Methods for in vivo identification of endogenous mrna targets of micrornas

Abstract

A method of generating a gene expression profile of noncoding regulatory RNA (ncRNA; e.g. a microRNA) in a cell in vivo, is carried out by: (a) partitioning from a cell at least one mRNA-protein (RNP) complex, the RNP complex comprising: (i) an RNA binding protein (RNABP) or RNA associated protein, (ii) at least one mRNA bound to or associated with said protein, and (iii) at least one ncRNA bound to or associated with said protein, and then (b) identifying at least one ncRNA in at least one RNP complex, thereby to produce a gene expression profile comprising the identity of an ncRNA in an RNP complex.

1 . A method of generating a gene expression profile of noncoding regulatory RNA (ncRNA) in a cell in vivo, comprising the steps of:
 (a) partitioning from a cell at least one mRNA-protein (RNP) complex, said RNP complex comprising: (i) an RNA binding protein (RNABP) or RNA associated protein, (ii) at least one mRNA bound to or associated with said protein, and (iii) at least one ncRNA bound to or associated with said protein, and then   (b) identifying at least one ncRNA in at least one RNP complex, thereby to produce a gene expression profile comprising the identity of an ncRNA in an RNP complex.   
     
     
         2 . The method of  claim 1 , wherein said ncRNA is a microRNA. 
     
     
         3 . The method of  claim 1 , said step of partitioning comprising:
 contacting a biological sample comprising said RNP complex from the cell with at least one ligand that specifically binds at least one component of the RNP complex;   separating the RNP complex by binding the ligand with an antibody specific for the ligand, wherein the antibody is attached to a solid support; and   collecting the RNP complex by removing the RNP complex from the solid support.   
     
     
         4 . The method of  claim 1 , wherein said mRNA in said RNP complex is predetermined. 
     
     
         5 . The method of  claim 1 , further comprising the step of:
 (c) identifying the mRNA in the RNP complex, thereby to produce a gene expression profile further comprising the identity of the mRNA associated with said miRNA.   
     
     
         6 . The method of  claim 1 , wherein said mRNA encodes a protein selected from the group consisting of amyloid protein, amyloid precursor protein, angiostatin, endostatin, METH-1, METH-2, Factor IX, Factor VIII, collagen, cyclin dependent kinase, cyclin D1, cyclin E, WAF1, cdk4 inhibitor, MTS1, cystic fibrosis transmembrane conductance regulator gene, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, erythropoietin, G-CSF, GM-CSF, M-CSF, SCF, thrombopoietin, BNDF, BMP, GGRP, EGF, FGF, GDNF, GGF, HGF, IGF-1, IGF-2, KGF, myotrophin, NGF, OSM, PDGF, somatotrophin, TGF-beta, TGF-alpha, VEGF, interferon, TNF-alpha, TNF-beta, cathepsin K, cytochrome p-450, famesyl transferase, glutathione-s transferase, heparanase, HMG CoA synthetase, n-acetyltransferase, phenylalanine hydroxylase, phosphodiesterase, ras carboxyl-terminal protease, telomerase, TNF converting enzyme, E-cadherin, N-cadherin, selectin, CD40, 5-alpha reductase, atrial natriuretic factor, calcitonin, corticotrophin releasing factor, glucagon, gonadotropin, gonadotropin releasing hormone, growth hormone, growth hormone releasing factor, somatotropin, insulin, leptin, luteinizing hormone, luteinizing hormone releasing hormone, parathyroid hormone, thyroid hormone, thyroid stimulating hormone, antibodies, CTLA4, hemagglutinin, MHC proteins, VLA-4, kallikrein-kininogen-kinin system, CD4, sis, hst, ras, abl, mos, myc, fos, jun, H-ras, ki-ras, c-fms, bcl-2, L-myc, c-myc, gip, gsp, HER-2, bombesin receptor, estrogen receptor, GABA receptor, EGFR, PDGFR, FGFR, NGFR, GTP-binding regulatory proteins, interleukin receptors, ion channel receptors, leukotriene receptor antagonists, lipoprotein receptors, opioid pain receptors, substance P receptors, retinoic acid and retinoid receptors, steroid receptors, T-cell receptors, thyroid hormone receptors, TNF receptors, tissue plasminogen activator; transmembrane receptors, calcium pump, proton pump, Na/Ca exchanger, MRP 1, MRP2, P170, LRP, cMOAT, transferrin, APC, brca1, brca2, DCC, MCC, MTS1, NF1, NF2, nm23, p53 and Rb. 
     
     
         7 . The method of  claim 1 , wherein said partitioning step comprises partitioning a plurality of RNP complexes; and wherein said identifying step comprises identifying a plurality of ncRNAs associated with said plurality of RNP complexes; said method further comprising:
 (c) identifying a plurality of mRNAs associated with said plurality of RNP complexes; thereby to produce a gene expression profile further comprising the identity of a subset of ncRNAs associated with a subset of mRNAs.   
     
     
         8 . The method of  claim 1 , wherein said cell is a plant cell. 
     
     
         9 . The method of  claim 1 , wherein said cell is an animal cell. 
     
     
         10 . The method of  claim 1 , wherein said cell is a bacterial cell. 
     
     
         11 . The method of  claim 1 , wherein said cell is a yeast cell. 
     
     
         12 . The method of  claim 1 , wherein said cell is a protozoal cell. 
     
     
         13 . A method of identifying and/or confirming mRNA target(s) of one or more microRNAs, the method comprising:
 (a) partitioning from a biological sample at least one RNP complex, said complex containing a subset of mRNAs associated with the RNP complex(es), and   (b) identifying a subset of microRNA associated with the RNP complex, thereby determining the association between a microRNA and an mRNA target.   
     
     
         14 . The method of  claim 13 , wherein the step of partitioning comprises capturing the RNP complex(es) on a solid support. 
     
     
         15 . The method of  claim 13 , further comprising assaying activity of at least one of the identified miRNA with respect to one or more of the identified mRNAs. 
     
     
         16 . The method of  claim 13 , further comprising predicting an mRNA target of a microRNA in silico. 
     
     
         17 . The method of  claim 13 , wherein the subset of mRNAs is represented by less than 75% of all mRNAs in the biological sample. 
     
     
         18 . The method of  claim 13 , wherein the subset of mRNAs comprises at least 2 mRNAs. 
     
     
         19 . The method of  claim 13 , wherein the subset of miRNAs is represented by less than 75% of all miRNAs in the biological sample. 
     
     
         20 . The method of  claim 13 , wherein the subset of miRNAs is comprises at least 2 miRNAs. 
     
     
         21 . The method of  claim 13 , wherein the subset of miRNAs and/or the subset of the cellular miRNAs is/are identified by using a nucleic acid array. 
     
     
         22 . The method of  claim 13 , wherein the step of partitioning may comprise contacting an mRNP complex with (i) an antibody that specifically binds at least one component of the mRNP complex or (ii) an ectopically expressed epitope-tagged RNA-binding protein or an RNA-associated protein. 
     
     
         23 . The method of  claim 19 , wherein the RNA-binding protein is a native or tagged Hu protein or poly(A)-binding protein (PABP). 
     
     
         24 . The method of  claim 13 , where the identified subset of the microRNAs includes an miRNA selected from the group consisting of miR-181a, miR-181b, miR-181c, miR-103, miR-107m miR-29c, miR-17-5p, miR-106a, miR-19b, miR-16, let-7a, let-7c, let-7d, and let-7f. 
     
     
         25 . A method of identifying and/or confirming mRNA target(s) of one or more microRNAs, the method comprising:
 (a) obtaining a biological sample comprising an mRNP complex;   (b) contacting the mRNP complex with (i) an antibody that specifically binds at least one component of the mRNP complex or (ii) an ectopically expressed epitope-tagged RNA-binding protein (RBP) or an RNA-associated protein (RAP),   (c) capturing the antibody, the RBP, or the RAP on a solid support, thereby partitioning from the biological sample at least one RNP complex, and   (d) identifying a subset of microRNA associated with the RNP complex(es),   thereby determining the association between a microRNA and an mRNA target.