Method for Identifying RNA Segments Bound by RNA-Binding Proteins or Ribonucleoprotein Complexes

Abstract

The present invention relates to a method for identifying a binding site on an RNA transcript, wherein the binding site binds to one or more binding moieties. The method includes, among other things, introducing a photoreactive nucleoside into living cells wherein the living cells incorporate the photoreactive nucleoside into RNA transcripts during transcription thereby producing modified RNA transcripts; reverse transcribing the RNA of isolated cross-linked segments thereby generating cDNA transcripts with one mutation wherein the photoreactive nucleoside is transcribed to a mismatched deoxynucleoside; amplifying the cDNA transcripts thereby generating amplicons; and analyzing the sequences of the amplicons aligned against the reference sequence so as to identify the binding site, wherein the sequences of each amplicon having a mutation resulting from the introduction of the photoreactive nucleoside is considered to be a valid amplicon comprising at least a portion of a binding site on the RNA transcript.

1 . A method for identifying a binding site on an RNA transcript, wherein the binding site binds to one or more binding moieties, the method comprising
 a) introducing a 2-thiouridine or 2,4-di-thiouridine photoreactive nucleoside into living cells wherein the living cells incorporate the photoreactive nucleoside into RNA transcripts during transcription thereby producing modified RNA transcripts;   b) irradiating said cells at a wavelength significantly absorbed by the photoreactive nucleoside to covalently cross-link a binding site on the modified RNA transcripts to one or more binding moieties;   c) removing all or part of the modified RNA transcripts which is not covalently cross-linked to the one or more binding moieties to form cross-linked segments having the photoreactive nucleoside, wherein the cross-linked segments comprise at least a portion of the binding site;   d) isolating the cross-linked segments thereby generating isolated cross-linked segments;   e) reverse transcribing the RNA of isolated cross-linked segments thereby generating cDNA transcripts with a deoxyguanosine (G) to deoxyadenosine (A) mutation wherein the photoreactive nucleoside is transcribed to a mismatched deoxynucleoside;   f) amplifying the cDNA transcripts thereby generating amplicons with a deoxythymidine (T) to deoxycytidine (C) mutation;   g) determining the nucleotide sequences of the amplicons having at least 15 nucleotides;   h) aligning the sequences of the amplicons against a reference sequence; and   i) analyzing the sequences of the amplicons aligned against the reference sequence so as to identify the binding site, wherein the sequences of each amplicon having the T to C mutation resulting from the introduction of the photoreactive nucleoside is considered to be a valid amplicon comprising at least a portion of a binding site on the RNA transcript.   
     
     
         2 . The method according to  claim 1  further comprising removing the binding moiety from the isolated cross-linked segments thereby generating isolated segments prior to step (e). 
     
     
         3 . The method of  claim 1  further comprising determining the sequence of a consensus motif, wherein the determination comprises using the mutation as an anchor and comparing the sequence surrounding the mutation to the reference sequence, wherein the mutation is within a sequence window that includes the mutation plus at least one nucleotide on either side of the mutation. 
     
     
         4 . The method of  claim 3  wherein the sequence window includes five to twenty nucleotides on either side of the mutation. 
     
     
         5 . The method of  claim 3  wherein the mutation is at the center of the sequence window. 
     
     
         6 . The method according to  claim 1  wherein the reference sequence is a genomic sequence. 
     
     
         7 . The method according to  claim 6  wherein the genomic sequence is a sequence that produced the RNA transcript. 
     
     
         8 . The method according to  claim 1  wherein the reference sequence is a synthetic RNA sequence. 
     
     
         9 . The method according to  claim 1  wherein the reference sequence is derived from an expressed sequence tag database. 
     
     
         10 . The method according to  claim 1  further comprising identifying a feature required for interaction of the binding site and the binding moiety. 
     
     
         11 . The method of  claim 1  wherein the binding moiety is a protein or a protein complex. 
     
     
         12 . siRNA, endo-siRNA, snoRNA, snRNA, tRNA, rRNA or a combination thereof. 
     
     
         13 . The method of  claim 11  wherein the protein is an RNA-binding protein, an RNA-associated protein or a combination thereof. 
     
     
         14 . The method of  claim 1  wherein the binding site is a coding transcript. 
     
     
         15 . The method of  claim 1  wherein the binding moiety is epitope-tagged. 
     
     
         16 . The method of  claim 1  wherein step (c) comprises (i) forming a soluble extract of the cells; and (ii) treating the extract with a nuclease thereby forming the cross-linked segments. 
     
     
         17 . The method of  claim 16  wherein the nuclease is ribonuclease—T1. 
     
     
         18 . The method of  claim 17  wherein aligning the sequences of the amplicons comprises determining which amplicons have the characteristic that, when aligned with the reference sequence, the reference sequence has a guanosine one nucleotide upstream from the 5′ end of the amplicons. 
     
     
         19 . The method of  claim 17  wherein analyzing the sequences of the amplicons comprises determining which amplicons have the characteristic that, when aligned with the genomic sequence, the genomic sequence has a guanosine one nucleotide upstream from the 5′ end of the amplicons, wherein such amplicons are valid amplicons. 
     
     
         20 . (canceled) 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 1  wherein the thiouridine analog is substituted at the 5 and/or 6 position substituents selected from the group consisting of methyl, ethyl, halo, nitro, NR 1 R 2  and OR 3  wherein R 1 , R 2  and R 3  independently represent hydrogen, methyl or ethyl. 
     
     
         23 . The method of  claim 1  wherein aligning the sequences of the amplicons comprises determining which amplicons have a mutation wherein a deoxythymidine of the reference sequence is replaced by a deoxycytidine in the amplicons. 
     
     
         24 . The method of  claim 1  wherein analyzing the sequences of the amplicons comprises determining which amplicons have only one mutation wherein a deoxythymidine of the reference sequence is replaced by a deoxycytidine in the amplicons, wherein such amplicons are valid amplicons. 
     
     
         25 . (canceled) 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . The method of  claim 15  wherein isolating the cross-linked segments comprises immunoprecipitation. 
     
     
         30 . The method of  claim 29  further comprising separating the cross-linked segments by length after immunoprecipitation. 
     
     
         31 . The method of  claim 29  further comprising treating the cross-linked segments with nuclease after immunoprecipitation. 
     
     
         32 . The method of  claim 1  wherein removing the binding moiety comprises digesting the binding moiety with a protease. 
     
     
         33 . The method of  claim 1  wherein the wavelength is greater than 300 nm. 
     
     
         34 . The method of  claim 1  wherein the living cell is part of a cell culture, a cell extract, whole tissue, or a whole organ. 
     
     
         35 . A kit for identifying a binding site on an RNA transcript, the kit comprising:
 a) a 2-thiouridine or 2,4-di-thiouridine supplemented tissue culture medium,   b) reagents for nuclease digestion,   c) reagents for immunoprecipitation, and   d) adapters and primers for small RNA cloning.