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. 2015 May 7;161(4):762-73.
doi: 10.1016/j.cell.2015.03.020.

Non-coding RNA Generated following Lariat Debranching Mediates Targeting of AID to DNA

Simin Zheng  1 , Bao Q Vuong  2 , Bharat Vaidyanathan  1 , Jia-Yu Lin  3 , Feng-Ting Huang  3 , Jayanta Chaudhuri  4
Affiliations

Non-coding RNA Generated following Lariat Debranching Mediates Targeting of AID to DNA

Simin Zheng et al. Cell. .

Abstract

Transcription through immunoglobulin switch (S) regions is essential for class switch recombination (CSR), but no molecular function of the transcripts has been described. Likewise, recruitment of activation-induced cytidine deaminase (AID) to S regions is critical for CSR; however, the underlying mechanism has not been fully elucidated. Here, we demonstrate that intronic switch RNA acts in trans to target AID to S region DNA. AID binds directly to switch RNA through G-quadruplexes formed by the RNA molecules. Disruption of this interaction by mutation of a key residue in the putative RNA-binding domain of AID impairs recruitment of AID to S region DNA, thereby abolishing CSR. Additionally, inhibition of RNA lariat processing leads to loss of AID localization to S regions and compromises CSR; both defects can be rescued by exogenous expression of switch transcripts in a sequence-specific manner. These studies uncover an RNA-mediated mechanism of targeting AID to DNA.

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Conflict of interest statement

The authors declare that they have no competing financial interests.

Figures

Figure 1
Figure 1. Switch RNA can bind AID
(A) In vitro transcribed (IVT) telomeric and switch RNAs bind to AID. IVT biotinylated RNAs were folded and incubated with whole cell extracts from stimulated CH12 cells, followed by pull-down with streptavidin beads. Proteins recovered were analyzed by immunoblot with AID or Apobec3 antibodies; while bound RNAs were analyzed by dot blot using streptavidin-HRP. Input RNAs were verified to be biotinylated by streptavidin-HRP-northern blot. SμF, SαF; forward/sense switch μ and α RNA. SμR, SαR; reverse/anti-sense switch μ and α RNA. Result shown is representative of three independent pull-down experiments. (B) Switch RNA interacts with AID in vivo. CH12 cells stably expressing S1-aptamer tagged Sα transcripts, in either the forward/sense (S1SαF) or the reverse/anti-sense (S1SαR) orientation, were stimulated. Untagged SαF and SαR expressing cells were used as controls. The S1-aptamer tag has affinity for streptavidin and ribonucleoprotein complexes were isolated on streptavidin beads. RNA in the eluates was reverse transcribed and analyzed by qPCR (RT-qPCR) for amounts of Sα transcripts relative to S1SαF, while proteins in the eluates were analyzed by immunoblot. Result shown is representative of three independent pull-down experiments. (C) Competition RNA binding assay. RNA pull-down was performed with 1 nM biotinylated SμF RNA and 100 ng MBP-AID(WT) protein, in the presence of increasing concentrations of non-biotinylated competitor RNAs. Bound MBP-AID(WT) recovered by pull-down with streptavidin beads were analyzed by immunoblot with an AID antibody. Data shown is representative of three experiments.
Figure 2
Figure 2. G-quadruplex structures in switch RNA
(A) Sense switch RNAs and telomere RNA are predicted to form G-quadruplex structures. QGRS Mapper software was used to assess the G-quadruplex forming potential of the RNA sequences. The probability of G-quadruplex structure formation is reported as a G-score and represented over the corresponding regions in blue. (B) Sequences of synthetic RNA oligonucleotides. Sμ4G, four Sμ repeats in tandem; mutant Sμ4G (Sμ4Gmut), with G-to-C mutations that abolish guanine tracts. Guanine tracts in Sμ4G and corresponding regions in Sμ4Gmut are underlined. (C) Sμ4G was resolved on a denaturing gel, or folded in either KCl- or LiCl-containing buffer and resolved on a native gel, and stained with SYBR GOLD following electrophoresis. (D) Sμ4G associates with AID while Sμ4Gmut does not. RNA pull-down was performed with biotinylated Sμ4G and Sμ4Gmut (folded in either KCl- or LiCl-containing buffer) and stimulated CH12 extracts. Recovered proteins were analyzed by immunoblot and bound RNAs by streptavidin-HRP blot. Result shown is representative of three independent pull-down experiments. (E) Sμ4G interacts with AID in vivo. CH12 cells stably expressing S1-aptamer tagged Sμ4G or Sμ4Gmut transcripts were stimulated and ribonucleoprotein complexes were isolated on streptavidin beads. CH12 cells expressing anti-sense S1Sμ4G, which is unable to bind streptavidin, were used as control. RNA in the eluates was analyzed by RT-qPCR for amounts of exogenous transcripts relative to S1Sμ4G, while proteins in the eulates were analyzed by immunoblot. Result shown is representative of two independent pull-down experiments. (F) Circular dichroism (CD) analysis of G-quadruplex structures. CD spectra of Sμ4G, Sμ4Gmut, SμF and SμR RNAs (folded in either KCl- or LiCl-containing buffer). Wavelengths of observed peaks are indicated. Peaks characteristic of parallel G-quadruplexes are (positive- ~260 nm, negative- ~240 nm). (G) Colorimetric assay of G-quadruplexes. RNAs were folded and incubated with hemin. G-quadruplexes bind hemin and the resultant complex exhibits peroxidase-like activity, which can be detected as an increase in absorbance around 420 nm when substrate is added (Haeusler et al., 2014; Li et al., 2013). See also Figure S1.
Figure 3
Figure 3. Glycine-133 of AID is critical for RNA binding and CSR
(A) Purified recombinant MBP-tagged, wild-type (WT) or mutant AID(G133V) proteins were analyzed by Coomassie stain and immunoblot with AID antibody. Arrow indicates the size corresponding to full-length MBP-AID proteins on the Coomassie stained gel. (B) Purified proteins were used in the RNA pull-down assay with IVT biotinylated switch RNAs, followed by analysis of recovered proteins by immunoblot with an AID antibody, and bound RNAs by streptavidin-HRP dot blot. Result shown is representative of three independent pull-down experiments. (C) Enzymatic activity of purified proteins was determined by a deamination assay. The rate of deamination was determined as a function of protein concentration as described in Experimental Procedure. The average ± SD of three independent protein preparations is shown; NS, p=not significant, p≥0.05 at 25, 50 and 100 pmoles enzyme concentrations. (D) The G133V mutation does not affect binding of AID to single-stranded DNA (ssDNA). Purified proteins were incubated with biotinylated ssDNA, followed by pull-down with streptavidin beads and analysis by immunoblot with an AID antibody. Data shown is representative of three experiments. (E–H) Splenic B cells were isolated from AID−/− mice and transduced with vector control (pMIG), or vectors to express AID(WT) or AID(G133V). (E) Expression of AID proteins was verified by immunoblot with AID or GAPDH (control) antibodies. (F) CSR to IgG1 was determined by flow cytometry. A representative experiment is shown. The numbers in the corners indicate the percentage of cells in each quadrant, while the numbers in parentheses indicate the percentage of IgG1+ cells within the GFP+ gate. (G) The average percentage of IgG1+ cells within the GFP+ gate from three independent experiments ± SD is shown. (H) Localization of AID proteins to S regions was determined by ChIP, using AID or H3 antibodies. Sμ and Sγ1 DNA in ChIP samples was measured by qPCR, normalized to input DNA and the AID(WT) control. The mean of three independent experiments ± SD is shown. See also Figure S2.
Figure 4
Figure 4. DBR1+/− splenic B cells exhibit reduced CSR upon ex vivo stimulation
Splenic B cells were isolated from heterozygous (+/−) DBR1 gene-trapped mice and wild-type (+/+) littermate controls, and stimulated in culture with anti-CD40 and IL-4 for 72 h. (A) Expression of full length DBR1 is reduced in DBR1+/− B cells. The level of DBR1 mRNA was measured by RT-qPCR using primers downstream of the gene trap insertion, normalized to β-actin mRNA and DBR1+/+ control. The average of four pairs of DBR1+/− mice and their littermate DBR1+/+ controls ± SD are shown; *p<0.05. (B) CSR to IgG1 was determined at 72h post-stimulation by flow cytometry. Data shows the mean of 10 pairs of DBR1+/− mice and their DBR1+/+ littermates ± SD. *p<0.05. (C–E) Splenic B cells from DBR1+/+ and DBR1+/− mice were transduced with retroviral vector control (pMIR) or vector expressing Xpress-tagged DBR1 (XpDBR1). (C) Expression of XpDBR1 and AID were verified by immunoblot by anti-Xpress, anti-AID and anti-GAPDH (control) antibodies. (D) CSR to IgG1 was determined by flow cytometry. The average percentage of IgG1+ cells within the mCherry+ gate from three independent experiments ± SD is shown. *p<0.05. (E) Expression of XpDBR1 does not adversely affect levels of μ- and γ1-germline switch transcripts (GLT) compared to pMIR. Levels of μ- and γ1-GLT were determined by qRT-PCR, normalized to β-actin mRNA and pMIR control. Data represents the average of three independent experiments ± SD; NS, p=not significant, p≥0.05. See also Figure S3.
Figure 5
Figure 5. Debranching of intronic RNA lariats is required for targeting of AID to S regions and efficient CSR
(A–F) Knockdown of DBR1 impairs CSR and AID localization to S regions. CH12 cells were transduced with shRNA against DBR1 or scrambled control shRNA. (A) Knockdown of DBR1 transcripts was determined by RT-qPCR. (B) Accumulation of Sμ lariats following DBR1 knockdown was determined by RT-qPCR using primers (arrows) positioned across the branchpoint as shown (inset). N.D., not detected. Data in (A) and (B) were normalized to β-actin mRNA and the scrambled control; the average of at least three independent knockdown experiments ± SD are shown; *p<0.05. (C) CSR to IgA was assayed by flow cytometry at indicated times following stimulation. Data shows the mean of three independent knockdown experiments ± SD. *p<0.05. (D) AID protein expression was analyzed at indicated times following stimulation by immunoblot with AID or GAPDH (control) antibodies. (E) Localization of AID to S regions was determined by ChIP on cells 48 h post-stimulation using AID or H3 antibodies. Sμ and Sα DNA in ChIP samples were measured by qPCR and normalized to input DNA and the scrambled control. Data represents mean of three independent knockdown experiments ± SD. *p<0.05. (F) R-loop formation is unaffected by DBR1 knockdown. Genomic DNA was prepared from cells 24 h post-stimulation and treated with the bisulfite modification assay. Sα was examined and the number of molecules containing R-loops is represented as a percentage of the total number of DNA amplicons sequenced. The mean of three independent knockdown experiments ± SD is shown; NS, p=not significant, p≥0.05. See also Figure S4, S5.
Figure 6
Figure 6. Localization of AID to S regions can be restored by exogenous expression of switch transcripts
(A) Experimental design. CH12 cells were transduced with empty vector (pEF) or vectors to express forward/sense or reverse/anti-sense switch α RNA (pEF-SαF or pEF-SαR, respectively). Transduced cells were sorted, infected with scrambled control shRNA or shRNA to knockdown DBR1, and stimulated with CIT. (B–C) Expression of exogenous SαF rescues AID localization to Sα at the endogenous IgH locus, but not to the non-complementary Sμ. ChIP was performed on cells 48 h post stimulation by immunoprecipitation with anti-AID antibodies. Sα (B) and Sμ (C) DNA in ChIP samples was quantified by qPCR, and normalized to input and scrambled control. (D) Expression of exogenous SαF does not rescue CSR in DBR1 knockdown cells. CSR to IgA was assayed 72 h after stimulation by flow cytometry. Data in (B)–(D) represent the mean of three independent experiments ± SD. *p<0.05; NS, p=not significant, p≥0.05. See also Figure S6.
Figure 7
Figure 7. Co-expression of sense Sμ and Sα transcripts rescues CSR to IgA in DBR1 knockdown cells
(A) Experimental design. CH12 cells were transduced with empty vectors (EF) or vectors expressing forward/sense (For) or reverse/anti-sense (Rev) switch transcripts. Doubly transduced cells were sorted, infected with scrambled control shRNA or shRNA to knockdown DBR1, and stimulated with CIT. (B) CSR to IgA was assayed by flow cytometry 72 h after stimulation. The average of three independent experiments ± SD is shown. *p<0.05; NS, p=not significant, p≥0.05. (C) Model for RNA-mediated targeting of AID during CSR. When B cells are stimulated to undergo class switching, transcription occurs at each of the recombining S regions to produce primary switch transcripts. Primary transcripts are spliced, with the intronic switch region sequences (Sx) spliced out as a lariat intermediate. Debranching enzyme (DBR1) catalyzes the release of the lariat and debranches the switch transcript into its linear form. The linear switch transcript, free of exonic sequences, can function as guide RNAs by forming G-quadruplex or G-quadruplex-like structures, which allows association with AID. AID, bound to guide RNAs, can be targeted specifically to the complementary S region DNA based on sequence information provided by the guide RNAs. See also Figure S6.
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