ResearchDefining critical roles for RNA-binding proteins (RBPs) in B cells undergoing antibody gene diversification
B cells are crucial to immune defence by producing large quantities of antibodies with pathogen neutralizing capacities, when terminally differentiated into plasma cells. Effective antibody responses are tailored and can be made to target a limitless range of antigens with exquisite specificity. This remarkable ability of the adaptive immune system relies on the generation of a diverse repertoire of antibodies, which goes hand-in-hand with B cell development.
Antibodies are encoded by immunoglobulin heavy (IgH) and light (IgL) chain loci, which go through processes of DNA deletion-recombination – V(D)J recombination, class switch recombination (CSR) - and somatic hypermutation (SHM), at different stages of B cell development. Our research focuses on the role of RBPs at B cell developmental-stages when V(D)J recombination and CSR occur. Control over mRNA metabolism or regulation of RNA function by RBPs is particularly relevant at these steps, as important cellular decisions – programmed induction of DNA mutation/breaks at immunoglobulin genes, cell proliferation, cell differentiation – rely on tight regulation of gene expression and the maintenance of genome stability.
Understanding the molecular mechanisms regulating antibody production is important, as impaired ability to generate a diverse antibody repertoire often results in immunodeficient or autoimmune conditions. Our work also has potential implications for other pathological outcomes associated with antibody gene diversification, as for example the development of B cell lymphomas. In the future, RBPs may constitute ideal targets to modulate B cell function during vaccination or disease, and this will require a better understanding of their roles in B cell biology.
Antibody gene diversification is coordinated with B cell development
During progenitor (pro-) and precursor (pre-) B cell stages in the bone-marrow, immunoglobulin loci undergo random assembly of V, D and J gene segments, which encode for the variable region of IgH and IgL chain proteins. This process is known as V(D)J recombination and the large number of possible different combinations between gene segments accounts for a wide range of antigen binding specificities in the antibody repertoire (see figure below for the mouse IgH locus).
V(D)J recombination at the IgH locus is initiated at the pro-B cell stage, and the expression of a functional IgH chain is monitored through pairing with surrogate light-chain components to form a pre-B cell receptor (pre-BCR) on the membrane of large pre-B cells. The pre-BCR acts as a key checkpoint in B cell development, to drive the clonal expansion and survival of large pre-B cells. Pre-BCR signals are also required for developmental progression into small pre-B cells, which occurs concomitantly with cessation of proliferation and the initiation of Igκ or Igλ loci V(D)J recombination. Successful rearrangement of IgL loci results in the assembly of the BCR on the membrane of immature B cells.
When mature B-cells encounter antigen in peripheral lymphoid organs, they are activated to proliferate, undergo CSR and differentiate to antibody-secreting plasma cells. CSR mechanisms occur at the IgH locus, and replace the default Cμ exon (encoding for IgM) by one of a limited set of CH exons encoding other IgH chain isotypes (IgG3, IgG1, IgG2b, IgG2c, IgE or IgA) (see figure below). These exons encode for the constant region of IgH chain proteins and determine the effector function of antibodies (e.g. activation of different immune effector cells or the complement system). Mature B-cell activation is also associated with the formation of germinal centres (GCs), specialized cellular microenviroments where IgH and IgL loci undergo somatic hypermutation (SHM) at the V(D)J exon. Together with selection mechanisms occurring at the GC, SHM allows for the production of antibodies with increased affinity for antigen.
A role for DEAD-box RNA helicase 1 (DDX1) in class switch recombination
CSR does not occur at random and recombination is directed to a particular CH exon by cytokine-induced, non-coding RNA transcription initiated upstream of that exon. These long non-coding RNAs are termed germline transcripts (GLTs) or switch RNA, and include a 1-10 Kb long first intron characterized by a G-rich and highly repetitive sequence. On the DNA, these sequences are known as switch (S-)regions as they contain a high density of AGCT motifs recognized by activation-induced cytidine deaminase (AID), the enzyme that initiates CSR. Transcription of switch RNA promotes the formation of RNA:DNA hybrid structures behind the elongating RNA polymerase II. These structures, known as R-loops, result in the displacement of the non-template DNA as a single strand that may act as a substrate for AID to deaminate cytidines to uracils. The resulting U:G mismatches are subsequently processed into DNA double-strand breaks (DSBs) and two distinct S-regions are ligated by non-homologous end-joining proteins.
Recent evidence supports a role for the switch RNA itself in targeting AID to complementary S-region DNA. AID was demonstrated to bind G-quadruplex (G4) structures present in the first intron of switch RNAs once spliced, and an AID mutant unable to bind G4 RNA impairs CSR. We have recently demonstrated that DEAD-box RNA-helicase 1 (DDX1) is part of the molecular machinery that remodels switch RNA structures in B-cells undergoing CSR (Ribeiro de Almeida et al, 2018). DDX1 binds to G4 RNA and utilizes ATP to convert them into RNA:DNA-hybrid structures over complementary S-region DNA. DDX1 depletion in B cells results in decreased AID targeting to S-regions and impaired CSR. Notably R-loop levels over S-regions are diminished by chemical stabilization of G4 RNA or by the expression of a DDX1 ATPase deficient mutant that acts as a dominant-negative protein to reduce CSR efficiency.
So far R-loops were believe to exclusively form co-transcriptionally when RNA exits RNA polymerase II during transcription and anneals with the template DNA strand. We propose that R-loops over IgH S-regions can be formed downstream of G4 RNA, via DDX1-dependent mechanisms. It is possible that both mechanisms cooperate to allow efficient CSR. Understanding how G4 RNA/DDX1-dependent mechanisms impact on AID activity in B cells is one of our current topics of research.