Targeted RNA N 6‐Methyladenosine Demethylation Controls Cell Fate Transition in Human Pluripotent Stem Cells

Abstract Deficiency of the N 6‐methyladenosine (m6A) methyltransferase complex results in global reduction of m6A abundance and defective cell development in embryonic stem cells (ESCs). However, it's unclear whether regional m6A methylation affects cell fate decisions due to the inability to modulate individual m6A modification in ESCs with precise temporal control. Here, a targeted RNA m6A erasure (TRME) system is developed to achieve site‐specific demethylation of RNAs in human ESCs (hESCs). TRME, in which a stably transfected, doxycycline‐inducible dCas13a is fused to the catalytic domain of ALKBH5, can precisely and reversibly demethylate the targeted m6A site of mRNA and increase mRNA stability with limited off‐target effects. It is further demonstrated that temporal m6A erasure on a single site of SOX2 is sufficient to control the differentiation of hESCs. This study provides a versatile toolbox to reveal the function of individual m6A modification in hESCs, enabling cell fate control studies at the epitranscriptional level.

Lentivirus production. HEK293T cells were seeded in 10 cm dishes. At about 90% confluency, cells were transfected with 16 μg lentiviral vector, 4 μg pMD2.G and 12.5 μg psPAX2 using BES buffered saline (Sigma). The lentivirus supernatant was harvested at 48 and 72 hours after transfection, mixed together, centrifuged at 600 g for 10 min, passed through a 0.45 μm filter and stored at -80 °C.
Construction of the TRME hESC lines. NKX2-5 eGFP/w hESCs were enzymatically dissociated into single cells by Accutase (STEMCELL Technologies) and nucleofected with TRME editor plasmid (or the corresponding negative control plasmids) and transposase plasmid at a mass ratio of 1,000:1 using the Neon® Transfection System (Thermo Fisher Scientific). Then cells were seeded onto 6-well plates in E8 medium supplemented with Y-27632 (5 μM). After nucleofection, puromycin (1 µg ml -1 , Selleck) and doxycycline (1 µg ml -1 , Sigma) were added for transfected cell selection until stable colonies appeared. Fluorescence-activated cell sorting (FACS) was carried out to isolate GFP positive cells which were then seeded onto 6-well plate at a density of 2 × 10 5 cells per well and infected with crRNA-expressing lentiviruses at 30% confluency for 6 hours. After two rounds of transduction, TRME hESCs expressing both GFP and mCherry were sorted by FACS and expanded in following cell culture.
Immunofluorescence staining. TRME hESCs were washed with PBS and fixed by 4% paraformaldehyde (Meilunbio) for 15 min at room temperature. The fixed cells were then washed three times with PBS and treated with 0.3% Triton X-100 (Sigma) at room temperature for 30 min for permeabilization followed by incubation with 3% BSA (Solarbio) for 1 hour.
After blocking, cells were incubated with primary antibodies overnight at 4 ºC and then stained with isotype-matched Alexa Fluorescence-conjugated secondary antibodies (Thermo Fisher Scientific) for 1 hour at room temperature. DAPI (1:1,000 dilution)-stained compartments serve as markers of the nuclei. Image acquisition was performed on Operetta CLS and processed with Western blot. Cells were digested by 0.5 mM EDTA and washed twice in PBS. Then cells were pelleted, and re-suspended in RIPA buffer (CST) containing the protease inhibitor cocktail (MCE) for 30 min on ice. Lysed samples were centrifuged (12,000 g for 2 min at 4 °C) and supernatant collected. Protein concentration was quantified with BCA Protein Assay Kit (Beyotime) followed by heating for 15 min at 95 °C. Boiled samples were loaded on SDS-PAGE gels and separated by electrophoresis for about 2 hours. Gels were then removed from plates and transferred onto the Immobilon-P membranes (Millipore) and the polyvinylidene difluoride membranes were blocked in TBST buffer containing 5% skimmed milk (BD Biosciences) for 1 hour. Subsequently, the membranes were incubated with primary antibodies overnight at 4 °C followed by blotting with secondary antibodies at room temperature for 2 hours. Blotting signal was visualized after reaction with enhanced chemiluminescence (ECL Plus, GE Healthcare). Primary antibodies are listed as followed: HA (66006-1-Ig, Proteintech; 1: 1,000); β-TUBULIN (AF1216, Beyotime; 1: 1,000); SOX2 (AB5603, Millipore; 1:1,000); β-ACTIN (AF0003, Beyotime; 1: 1,000).
Alkaline phosphatase (AP) staining. AP staining was performed using the Alkaline Phosphatase Detection Kit (Sigma-Aldrich) following the manufacturer's instruction. Cell proliferation assays. For the growth curve assay, cells were seeded on 96-well plates at a density of 2 × 10 4 cells per well. After the indicated incubation time, CCK8 (GLPBIO) was added to each well and further incubated for 2 hours at 37 °C. OD450 absorbance was measured with a microplate reader to detect the number of cells.

Cell viability assays.
SELECT for detection of m 6 A. SELECT qPCR was conducted by following Xiao's protocol. [2] Briefly, total RNA was isolated from PBS-washed cells using FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme) according to the manufacturer's guide. RNA isolation and purification. Cells were washed with PBS and lysed with TRIzol (Thermo Fisher Scientific). Then trichloromethane (Guangzhou Chemical Reagent Factory) was used to extract the total RNA from the TRIzol lysate. Isopropanol (Sangon Biotech) was used to precipitate RNA from the mixture. mRNA was purified from the total RNA by using the Dynabeads™ mRNA Purification Kit (Thermo Fisher Scientific).  10 mM Tris-HCl, pH 7.4; 0.1% Igepal CA-630; 6.7 mM m 6 A in nuclease-free H2O) at 37 °C, 1,000 rpm for 1.5 hours and purified by alcohol precipitation. Eluted RNA was used for further RT-qPCR.

RNA binding protein immunoprecipitation (RIP
MeRIP-seq. The m 6 A immunoprecipitation method was the same as above. Input samples and IP samples were applied for NGS library construction by using the NEBNext® Ultra II Directional RNA Library Prep (NEB) according to the manufacturer's guide. The adaptor ligated DNAs amplified by PCR for 15 cycles were sequenced on the HiSeqX sequencing platform. MeRIP-seq data were deposited in the NCBI's Gene Expression Omnibus (GEO) database and can be accessed by the accession number GSE158421.
Analysis of MeRIP-seq data. Quality control was performed on raw data with FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Cutadapt (pypi.org/project/cutadapt/) was used to find and remove adapter sequences. Clean data was mapped to human reference genome (hg19) using HISAT2 [3] with default parameters. Multiple-alignment reads were filtered using Samtools to retain reads with map quality (MapQ) scores above 30 and with properly oriented read mates. Bam files were converted to bed files using in-house python scripts. The m 6 A peaks were identified using MACS2 (-f BEDPE --nomodel --keep-dup all -q 0.01). [4] FPKM value was calculated to represent m 6 A peak intensity. The identified m 6 A peak regions were further filtered to assess TRME's specificity. In detail, the m 6 A peak region intensity in IP sample is at least 20% higher than that in input sample, and m 6 A peak intensity (CUGUCCGGCCCUCACAUGUGUGAGAGGG) was scanned across the transcript sequence covered by final 2643 m 6 A peaks for searching mismatched loci with Cas-OFFinder [5] allowing less than or equal to 8 mismatches.
Isolation of cytoplasmic and nuclear RNAs. The cytoplasmic and nuclear RNAs fractionation procedure was performed as described previously with some modifications. [6] Briefly, cells were trypsinized and washed once with cold PBS, and then resuspended in 1 volume of HLB+N  Samples that were more than two standard deviations from the mean were excluded from the analyses. Differences were considered significant when p-value < 0.05; *p < 0.05, **p < 0.01; NS, no significant.