Transcriptional mutagenesis reduces splicing fidelity in mammalian cells

Splicing fidelity is essential to the maintenance of cellular functions and viability, and mutations or natural variations in pre-mRNA sequences and consequent alteration of splicing have been implicated in the etiology and progression of numerous diseases. The extent to which transcriptional errors or lesion-induced transcriptional mutagenesis (TM) influences splicing fidelity is not currently known. To investigate this, we employed site-specific DNA lesions on the transcribed strand of a minigene splicing reporter in normal mammalian cells. These were the common mutagenic lesions O6-methylguanine (O6-meG) and 8-oxoguanine (8-oxoG). The minigene splicing reporters were derived from lamin A (LMNA) and proteolipid protein 1 (PLP1), both with known links to human diseases that result from deregulated splicing. In cells with active DNA repair, 1-4% misincorporation occurred opposite the lesions, which increased to 20-40% when repair was compromised. Furthermore, our results reveal that TM at a splice site significantly reduces in vivo splicing fidelity, thereby changing the relative expression of alternative splicing forms in mammalian cells. These findings suggest that splicing defects caused by transcriptional errors can potentially lead to phenotypic cellular changes and increased susceptibility to the development of disease.