MRE11 proximal polyadenylation site-mediated looping impacts transcription and genomic stability

Area of Science:

• Molecular Biology
• Genetics
• Genomics

Background:

• Alternative polyadenylation (APA) generates transcript isoforms with variable 3' untranslated regions (UTR) lengths.
• The role of APA in DNA damage response (DDR) genes remains largely unexplored.
• The MRE11-RAD50-NBS1 (MRN) complex is crucial for DNA repair and genome stability.

Purpose of the Study:

• To investigate the function of the proximal polyadenylation site (pPAS) of MRE11 in DNA damage response.
• To elucidate the mechanism by which MRE11 pPAS regulates MRE11 transcription and MRN complex levels.
• To determine the broader implications of PAS-promoter looping in other DDR genes.

Main Methods:

• Deletion of MRE11 pPAS in cells.
• Analysis of MRE11 transcription and MRN complex levels.
• 5-ethynyl-2'-deoxyuridine sequencing (EdU-seq) to assess DNA synthesis.
• MRE11 chromatin immunoprecipitation sequencing (ChIP-seq) to evaluate MRE11 binding.
• Investigation of PAS-promoter looping in other DDR genes.

Main Results:

• Deletion of MRE11 pPAS disrupts PAS-promoter looping, reduces MRE11 transcription, and impairs MRN complex levels.
• MRE11pPAS knockout cells exhibit ectopic DNA replication and reduced viability.
• Aberrant DNA synthesis occurs in intronic and intergenic regions, correlating with decreased MRE11 binding.
• PAS-promoter looping is identified as a regulatory mechanism in multiple DDR genes.

Conclusions:

• The MRE11 pPAS is a critical noncoding element regulating MRE11 transcription and genome stability.
• PAS-promoter looping is a novel mechanism maintaining genome integrity.
• This mechanism extends to other DNA damage response genes, suggesting a conserved regulatory pathway.