R-Loop Depletion by Over-expressed RNase H1 in Mouse B Cells Increases Activation-Induced Deaminase Access to the Transcribed Strand without Altering Frequency of Isotype Switching

Robert W. Maul, Hyongi Chon, Kiran Sakhuja, Susana M. Cerritelli, Lina A. Gugliotti, Patricia J. Gearhart, Robert J. Crouch

Research output: Contribution to journalArticle

Abstract

R-loops, three-strand structures consisting of mRNA hybridized to the complementary DNA and a single-stranded DNA loop, are formed in switch regions on the heavy-chain immunoglobulin locus. To determine if R-loops have a direct effect on any of the steps involved in isotype switching, we generated a transgenic mouse that over-expressed RNase H1, an enzyme that cleaves the RNA of RNA/DNA hybrids in B cells. R-loops in the switch μ region were depleted by 70% in ex vivo activated splenic B cells. Frequencies of isotype switching to IgG1, IgG2b, IgG2c, and IgG3 were the same as C57BL/6 control cells. However, somatic hypermutation was increased specifically on the transcribed strand from μ-γ joins, indicating that R-loops limit activation-induced (cytosine) deaminase access to the transcribed DNA strand. Our data suggest that, in the normal G + C-rich context of mammalian class switch recombination regions, R-loops are obligatory intermediates. Processing of the R-loops is needed to remove RNA allowing activation-induced (cytosine) deaminase to promote somatic hypermutation on both DNA strands to generate double-strand DNA breaks for efficient class switch recombination. One of the two cellular RNases H may assist in this process.

Original languageEnglish (US)
JournalJournal of Molecular Biology
DOIs
StateAccepted/In press - Oct 30 2016
Externally publishedYes

Fingerprint

Immunoglobulin Class Switching
Cytosine Deaminase
B-Lymphocytes
RNA
Genetic Recombination
DNA
Immunoglobulin G
Ribonuclease H
Immunoglobulin Heavy Chains
Double-Stranded DNA Breaks
Single-Stranded DNA
Transgenic Mice
Complementary DNA
Messenger RNA
Enzymes
ribonuclease HI
AICDA (activation-induced cytidine deaminase)

Keywords

  • Class switch recombination
  • R-loops
  • RNase H1
  • Somatic hypermutation
  • Transgenic mouse

ASJC Scopus subject areas

  • Molecular Biology

Cite this

R-Loop Depletion by Over-expressed RNase H1 in Mouse B Cells Increases Activation-Induced Deaminase Access to the Transcribed Strand without Altering Frequency of Isotype Switching. / Maul, Robert W.; Chon, Hyongi; Sakhuja, Kiran; Cerritelli, Susana M.; Gugliotti, Lina A.; Gearhart, Patricia J.; Crouch, Robert J.

In: Journal of Molecular Biology, 30.10.2016.

Research output: Contribution to journalArticle

Maul, Robert W. ; Chon, Hyongi ; Sakhuja, Kiran ; Cerritelli, Susana M. ; Gugliotti, Lina A. ; Gearhart, Patricia J. ; Crouch, Robert J. / R-Loop Depletion by Over-expressed RNase H1 in Mouse B Cells Increases Activation-Induced Deaminase Access to the Transcribed Strand without Altering Frequency of Isotype Switching. In: Journal of Molecular Biology. 2016.
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abstract = "R-loops, three-strand structures consisting of mRNA hybridized to the complementary DNA and a single-stranded DNA loop, are formed in switch regions on the heavy-chain immunoglobulin locus. To determine if R-loops have a direct effect on any of the steps involved in isotype switching, we generated a transgenic mouse that over-expressed RNase H1, an enzyme that cleaves the RNA of RNA/DNA hybrids in B cells. R-loops in the switch μ region were depleted by 70{\%} in ex vivo activated splenic B cells. Frequencies of isotype switching to IgG1, IgG2b, IgG2c, and IgG3 were the same as C57BL/6 control cells. However, somatic hypermutation was increased specifically on the transcribed strand from μ-γ joins, indicating that R-loops limit activation-induced (cytosine) deaminase access to the transcribed DNA strand. Our data suggest that, in the normal G + C-rich context of mammalian class switch recombination regions, R-loops are obligatory intermediates. Processing of the R-loops is needed to remove RNA allowing activation-induced (cytosine) deaminase to promote somatic hypermutation on both DNA strands to generate double-strand DNA breaks for efficient class switch recombination. One of the two cellular RNases H may assist in this process.",
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