Additional file 1 of RNA 2'-O-methylation promotes persistent R-loop formation and AID-mediated IgH class switch recombination
Creators: name: Kassab, Muzaffer Ahmad
name: Chen, Yibin
name: Wang, Xin
name: He, Bo
name: Brown, Eric J.
name: Yu, Xiaochun
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Figures S1-S8. Fig. S1. IgM contains 2′-OMe and 2′-OMe is not a universal feature of R loops. (A) 2′-O-methylation is present in IgM RNA. The diagram shows LC–MS/MS analysis of enriched IgM RNA from CH12F3 cells. The cells were stimulated with CIT, and the RNA was enriched using the ChIRP protocol. The extracted ion chromatogram (EIC) of m/z 113.03 (uridine) was derived from the MS/MS scan of 2′-O-methyluridine (Um) at m/z 259.09 (test: blue color). The chemical structure and the mass spectral properties of chemically synthesized Um (standard: green color) eluted at 1 min are shown and overlapped with the test sample. (B) Left: Schematic representation of Primer extension performed on an enriched IgA RNA from stimulated cells. Right: Primary sequence data of a clone, the position of Um1581, the primer used for RT and the full-length cDNA product are labeled. Reverse transcription of the IgA RNA stopped at the G residue marked with a stop sign, due to 2′-OMe of the upstream U residue. The TSO primer then hybridized to the truncated cDNA end which was then cloned into a TA cloning vector and sequenced. (C) 2′-O-methylation is not a universal feature of R-loops. Total cellular R-loops were isolated from stimulated CH12F3 cells using DRIP. The DNA within the R-loops was digested and the RNA was purified. The purified RNA was digested and subjected to LC MS/MS. The extracted ion chromatogram (EIC) of m/z 113.03 (uridine) was derived from the MS/MS scan of Um at m/z 259.09 (test: blue color). Uridine spectral peaks m/z 259.09 were present in the RNA obtained from stimulated CH12F3 cells but the spectral peaks m/z 259.09 corresponding to Um were absent in the RNA. The chemical structure and the mass spectral properties of chemically synthesized Um (standard: green color) eluted at 1 min are shown and overlapped with the test sample. (D) siRNA mediated knockdown of 2′-O-methyltransferases. Top panel: Total cellular RNA from CH12F3 cells was assayed for the levels of FBL, FTSJ2, FTSJ3 and CMTR1 via qPCR after gene-specific siRNA-mediated knockdown. A scrambled siRNA sequence was used in the control (Con) samples. The mean ± standard deviation of three experiments is shown. Right panel: Protein expression analysis after siRNA-mediated knockdown of methyltransferases using western blot. To confirm knockdown efficiency, stimulated cells were transfected with the respective siRNAs or a control (Con) siRNA and grown for 24 h. The cells were then stimulated with CIL and allowed to grow for 48 h before protein levels were measured using Western blot analysis. The Statistical significance was determined using a two-tailed Student’s t-test and error bars represent ± standard error of the mean. (E) FBL expression analysis in splenic B cells after siRNA-mediated knockdown of FBL. Splenic B cells were stimulated with CIT and allowed to grow for 24 h. The next day, FBL siRNA electroporation was done and cells were allowed to grow for the 24 h and FBL levels were measured using Western blot analysis. Right panel: bar graph shows quantification of FBL protein from three replicates. The Statistical significance was determined using a two-tailed Student’s t-test and error bars represent ± standard error of the mean. Fig. S2. Specificity of siRNA mediated FBL knockdown. (A) Knockdown of AID upon siRNA Transfection (B) CSR efficiency in CH12F3 cells after siRNA mediated AID knockdown. Two additional experiments gave similar results. The cells were transfected with AID and then stimulated with CIT for 24 h and the surface expression of IgM and IgA was measured using flow cytometry. The right panel shows the mean levels of class switching to IgA in CH12F3 cells after AID knockdown from three independent experiments,**:p ≤ 0.01. (C) Overlay histogram of carboxyfluorescein succinimidyl ester (CFSE) intensity (i.e. proliferation) of CH12F3 cells transfected with FBL siRNAs or control (Con) siRNA, grown for 24/48/72 h post transfection. (D) Effect of siRNA-mediated FBL knockdown on cellular translation. CH12F3 cells were transfected using FBL siRNA or control (Con) siRNA, grown for 24 h and stimulated with CIT for 48 h. The cell lysate was fractionated in a 7–47% sucrose gradient. The fractions were collected, and absorbance was measured at 260 nm. Error bars are representing standard deviation of the three independent experiments. The Statistical significance was determined using a two-tailed Student’s t-test. Fig. S3. The figure shows the expression of DNA-RNA hybrids (DRIP) in stimulated CH12F3 cells at the IgA locus 24 h after FBL and AID knockdown. One set of samples was treated with RNase H prior to immunoprecipitation using S9.6 antibodies. Pull down efficiency was calculated using total input DNA as a normalization control. Error bars represent standard deviation of three independent experiments. The Statistical significance was determined using a two-tailed Student’s t-test. Fig. S4. FBL associates with AID during CSR. (A) In vitro FBL and AID interaction. We transfected FLAG-tagged AID and HA-tagged FBL into HEK293T using Lipofectamine-2000 and the reversible co-IP was performed on the cell lysates 24 h after transfection. (B) Reversible co-IP of endogenous AID and FBL in activated CH12F3 cells. CH12F3 cells were stimulated with CIT for 48 h, and the cell lysate was analyzed by western blot. (C) In-vitro AID association with NOP56 and NOP58. FLAG-tagged AID and HA-tagged NOP56, NOP58 and FBL were transfected into HEK293T cells using Lipofectamine 2000 and reversible co-immunoprecipitation (co-IP) was performed on the cell lysates 24 h after Transfection. (D) Ectopic expression of WT and D2 mutant FBL in CH12F3 cells. The plasmids constructs were transfected into CH12F3 cells using electroporation and the protein expression was checked by western blotting. (E) Ectopic expression of WT and ΔC mutant AID in CH12F3 cells. The plasmids constructs were transfected into CH12F3 cells using electroporation and the protein expression was checked by western blotting. Fig. S5. SNORD1C is required for AID and FBL mediated 2′-O-methylation. (A) Schematic representation of the IgA RNA and aSNORD1C showing the positions of complementarity. The encompassing Um1581 is highlighted by an asterisk. (B) Directional RT-PCR in HEK293T and CH12F3 to check the presence of sense and antisense SNORD1C. Sense (Sen), antisense (Ant) or no primer (NT) was used to reverse transcribe the RNA and the resulting cDNA was amplified using both primers together. (C) Generation of AID stable cell line in HEK293T. The cDNA sequence of human AID was cloned in pIRES2-EGFP vector, transfected into HEK293T cells and the stable cells were selected using G418. The recombinant protein was detected by Western blot using anti-Flag antibodies. Mock: parental HEK293T cells, AID: clone with a positive EGFP signal, Western blot with actin antibodies served as a loading control. (D) To confirm the knockdown efficiency of ASOs, stimulated CH12F3 cells were transfected with either SNORD1C ASO or a control (Con) ASO and grown for 24 h. The cells were then stimulated with CIT and allowed to grow for 48 h, and the levels of FBL were measured using qPCR. The change in gene expression of the target genes was quantified with respect to snoRNA U16. Error bars are representing standard deviation of the three independent experiments. (E) aSNORD1C knockdown does not affect 28S rRNA methylation at G4044. CH12F3 cells were transfected with ASO’s against aSNORD1C and the levels of 28S rRNA methylation at G4044 were determined using RTLP. The Statistical significance was determined using a two-tailed Student’s t-test and error bars represent ± standard error of the mean. Fig. S6. snoRNAs can guide global S region. (A) List of IgA and IgG S region complementary snoRNAs. (B-E) snoRNA with “GGGUCGGG” sequence including SNORD23 and SNORA5 share homology across IgA, IgG, IgM and IgE S regions. (F) Knockdown of AID does not impair FBL recruitment. The top panel shows schematic representation of the Igμ and Igα loci and the position of primers (A and B) within the respective switch regions used for PCR amplification. CH12F3 cells were transfected with respective siRNAs for 24 h. The cells were then CIT stimulated and the immunoprecipitates were collected 24 h later ChIP assay was done using anti-FBL antibodies or anti IgG antibodies. The immunoprecipitates were analyzed by qPCR and relative enrichment was calculated using immumoprecipitate/relative to the input samples. The data represents mean ± standard deviation of three biologically independent experiments *:p &lt; 0.05. (G) Knockdown of FBL, AID and aSNORD1C does not affect germline transcription. CH12F3 cells were transfected with respective siRNAs and stimulated with CIT for 48 h and the levels of IgA transcription was determined using nuclear run-on assays. (H) Knockdown of AID, FBL and aSNORD1C is specific. FBL, AID and aSNORD1C was knocked down using the respective siRNAs and the levels of AID and FBL was determined in each case using western blot. The Statistical significance was determined using a two-tailed Student’s t-test and error bars represent ± standard error of the mean. Fig. S7. FBL- aSNORD1C promote AID recruitment at IgH locus and the complex (AID-aSNORD1C-FBL) deposits 2'-O-Me on the R-loops thereby promoting persistent R-loop formation. Suggested model depicting the possible mechanism involved in the formation of modified R-loop during CSR. Following the appearance of R-loops at the IgH locus, FBL bound AID recruitment is promoted at the R loop by aSNORD1C. FBL in association with its interacting partners (AID and aSNORD1C) deposits 2′-OMe marks on the RNA. The 2′-O-methylated R-loops now resist RNase H mediated digestion. This phenomenon ensures selective persistence of IgH-associated R-loops for an optimal CSR. Fig. S8. AID does not deaminate 2′-O-methylated RNA. (A) Recombinant human AID was purified from Sf9 insect cells, run on a polyacrylamide gel, and stained with Coomassie blue. A size marker and the GST tag are depicted. (B) The top panel shows schematic representation of AID mediated deamination of dC to dU on a ssDNA. The ssDNA containing dC is incubated with the purified AID. T7 sequenase is then used to polymerize a complimentary DNA strand on the synthetic DNA in presence of three dNTP and either in the presence of ddA or ddG. When the template contains dC, T7 polymerization of that template in presence of ddG leads to chain termination and a strong DNA band is formed, however; when AID converts dC to dU, T7 sequenase can read through the dU and a full-length DNA band is formed. The bottom panel shows the results of AID mediated deamination and the sequence of the DNA used in the assay is shown on the right side of the gel. The position of primer used for chain termination reaction and the conversion of dC to dU is shown. (C) The top panel shows schematic representation of AID mediated deamination of Cm to Um on an RNA. Synthetic ssRNA containing Cm or ssRNA-DNA duplex is used as a substrate for AID mediated deamination as described for DNA in Fig. S7B. The RNA is then reverse transcribed into cDNA using AMV RT enzyme. If the Cm on the template RNA was unaltered upon AID treatment, RT reaction at the Cm will be terminated in presence of ddG leading to the formation of an incomplete cDNA band. However, if AID converts Cm to Um, the RT enzyme will read through the now formed Um and a full length cDNA will be formed in the ddG lane. The bottom panel shows the results of deamination obtained with AID mediated RNA deamination. The sequence of the RNA used in the assay is shown on the right side of the gel.