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Alu insertion-mediated dsRNA structure formation with pre-existing Alu elements as a disease-causing mechanism.

Emmanuelle Masson1, Sandrine Maestri1, Valérie Bordeau2

  • 1Univ Brest, Inserm, EFS, UMR 1078, GGB, 29200 Brest, France; CHRU Brest, 29200 Brest, France.

American Journal of Human Genetics
|September 12, 2024
PubMed
Summary

A homozygous Alu insertion in SPINK1 causes pancreatic insufficiency by forming double-stranded RNA (dsRNA) structures with intronic Alu elements, disrupting gene expression. Restoring SPINK1 mRNA requires aligning these Alu sequences.

Keywords:
3′-UTR reporter assayFLGEARNA secondary structureSPINK1aberrant splicingdouble-stranded RNA structuredsRNAfull-length gene expression assayinverted Alu elements

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Area of Science:

  • Genetics
  • Molecular Biology
  • Human Disease

Background:

  • A homozygous Alu insertion variant (Alu_Ins) in the SPINK1 3'-untranslated region (3'-UTR) causes severe infantile exocrine pancreatic insufficiency.
  • The precise mechanism by which Alu_Ins leads to complete loss of SPINK1 mRNA expression was previously unknown.

Purpose of the Study:

  • To elucidate the molecular mechanisms underlying SPINK1 mRNA loss caused by the Alu_Ins variant.
  • To investigate the role of double-stranded RNA (dsRNA) structures formed by Alu elements in gene regulation.

Main Methods:

  • Luciferase reporter assays to assess the impact of Alu_Ins on 3'-UTR function.
  • Bioinformatic analysis (RepeatMasker) to identify Alu elements within the SPINK1 gene.
  • RNA structure prediction (RNAfold) and gene expression assays to study Alu-Alu interactions.

Main Results:

  • Alu_Ins alone caused only a ~50% reduction in reporter gene activity, insufficient to explain the phenotype.
  • Extensive dsRNA structures formed between Alu_Ins and intronic Alu elements, dependent on their orientation.
  • Restoration of SPINK1 mRNA expression was observed when all three Alu elements were in the same orientation.

Conclusions:

  • The severe exocrine pancreatic insufficiency is caused by dsRNA formation between the SPINK1 3'-UTR Alu insertion and intronic Alu repeats.
  • Alu element orientation is critical for regulating gene expression and disease association.
  • Findings highlight the significance of Alu insertions in genetic disease and the need for careful interpretation.