DNA damage regulates alternative splicing through inhibition of RNA polymerase II elongation

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA damage triggers apoptosis, a process often regulated by alternative splicing (AS).
• The precise control mechanisms linking DNA damage, AS, and apoptosis remain largely unelucidated.
• The role of RNA polymerase II phosphorylation in regulating AS during DNA damage is not well understood.

Purpose of the Study:

• To investigate the impact of ultraviolet irradiation (UV) on cotranscriptional alternative splicing (AS).
• To elucidate the role of RNA polymerase II carboxy-terminal domain (CTD) phosphorylation in UV-induced AS.
• To confirm the relevance of UV-induced AS in p53-independent apoptosis.

Main Methods:

• In vivo and real-time estimation of transcriptional elongation.
• Utilizing phosphomimetic and nonphosphorylatable CTD mutants of RNA polymerase II.
• Employing splicing-sensitive microarrays to analyze gene expression and AS changes.
• Assessing the effect of bcl-x alternative splicing reversion on UV-induced apoptosis.

Main Results:

• UV irradiation inhibits transcriptional elongation and alters cotranscriptional AS in a p53-independent manner.
• Hyperphosphorylation of the RNA polymerase II CTD is identified as a key mediator of UV-induced AS.
• CTD mutants mimicking phosphorylation replicate UV's effect on AS, while nonphosphorylatable mutants prevent it.
• Reverting AS changes in the bcl-x gene prevents UV-induced apoptosis in p53-deficient cells.
• A significant overlap exists between genes with altered AS and reduced expression following UV exposure.

Conclusions:

• UV-induced DNA damage regulates cotranscriptional AS via RNA polymerase II CTD hyperphosphorylation and inhibited elongation.
• This mechanism plays a crucial role in p53-independent apoptosis.
• Transcriptional coupling to AS is a significant feature of the cellular DNA damage response.