Nonsense mediated decay (NMD) leads to the degradation of pre-mRNAs which contain a premature termination codon (PTC). Recent reports suggest that nuclear NMD may involve a pre-splicing proofreading mechanism, nuclear translation. We propose that NMD affects the rate of splicing of the introns upstream or downstream from exons containing PTCs. Quantitative RT-PCR will be used to measure the abundance of introns in wildtype, nonsense codon-containing, and missense-codon containing precursor mRNAs since it has been proven mathematically that the introns which are excised most quickly will be less abundant within the pre-mRNA population. Two mRNAs will be tested: the somatically rearranging Ig-mu and the non-rearranging DHFR. Finally, several different systems will be established in which only nuclear or cytoplasmic translation can occur. Then, the rate and order of intron removal will be tested under conditions which will demonstrate that any change in splicing is due to nuclear translation.
| Lytle, J Robin; Yario, Therese A; Steitz, Joan A (2007) Target mRNAs are repressed as efficiently by microRNA-binding sites in the 5'UTR as in the 3'UTR. Proc Natl Acad Sci U S A 104:9667-72 |
| Lytle, J Robin; Steitz, Joan A (2004) Premature termination codons do not affect the rate of splicing of neighboring introns. RNA 10:657-68 |
