Methods for identifying novel nucleic acid regulatory elements and compounds that affect the regulation

Abstract

Described herein are methods for identifying novel nucleic acid regulatory elements and compounds that modulate the regulation of such elements. Also described herein are nucleic acid sequence identified as novel nucleic acid regulatory elements and host cells containing such nucleic acid regulatory elements in a vector.

1 . A method of identifying a nucleic acid sequence, derived from the mRNA untranslated region (UTR) of a gene, that has a physiologically relevant post-transcriptional regulatory function, said method comprising: 
 i) identifying a 5′ or 3′ UTR sequence from a gene of interest;    ii) selecting host cells which express the gene that is endogenously associated with the UTR;    iii) stably transfecting one set of host cells with a test expression vector, said test vector comprising said 5′ or 3′ UTR sequence linked to, and positioned upstream or downstream, respectively, of a reporter gene construct (UTR/reporter), and stably transfecting another set of host cells with a control expression vector, said control vector comprising said reporter gene and lacking said UTR sequence (control/reporter);    iv) identifying whether said UTR sequence has a post-transcriptional regulatory function by assessing whether a first mRNA function of the UTR/reporter transcript is changed compared to the control/reporter transcript; and    v) determining whether the UTR regulatory function identified in step (iv) is physiologically relevant by assessing whether said changed first mRNA function of the UTR/reporter transcript corresponds to the first mRNA function of the gene endogenously associated with the UTR.    
   
   
       2 . The method  claim 1 , further comprising confirming the physiological relevance of said UTR regulatory function by assessing whether a second mRNA function of the UTR/reporter transcript corresponds to the second mRNA function of the UTR-associated endogenous gene.  
   
   
       3 . The method of  claim 1 , wherein said mRNA function is selected from the group consisting of pre-mRNA processing, mRNA stability, mRNA translational efficiency, mRNA localization, mRNA sequestration, and mRNA editing and splicing.  
   
   
       4 . The method of  claim 1 , wherein said UTR/reporter further comprises a second UTR sequence from said gene of interest, wherein said UTR/reporter comprises both the 5′ UTR and the 3′ UTR sequences of said gene, positioned upstream and downstream, respectively, of said reporter gene.  
   
   
       5 . The method of  claim 1 , wherein said UTR sequence is the full length untranslated sequence derived from the mRNA untranslated region of a gene.  
   
   
       6 . A population of host cells comprising at least one cell stably transfected with a test expression vector, said test vector comprising a UTR sequence linked to a reporter gene construct (UTR/reporter), said population further comprising at least one other host cell stably transfected with a control expression vector, said control vector comprising said reporter gene and lacking said UTR sequence (control/reporter), wherein said host cells endogenously express the gene associated with the UTR.  
   
   
       7 . A nucleic acid sequence identified as having a physiologically relevant post-transcriptional regulatory function by the method of  claim 1 .  
   
   
       8 . A method of identifying candidate compounds having an effect on expression of a gene of interest, said method comprising: 
 i) identifying UTR sequences having post-transcriptional regulatory function by the method of  claim 1;     ii) contacting said compound with an mRNA molecule comprising said UTR sequence;    iii) measuring whether said compound alters a UTR-mediated effect.    
   
   
       9 . The method of  claim 8 , wherein said compound is contacted with a cell that expresses said mRNA molecule.  
   
   
       10 . The method of  claim 9 , wherein said measured UTR-mediated effect is the expression of a gene linked to said UTR sequence.  
   
   
       11 . The method of  claim 8 , wherein said UTR-mediated effect is a change in mRNA function.  
   
   
       12 . The method of  claim 8 , wherein said compound is contacted with said RNA molecule in vitro, and further wherein said UTR-mediated effect is an interaction between an mRNA transcript comprising said UTR and an RNA binding protein.