Nonsense suppression and genetic codon alteration by targeted modification
Methods for affecting mRNA expression or translation through the modification of pre-mRNA or mRNA transcripts are described. In one embodiment of the methods of the present invention, the branch point adenosine of a pre-mRNA transcript is 2′-O-methylated to block splicing and subsequent expression of the protein encoded by the transcript. In another embodiment, a uridine residue in a nonsense stop codon may be modified to pseudouridine, causing the translation machinery to read through the nonsense stop codon and translate a full length protein.
1. A method for causing a stop codon in an mRNA transcript to be read as a coding codon, comprising: causing a uridine residue in the codon to be modified to a pseudouridine; wherein the presence of the pseudouridine in the stop codon causes the stop codon to be translated as a coding codon.
2. The method of claim 1 , wherein the uridine to pseudouridine modification comprises contacting the mRNA to be modified with an engineered RNA.
3. The method of claim 2 , wherein the engineered RNA is a small nucleolar RNA.
4. The method of claim 3 , wherein the small nucleolar RNA is a box H/ACA small nucleolar RNA.
5. The method of claim 1 , wherein the stop codon is a nonsense codon.
6. The method of claim 1 , wherein the method is performed in vivo.