Transcriptional mutagenesis reduces splicing fidelity in mammalian cells

Abstract

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.

O 6 -methylguanine–induced transcriptional mutagenesis reduces p53 tumor-suppressor function

“…In agreement with earlier studies performed in vitro and in vivo ( 7 , 11 , 12 , 32 , 33 ), this work demonstrates that O 6 -meG is mutagenic during transcription. Furthermore, DNA repair wields a significant impact on the frequency of TM induced by O 6 -meG, an observation that has been made for other DNA lesions ( 9 , 12 , 33 , 34 ). As reported here, RNA-seq analysis indicated that 14.7% of p53 transcripts from cells lacking AGT activity contained a uridine misincorporation at codon 248, a number that was 100-fold lower in cells exhibiting AGT activity.…”

“…As reported here, RNA-seq analysis indicated that 14.7% of p53 transcripts from cells lacking AGT activity contained a uridine misincorporation at codon 248, a number that was 100-fold lower in cells exhibiting AGT activity. These numbers are lower than previously reported levels in vivo ( 11 , 12 ), which could be explained by the different cell systems used and/or the DNA sequence context of the lesion ( 35 ). The low levels of misincorporation (<1%) observed when AGT is active could reflect errors occurring during library preparation and sequencing ( 36 ).…”

“…However, DNA lesions also influence RNA synthesis, not only by causing transcriptional arrest, which in turn activates transcription-coupled DNA repair ( 2 , 3 ), but also by inducing transcriptional misincorporation into nascent RNA, an event referred to as “transcriptional mutagenesis” (TM) ( 4 ). TM, which has been observed in vitro ( 5 – 8 ) and in vivo ( 9 – 12 ), results most often from transcriptional bypass of small lesions formed by oxidation or alkylation of the bases in DNA. Although TM has been shown to induce phenotypic changes in mammalian cells by inducing altered protein function ( 9 – 11 ), its role in disease development remains unclear.…”

“…O 6 -meG is repaired by the protein alkylguanine–DNA alkyltransferase (AGT) that transfers the methyl group from the damaged base to a cysteine residue within the protein, resulting in restoration of guanine in DNA. However, O 6 -meG that escapes repair is readily bypassed by RNA polymerases, which can lead to significant levels of TM when uridine rather than cytidine is misincorporated into RNA ( 7 , 11 , 12 ). In fact, TM induced by O 6 -meG can result in changes in protein function in vivo ( 11 ) and can attenuate the splicing fidelity of G-rich exon–intron junctions in mammalian cells ( 12 ).…”

DNA oxidation induced by fetal exposure to BPA agonists impairs female meiosis

“…A. Paredes et al, 2017). For these reasons, we speculated that induction of oxidative stress by bisphenols could be responsible of observed transcriptional alteration as well as RNA splicing modification during meiosis initiation leading to prophase I defects.…”