Methods of assaying for compounds that inhibit premature translation termination and nonsense-mediated RNA decay

Abstract

The present application provides methods of assaying for compounds that inhibit premature translation termination and nonsense mediated RNA decay in cells.

1 . A method of in vitro screening for compounds that modulate premature translation termination and nonsense-mediated mRNA decay, the method comprising the steps of: 
 (i) incubating a translation assay, the assay comprising an in vitro translation cellular extract; a nucleic acid encoding a polypeptide, wherein the coding sequence for the polypeptide comprises a premature stop codon; and a candidate modulator compound; and    (ii) detecting the polypeptide translated from the nucleic acid.    
   
   
       2 . The method of  claim 1 , wherein the nucleic acid encodes an enzyme.  
   
   
       3 . The method of  claim 1 , wherein the nucleic acid encodes an immunoglobin.  
   
   
       4 . The method of  claim 1 , wherein the nucleic acid encodes luciferase, green fluorescent protein, red fluorescent protein, phosphatase, peroxidase, kinase, chloramphenicol transferase, or β-galactosidase.  
   
   
       5 . (canceled)  
   
   
       6 . The method of  claim 1 , wherein the cellular extract is from yeast, plants, mammals, or amphibians.  
   
   
       7 . The method of  claim 1 , wherein the cellular extract is a eukaryotic reticulocyte lysate.  
   
   
       8 . (canceled)  
   
   
       9 . The method of  claim 1 , wherein the cellular extract is a mammalian tissue culture cell extract.  
   
   
       10 . (canceled)  
   
   
       11 . The method of  claim 1 , wherein the polypeptide is detected by ELISA, light emission, colorimetric measurements, enzymatic activity, or radioactivity.  
   
   
       12 . The method of  claim 1 , wherein the assay is performed in a well of a microtiter dish.  
   
   
       13 - 15 . (canceled)  
   
   
       16 . The method of in vivo screening for compounds that modulate premature translation termination and nonsense-mediated mRNA decay, the method comprising the steps of: 
 (i) expressing in a cell a nucleic acid encoding a polypeptide, wherein the coding sequence for the polypeptide comprises a premature stop codon;    (ii) contacting the cell with a candidate modulator compound; and    (iii) detecting either the polypeptide translated from the nucleic acid or RNA transcribed from the nucleic acid.    
   
   
       17 . The method of  claim 16 , wherein the nucleic acid comprises a promoter operably linked to a heterologous nucleic acid encoding the polypeptide.  
   
   
       18 . The method of  claim 17 , wherein the heterologous nucleic acid encoding the polypeptide comprises an intron and at least two exons comprising coding sequence.  
   
   
       19 . (canceled)  
   
   
       20 . The method of  claim 18 , wherein the heterologous nucleic acid encodes a chimeric polypeptide.  
   
   
       21 . (canceled)  
   
   
       22 . The method of  claim 17 , wherein the nucleic acid encodes an enzyme.  
   
   
       23 . The method of  claim 17 , wherein the nucleic acid encodes luciferase, green fluorescent protein, red fluorescent protein, phosphatase, peroxidase, kinase, chloramphenicol transferase, or β-galactosidase.  
   
   
       24 . The method of  claim 17 , wherein the nucleic acid encodes an immunoglobin.  
   
   
       25 . The method of  claim 16 , wherein the nucleic acid is an endogenous gene.  
   
   
       26 . The method of  claim 25 , wherein the endogenous gene is α-globin, β-globin, factor VIII, factor IX, vWF, p53, dystrophin, CFTR, Rb, MSH1, MSH2, APC, Wt1, hexosaminidase A, neurofibromin 1, or neurofibromin 2.  
   
   
       27 . The method of  claim 25 , wherein the endoegenous gene encodes an immunoglobin.  
   
   
       28 . The method of  claim 16 , wherein the polypeptide is detected by ELISA, light emission, colorimetric measurements, enzymatic activity, drug resistance, FACS, or radioactivity.  
   
   
       29 . The method of  claim 16 , wherein the assay is performed in a well of a microtiter dish.  
   
   
       30 - 34 . (canceled)  
   
   
       35 . The method of  claim 16 , wherein the cell is a human cell or a mouse cell.  
   
   
       36 . The method of  claim 16 , wherein the cell is stably transfected with the nucleic acid.  
   
   
       37 . A kit for screening for compounds that modulate translation termination and nonsense-mediated mRNA decay, the kit comprising a nucleic acid encoding a polypeptide, wherein the polypeptide coding sequence comprises a premature stop codon; instructions for practicing a method of screening for compounds that inhibit translation termination at premature stop codons and nonsense mediated RNA decay; and a control compound that inhibits nonsense-mediated RNA decay.  
   
   
       38 - 43 . (canceled)