding protein-RNA complex containing said RNA binding protein of interest with an agent that creates a covalent bond between two components of said RNA binding protein-RNA complex;    b cleaving an RNA molecule of said RNA binding protein-RNA complex by contacting said RNA binding protein-RNA complex with an agent capable of cleaving a bond of said RNA molecule, thereby generating a fragment of said RNA molecule, wherein said fragment is at least 22 nucleotide bases in length;    c. selecting said RNA binding protein-RNA complex with a molecule that specifically interacts with a component thereof;    d purifying said RNA binding protein-RNA complex, wherein said purifying comprises a chromatographic method; and    e. assessing a presence or amount of said RNA transcript of interest or a fragment thereof in said plurality of RNA molecules,    thereby assessing a level of association of an RNA transcript of interest with an RNA binding protein of interest.    
     
     
         113 . (canceled)  
     
     
         114 . A method of assessing a level of association of an RNA transcript of interest with an RNA binding protein of interest, comprising the steps of: 
 a. contacting an RNA binding protein-RNA complex containing said RNA binding protein of interest with an agent that creates a covalent bond between two components of said RNA binding protein-RNA complex;    b. cleaving an RNA molecule of said RNA binding protein-RNA complex with an agent capable of cleaving a bond of said RNA molecule, thereby generating a fragment of said RNA molecule, wherein said fragment is at least 22 nucleotide bases in length;    c. selecting said RNA binding protein-RNA complex with a molecule that specifically interacts with a component thereof;    d. purifying said RNA binding protein-RNA complex, wherein said purifying step comprises an agent that disrupts an intermolecular interaction; and    e. assessing a presence or amount of said RNA transcript of interest or a fragment thereof in said plurality of RNA molecules,    thereby assessing a level of association of an RNA transcript of interest with an RNA binding protein of interest.    
     
     
         115 . (canceled)  
     
     
         116 . A method of screening a test compound for its ability to modulate a level of association between an RNA binding protein and an RNA transcript, comprising the steps of: 
 a. assessing a first level of association between said RNA binding protein and said RNA transcript in a first cell by the method of  claim 112 , wherein said first cell has been contacted with said test compound;    b. assessing a second level of association between said RNA binding protein and said RNA transcript in a second cell by the method of  claim 112 , wherein said second cell has not been contacted with said test compound; and    c. comparing said first level of association with said second level of association,    wherein a difference between said first level of association and said second level of association indicates an ability of said test compound to modulate a level of association between said RNA binding protein and said RNA transcript.    
     
     
         117 . A nucleotide linker comprising a nucleic acid sequence set forth in SEQ ID No 477-502.  
     
     
         118 . A method of amplifying a nucleic acid molecule, said method comprising the steps of: 
 a ligating said nucleic acid molecule to a first nucleotide linker, wherein said first nucleotide linker comprises a nucleic acid sequence set forth in SEQ ID No 477-502;    b. synthesizing a nucleic acid strand complementary to a product of step (a) using as a primer a second nucleotide linker, wherein said second nucleotide linker comprises a nucleic acid sequence set forth in SEQ ID No 477-502; and    c. amplifying a product of step (b) by a polymerase chain reaction, using as a primer a third nucleotide linker, wherein said third nucleotide linker comprises a nucleic acid sequence set forth in SEQ ID No 477-502,    thereby amplifying a nucleic acid molecule.    
     
     
         119 - 150 . (canceled)  
     
     
         151 . An isolated nucleic acid that comprises a sequence set forth in SEQ ID No 63, 64, 76, 77, 78, 84, or 292-335.  
     
     
         152 - 156 . (canceled)  
     
     
         157 . An isolated nucleic acid that comprises a sequence set forth in SEQ ID No 374, 377, 378, 380, 382, 384, 387, 394-396, 415, 416, and 421.  
     
     
         158 . An isolated peptide encoded by a nucleic acid sequence as set forth in SEQ ID No 374, 377, 378, 380, 382, 384, 387, 394-396,415, 416, and 421.  
     
     
         159 . A transgenic mouse comprising a mutation in a Nova-2 gene.  
     
     
         160 - 162 . (canceled)  
     
     
         163 . The transgenic mouse of  claim 159 , wherein said mutation is a null mutation.  
     
     
         164 . (canceled)  
     
     
         165 . A method for studying a symptom, disease or disorder associated with Nova-2 gene expression, comprising ascertaining the presence or absence of said symptom, disease or disorder in the transgenic mouse of  claim 159 .  
     
     
         166 . The method of  claim 163 , wherein said disorder is a neurological disorder.  
     
     
         167 . (canceled)  
     
     
         168 . The method of  claim 163 , wherein said disorder is an autoimmune disorder.  
     
     
         169 . The method of  claim 163 , further comprising introducing a wild-type Nova-2 gene into said transgenic mouse.  
     
     
         170 . A method of studying a Nova-2 protein function, comprising assessing a qualitative or quantitative change in a parameter in the transgenic mouse of  claim 159 , thereby studying a Nova-2 protein function.  
     
     
         171 . (canceled)  
     
     
         172 . The method of  claim 168 , further comprising isolating a nucleic acid molecule that interacts with said Nova-2 protein.  
     
     
         173 . A method for identifying a therapeutic agent for the treatment of a disease or disorder associated with Nova-2 gene expression comprising 
 a. contacting a cell of the transgenic mouse of  claim 159  with a candidate therapeutic agent;    b. assessing a parameter associated with said disease or disorder in said transgenic mouse in the presence of said candidate therapeutic agent; and    c. assessing a parameter associated with said disease or disorder in said transgenic mouse in the absence of said candidate therapeutic agent;    wherein amelioration of said parameter in said transgenic mouse is an indication of a therapeutic effect of said candidate therapeutic agent.    
     
     
         174 . The method of  claim 171 , wherein said disease or disorder is a neurological disorder.  
     
     
         175 . (canceled)  
     
     
         176 . The method of  claim 171 , wherein said disease or disorder is an autoimmune disorder.