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Induction and Assessment of Class Switch Recombination in Purified Murine B Cells
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RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination.

Claudia Ribeiro de Almeida1, Somdutta Dhir1, Ashish Dhir1

  • 1Sir William Dunn School of Pathology, University of Oxford, South Parks Road, OX1 3RE Oxford, UK.

Molecular Cell
|May 8, 2018
PubMed
Summary

This study reveals a post-transcriptional mechanism for class switch recombination (CSR) involving RNA helicase DDX1. DDX1 converts G-quadruplex structures in switch transcripts into R-loops, promoting AID targeting and CSR at the immunoglobulin heavy-chain locus.

Keywords:
DEAD-box RNA helicase 1G-quadruplexesR-loopsactivation-induced cytidine deaminaseclass switch recombination

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Area of Science:

  • Immunology
  • Molecular Biology
  • Genetics

Background:

  • Class switch recombination (CSR) is crucial for adaptive immunity, occurring at the immunoglobulin heavy-chain (IgH) locus.
  • CSR is associated with R-loop structures, which are RNA:DNA hybrids, often forming co-transcriptionally.
  • The precise mechanisms regulating R-loop formation and targeting key enzymes like AID remain incompletely understood.

Purpose of the Study:

  • To elucidate the role of RNA helicase DDX1 in the post-transcriptional formation of R-loops at the IgH locus.
  • To investigate how these R-loops facilitate the targeting of Activation-Induced Cytidine Deaminase (AID) for CSR.
  • To understand the interplay between G-quadruplex (G4) structures and R-loops in regulating CSR.

Main Methods:

  • Investigated R-loop formation in relation to CSR using molecular biology techniques.
  • Assessed the function of DDX1 in binding G-quadruplex structures and converting them into R-loops.
  • Utilized chemical stabilization of G4 RNA and dominant-negative DDX1 mutants to modulate R-loop levels and CSR efficiency.

Main Results:

  • Demonstrated that DDX1 mediates a post-transcriptional R-loop formation mechanism at IgH S-regions.
  • Showed that DDX1 binds to G-quadruplex structures in switch transcripts, converting them into R-loops.
  • Confirmed that R-loop formation is essential for targeting AID to S-regions, thereby promoting CSR.
  • Observed reduced R-loop levels and CSR efficiency upon G4 RNA stabilization or expression of a dominant-negative DDX1 mutant.

Conclusions:

  • Provided evidence for a novel post-transcriptional pathway in CSR involving DDX1-mediated G4 to R-loop conversion.
  • Highlighted the dynamic structural interconversion between G4 and R-loop motifs in switch transcripts as critical for CSR.
  • Established DDX1 as a key regulator linking RNA structure to DNA targeting for immunoglobulin diversification.