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XPC branch-point sequence mutations disrupt U2 snRNP binding, resulting in abnormal pre-mRNA splicing in xeroderma

Sikandar G Khan1, Koji Yamanegi, Zhi-Ming Zheng

  • 1Dermatology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA.

Human Mutation
|December 3, 2009
PubMed

Insights

Mutations in XPC gene splicing disrupt DNA repair, leading to xeroderma pigmentosum (XP) symptoms. This impacts U2 snRNP binding, reducing XPC protein and DNA repair capacity, affecting cell survival after UV exposure.

Area of Science:

  • Genetics
  • Molecular Biology
  • DNA Repair

Background:

  • Xeroderma pigmentosum (XP) is a genetic disorder characterized by defective DNA repair.
  • Mutations in the XPC gene are associated with XP, but the precise molecular mechanisms are not fully understood.
  • Branch-point sequences (BPS) in pre-mRNA are crucial for proper splicing.

Purpose of the Study:

  • To investigate the mechanism by which mutations in XPC gene branch-point sequences affect pre-mRNA splicing.
  • To determine the functional consequences of these splicing abnormalities on XPC protein levels and DNA repair.
  • To correlate cellular findings with clinical phenotypes in XP patients.

Main Methods:

  • Transfection of minigenes containing wild-type and mutated XPC sequences.
  • DNA oligonucleotide-directed RNase H digestion to assess U2 snRNP-BPS interaction.
  • Western blotting and immunofluorescence to quantify XPC protein levels and recruitment to DNA damage sites.
  • Post-UV survival assays and photoproduct analysis.

Main Results:

  • Mutated XPC minigenes exhibited aberrant pre-mRNA splicing, mimicking patient cells.
  • XPC BPS mutations were shown to disrupt the interaction between U2 snRNP and the branch-point sequence.
  • XP patients with BPS mutations showed reduced XPC protein levels (XP72TMA: 29% of normal) or undetectable levels (XP101TMA).
  • XP72TMA cells showed limited recruitment of XPC and other nucleotide excision repair (NER) proteins to UV-damaged DNA, while XP101TMA cells showed no recruitment.
  • XP101TMA cells exhibited a greater reduction in DNA repair capacity, lower post-UV survival, and impaired photoproduct removal compared to XP72TMA cells.

Conclusions:

  • Mutations in XPC gene branch-point sequences lead to defective pre-mRNA splicing by disrupting U2 snRNP binding.
  • This splicing defect results in reduced XPC protein levels and impaired DNA repair, contributing to the XP phenotype.
  • The degree of XPC protein reduction and NER impairment correlates with clinical severity, particularly skin cancer development.

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