Premature termination codon in the aggrecan gene of nanomelia and its influence on mRNA transport and stability

Aim. To analyze the influence of the premature termination codon on mRNA tr ansport and stability. Methods. Chondrocyte mRNA was isolated from homozygous and heterozygous nan omelic 17-days old embryos and examined by RT-PCR analysis. To analyze aggr ecan mRNA stability, mRNA synthesis was inhibited with DRB [5,6 dichloro-1- (-D-ribofuranosyl benzimidazole)], a specific inhibitor of RNA polymerase I I. Visualization of the aggrecan alleles was performed by in situ hybridiza tion. Results. The level of mutant aggrecan mRNA within the nucleus was equal to that of the control, but no mutant mRNA was observed in the cytoplasm. RT-P CR revealed that the mutant transcript was only detectable in the nucleus, compared with house-keeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH ) gene or collagen type II. A restriction site induced by premature termina tion codon TAA allowed the distinction of normal and mutant transcripts in chondrocytes derived from embryos heterozygous for the nanomelic mutation. After the treatment with DRB, identical decay rates were demonstrated for b oth transcripts within the heterozygous nucleus. In situ hybridization show ed no abnormal mRNA accumulation. Conclusion. This is the first evidence suggesting that the transcript of th e mRNA with the premature termination codon within an exon does exit the nu cleus.