This R21 grant application proposes to test the hypothesis that human colon and breast cancer cell lines may exhibit a global defect in NMRD (nonsense-mediated RNA decay) machinery. NMRD is responsible for degrading mRNA transcripts that contain premature termination (nonsense) mutations. If NMRD is deficient, stable mutant transcripts and truncated proteins would be predicted to accumulate. NMRD deficiency will be investigated using human breast and colon cancer cell lines by employing transfections of a minigene previously demonstrated to undergo NMRD, as well as transfection experiments using expression constructs of RENT1 (regulator of nonsense-transcripts; a novel human gene predicted to play a pivotal role in NMRD in humans based on an approximately 80% sequence conservation between it and the yeast UPF1 gene which has been shown to be required for NMRD in yeast).
Specific aims are: 1) To investigate the apparent NMRD deficiency in breast and colon cancer cell lines. This will be accomplished by transfection experiments using a minigene which has been shown to undergo normal NMRD when stably transfected into mammalian cells. If cancer cell lines have a general defect in NMRD, this minigene would be expected to be stable. 2) To investigate the role of RENT1 in NMRD. This will be accomplished by transfection of RENT1 minigenes into breast and colon cancer cells which have demonstrated stable tumor suppressor transcripts in the presence of nonsense mutations. 3) To investigate the apparent imperviousness of BRCA1 and APC transcripts with nonsense mutations to NMRD. This will be accomplished by constructing rearranged BRCA1 and APC minigenes and expressing them in normal breast and colon cells. Transfections with wild-type and mutant BRCA1 have already been reported. The experiments proposed here will use expression constructs in which regions of BRCA1 and APC are spliced into the minigene with demonstrated NMRD.
