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 - 174 . (canceled) 
     
     
         175 . A method for identifying an RNA molecule interacting with an RNA binding protein of interest in a biological sample, said method comprising the steps of:
 a. irradiating said biological sample to create 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, wherein said biological sample is selected from the group consisting of whole tissue biopsy, tissue sample biopsy and whole organ;   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 that is bound to the RNA binding protein, wherein said fragment is a transfer RNA, a small nuclear RNA, a ribosomal RNA, a messenger RNA, an anti-sense RNA, a small inhibitory RNA, a micro RNA or a ribozyme;   c. selecting said RNA binding protein-RNA complex with a molecule that specifically interacts with a component of said RNA binding protein-RNA complex;   d. purifying said RNA binding protein-RNA complex under stringent conditions comprising washing the complex with buffer at least 5 times; boiling said RNA binding protein-RNA complex in a denaturing ionic detergent; separating said RNA binding protein-RNA complex by SDS-PAGE; transferring said RNA binding protein-RNA complex to a substrate that preferentially binds RNA covalently bound to protein over RNA not covalently bound to protein; and digesting said RNA binding protein with a protease to liberate said fragment of said RNA molecule from said RNA binding protein-RNA complex; and   e. ligating nucleotide linkers to said fragment of said RNA molecule and amplifying said fragment of said RNA molecule,   thereby identifying an RNA molecule interacting with an RNA binding protein of interest.   
     
     
         176 . The method of claim  1 , wherein step (b) comprises utilization of a nuclease. 
     
     
         177 . The method of claim  1 , further comprising labeling said RNA molecule, wherein said method improves signaling from said label. 
     
     
         178 . The method of claim  1 , wherein said nucleotide linkers are directionally oriented.