Methods for isolating and characterizing endogenous mRNA-protein (mRNP) complexes

Abstract

Cellular mRNA-protein (mRNP) complexes are partitioned in vivo by contacting a biological sample with at least one ligand that specifically binds at least one component of a mRNP complex. Suitable biological samples comprise at least one mRNA-protein (mRNP) complex and include cell cultures, cell extracts, and whole tissue, including tumor tissue. Ligands include antibodies that specifically bind RNA-binding or RNA-associated proteins present in the mRNP complex. The mRNP complex is separated by binding the ligand with a binding molecule specific for the ligand, where the binding molecule is attached to a solid support. The mRNP complex is collected by removing the mRNP complex from the solid support. After collecting the mRNP complex, the mRNA bound within the complex may be characterized and identified. Subsets of the total mRNA population of a cell may accordingly be characterized, and a gene expression profile of the cell obtained.

1 - 33 . (canceled)  
     
     
         34 . A method of isolating a protein component of an RNA-protein complex, the method comprising the steps of: 
 obtaining a cDNA from an RNA of interest;    ligating the cDNA to a tagging DNA that encodes a binding partner for a ligand when the tagging DNA is expressed in a cell;    expressing the cDNA and the tagging DNA in a cell to produce a lysate comprising an RNA fusion molecule comprising the RNA of interest and the binding partner for the ligand;    contacting the lysate with the ligand, which ligand is attached to a solid support, thereby attaching the RNA fusion molecule to the solid support;    separating the RNA fusion molecule from the solid support; and    isolating at least one protein that has bound or associated with the RNA fusion molecule.    
     
     
         35 . A method of isolating a protein component of an RNA-protein complex, the method comprising the steps of: 
 obtaining an RNA fusion molecule comprising an RNA of interest and a binding partner for a ligand;    contacting a cell lysate with the RNA fusion molecule;    contacting the RNA fusion molecule with the ligand, which ligand is attached to a solid support, thereby attaching the RNA fusion molecule to the solid support;    separating the RNA fusion molecule from the solid support; and    isolating at least one protein that has bound or associated with the RNA fusion molecule.    
     
     
         36 . The method according to  claim 34 , further comprising the step of identifying the protein that has bound or associated with the RNA.  
     
     
         37 . The method according to  claim 36 , further comprising the step of generating an antibody against the identified protein.  
     
     
         38 . The method according to  claim 34 , wherein the RNA is an mRNA.  
     
     
         39 . The method according to  claim 34 , wherein the RNA is a mature mRNA.  
     
     
         40 . The method according to  claim 34 , wherein the tagging DNA is ligated to the 3′ end of the cDNA.  
     
     
         41 . The method according to  claim 34 , wherein the tagging DNA is ligated to the 5′ end of the cDNA.  
     
     
         42 . The method according to  claim 34 , wherein the tagging DNA is ligated internal to the cDNA.  
     
     
         43 . The method according to  claim 34 , wherein the ligand is a nucleic acid.  
     
     
         44 . The method according to  claim 34 , wherein the ligand is a protein.  
     
     
         45 . The method according to  claim 34 , wherein the ligand is an antibody.  
     
     
         46 . The method according to  claim 34 , wherein the ligand is a nucleic acid that binds to at least a portion of an RNA component of an RNA-protein complex.  
     
     
         47 . The method according to  claim 34 , wherein the ligand is an aptamer that binds to at least one component of an RNA protein complex.  
     
     
         48 . The method according to  claim 34 , wherein the ligand is a protein specific for at least one component of an RNA-protein complex.  
     
     
         49 . The method according to  claim 34 , wherein the ligand is an antibody specific for at least one component of an RNA-protein complex.  
     
     
         50 . The method according to  claim 34 , wherein the ligand is an antibody isolated from serum of a subject with cancer.  
     
     
         51 . The method according to  claim 34 , wherein the ligand is an antibody isolated from serum of a subject with an autoimmune disorder.  
     
     
         52 . The method according to  claim 34 , wherein the binding partner binds the ligand indirectly.  
     
     
         53 . The method according to  claim 34 , wherein the protein is an RNA binding protein.  
     
     
         54 . The method according to  claim 34 , wherein the protein is an RNA associated protein.  
     
     
         55 . The method according to  claim 34 , wherein the cell is treated with a compound.  
     
     
         56 . The method according to  claim 34 , wherein the cell is selected from the group consisting of a tumor cell, a primary cell, and a cultured cell.  
     
     
         57 . The method according to  claim 34 , further comprising cross-linking the RNA fusion molecule to one or more bound or associated proteins prior to contacting the RNA fusion molecule with the ligand.  
     
     
         58 . The method according to  claim 34 , wherein the solid support is selected from the group consisting of a bead, a plate, and a column.