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 one or more candidate compounds having an effect on the translation of a gene of interest, said method comprising:
 (a) identifying a 5′ or 3′ UTR sequence from a gene of interest that regulates translation;   (b) selecting host cells which express the gene that is endogenously associated with the UTR;   (c) stably transfecting one set of host cells with a test expression vector, said test vector comprising said 5′ or 3′ UTR sequence that regulates translation 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);   (d) identifying whether said UTR sequence has a translational function by assessing whether a first mRNA function of the UTR/reporter transcript is changed compared to the control/reporter transcript;   (e) determining whether the UTR regulatory function identified in step (d) 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;   (f) contacting said compound with an mRNA molecule comprising said UTR sequence; and   (g) measuring whether said compound alters a UTR-mediated translational effect.   
     
     
         2 . The method of  claim 1 , wherein said compound is contacted with a cell that expresses said mRNA molecule. 
     
     
         3 . The method of  claim 2 , wherein said UTR-mediated effect is the expression of a gene linked to said UTR sequence. 
     
     
         4 . The method of  claim 1 , wherein said UTR-mediated effect is a change in mRNA function. 
     
     
         5 . The method of  claim 1 , wherein said UTR-mediated effect is an increase in translation of the gene of interest. 
     
     
         6 . The method of  claim 1 , wherein said UTR-mediated effect is a decrease in translation of the gene of interest. 
     
     
         7 . The method of  claim 1 , wherein said compound is contacted with said mRNA 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. 
     
     
         8 . The method of  claim 1 , wherein said UTR-mediated effect is due to a change in translational initiation. 
     
     
         9 . A method for treating a physiological condition, such as cancer, inflammation, neurological disorders, diabetes, obesity, cardiovascular disorders, metabolic disorders, autoimmune diseases or cell proliferative conditions, in a mammal, by administrating to a mammal with a condition an effective amount of a compound identified using the method of  claim 1 . 
     
     
         10 . A method of identifying one or more candidate compounds having an effect on translation through the modulation of an intracellular pathway-mediated involved in UTR translational regulation, said method comprising:
 (a) identifying a 5′ or 3′ UTR sequence from a gene of interest that regulates translation;   (b) selecting host cells which express the gene that is endogenously associated with the UTR;   (c) stably transfecting one set of host cells with a test expression vector, said test vector comprising said 5′ or 3′ UTR sequence that regulates translation 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);   (d) identifying whether said UTR sequence has a translational function by assessing whether a first mRNA function of the UTR/reporter transcript is changed compared to the control/reporter transcript;   (e) determining whether the UTR regulatory function identified in step (d) 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;   (f) contacting said compound with an mRNA molecule comprising said UTR sequence; and   (g) measuring whether said compound alters an intracellular pathway-mediated UTR translational effect.   
     
     
         11 . The method of  claim 10 , wherein said compound is contacted with a cell that expresses said mRNA molecule. 
     
     
         12 . The method of  claim 11 , wherein said intracellular pathway-mediated UTR effect is measured by the expression of a gene linked to said UTR sequence. 
     
     
         13 . The method of  claim 10 , wherein the intracellular pathway-mediated UTR effect leads to a change in mRNA function. 
     
     
         14 . The method of  claim 10 , wherein said intracellular pathway-mediated UTR effect leads to an increase in translation of the gene of interest. 
     
     
         15 . The method of  claim 10 , wherein said intracellular pathway-mediated UTR effect leads to a decrease in translation of the gene of interest. 
     
     
         16 . The method of  claim 10 , wherein said intracellular pathway-mediated UTR effect is due to a change in translational initiation. 
     
     
         17 . A method for treating a physiological condition, such as cancer, inflammation, neurological disorders, diabetes, obesity, cardiovascular disorders, metabolic disorders, autoimmune diseases or cell proliferative conditions, in a mammal, by administrating to a mammal with a condition an effective amount of a compound identified using the method of  claim 10 .