RNA-YY1 Interactions

Abstract

Methods relating to obtaining libraries of YY1-binding long non-coding RNAs, libraries obtained thereby, and methods of use thereof.

1 . A method of preparing a library of nuclear ribonucleic acids (nRNAs) that specifically bind YY1, the method comprising:
 (a) contacting a sample containing nRNAs comprising at least 10 4  different nRNAs, with (i) YY1 protein and (ii) a YY1 binding agent, under conditions sufficient to form complexes between the nRNA, YY1 protein and the YY1 binding agent, and   (b) isolating the complexes.   
     
     
         2 . The method of  claim 1 , further comprising:
 (c) synthesizing cDNA complementary to the nRNA, and   (d) selecting cDNAs that (i) have RPKM above a desired threshold or (ii) are enriched compared to a control library, or both (i) and (ii).   
     
     
         3 . The method of  claim 1 , comprising:
 (a) providing the sample comprising nuclear ribonucleic acids, e.g., a wherein the sample comprises nuclear lysate, comprising nRNAs bound to nuclear proteins;   (b) contacting the sample with an antibody that binds specifically to YY1 protein, under conditions sufficient to form complexes between the agent and YY1 proteins such that the nRNAs remain bound to the YY1 proteins;   (c) isolating the complexes;   (d) synthesizing DNA complementary to the nRNAs to provide an initial population of cDNAs;   (e) PCR-amplifying the cDNAs using strand-specific primers;   purifying the initial population of cDNAs to obtain a purified population of cDNAs that are at least about 20 nucleotides (nt) in length;   (f) sequencing at least part of substantially all of the purified population of cDNAs; comparing the high-confidence sequences to a reference genome, and selecting those sequences that have at least 95% identity to sequences in the reference genome; and   (g) selecting those cDNAs that have (i) reads per kilobase per million reads (RPKM) above a desired threshold, and (ii) are enriched as compared to a control library;   thereby preparing the library of cDNAs.   
     
     
         4 . The method of  claim 1 , wherein the agent is an antibody and isolating the complexes comprises immunoprecipitating the complexes. 
     
     
         5 . The method of  claim 1 , wherein the cDNAs are synthesized using strand-specific adaptors. 
     
     
         6 . The method of  claim 1 , further comprising sequencing substantially all of the cDNAs. 
     
     
         7 . A library of cDNAs complementary to a pool of nuclear ribonucleic acids (nRNAs) prepared by the method of  claim 1 . 
     
     
         8 . The library of  claim 7 , wherein each of the cDNAs is linked to an individually addressable bead or area on a substrate. 
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 1 , wherein the sample is a cell-free sample. 
     
     
         11 . The method of  claim 1 , wherein the sample comprises a cell expressing the lncRNA and YY1. 
     
     
         12 . The method of  claim 1 , wherein the YY1, the lncRNA, or both, is labeled. 
     
     
         13 . The method of  claim 1 , further comprising isolating lncRNA-YY1 complexes from the sample. 
     
     
         14 . The method of  claim 13 , further comprising isolating unbound YY1 from the sample. 
     
     
         15 . The method of  claim 14 , wherein isolating lncRNA-YY1 complexes and unbound YY1 from the sample comprises contacting the sample with an anti-YY1 antibody, and isolating lncRNA-YY1-antibody complexes and unbound YY1. 
     
     
         16 . The method of  claim 15 , further comprising:
 (a) selecting a compound that disrupts binding of the lncRNA to YY1;   (b) contacting a tumor cell with the compound;   (c) measuring proliferation, survival, or invasiveness of the tumor cell in the presence and absence of the compound; and   (d) identifying as a candidate therapeutic compound a compound that inhibits proliferation, affects survival, e.g., induces or promotes cell death, or reduces or delays metastasis, of the tumor cell.   
     
     
         17 . The method of  claim 16 , further comprising administering the candidate compound to an animal model of cancer, and detecting an effect of the compound on cancer in the animal model. 
     
     
         18 . A method of identifying an RNA target for the treatment of cancer, the method comprising:
 (a) comparing (i) a library of nRNAs that specifically bind YY1 prepared from a normal cell with (ii) a library of nRNAs that specifically bind YY1 prepared from a cancerous cell, wherein the normal cell and cancerous cell are of the same tissue type; and   (b) identifying an nRNA that is differentially expressed between the libraries of (a)(i) and (a)(ii) as an RNA target for treatment of cancer.   
     
     
         19 . A method of identifying a therapeutic target for the treatment of cancer, the method comprising:
 (a) providing a population of nRNAs from a first cell type, by:   (1) providing a sample comprising nuclear ribonucleic acids from the first cell type;   contacting the sample with an antibody that binds specifically to YY1 protein, under conditions sufficient to form complexes between the agent and YY1 proteins such that the nRNAs remain bound to the YY1 proteins;   isolating the complexes; and   thereby providing a population of nRNAs from the first cell type;   (b) providing a population of nRNAs from a second cell type, by:   (1) providing a sample comprising nuclear ribonucleic acids bound to nuclear proteins, from the second cell type;   contacting the sample with an antibody that binds specifically to YY1 protein, under conditions sufficient to form complexes between the agent and YY1 proteins such that the nRNAs remain bound to the YY1 proteins;   isolating the complexes;   synthesizing DNA complementary to the nRNAs to provide an initial population of cDNAs;   (2) thereby providing a population of cDNAs from the second cell type;   wherein the first and second cell types are from the same type of tissue, and the first or second cell type is a tumor cell;   (c) contacting the population of nRNAs from the first cell type with the cDNAs from the second cell type, under conditions sufficient for the nRNAs to bind to complementary cDNAs; and   (d) identifying an nRNA that is differentially expressed in the first or second cell type as a therapeutic target for the treatment of cancer.