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MeRIP, RIP and RNA Sequencing of the H1299 stable cell lines which the endogenous YTHDF2 was silenced by a short hairpin RNA targeting YTHDF2 3’UTR (shYTHDF2) and then re-expressed Flag-tagged YTHDF2-WT orYTHDF2-K571R

ABSTRACT: SUMOylation affects many aspects of target proteins such as activity, stability, localization and protein-protein interactions. We have found that SUMOylation of YTHDF2 increased its binding activity with m6A-RNAs by using different experimental approaches. To confiremd this conclusion,the analysis of RIP-seq, MeRIP-seq and RNA-seq in H1299-shYTHDF2 cells re-expressing YTHDF2-WT and YTHDF2-K571R was performed. MeRIP+RIP targets showed lower binding affinities in the mutant YTHDF2-K571R when compared with YTHDF2-WT. Compared to the control group, the binding capacities of YTHDF2 to RIP targets in treated group with either 2-D08 or GA were decreased, especially to MeRIP+RIP targets. Moreover, SUMOylated YTHDF2 promoted m6A-RNAs degradation. Combined analysis of RNA-seq, RIP-seq and MeRIP-seq showed that the mRNA levels were up-regulated in shYTHDF2 stable cells re-expressing YTHDF2-K571R compared with those in re-expressing YTHDF2-WT.

ORGANISM(S): Homo sapiens

PROVIDER: GSE164917 | GEO | 2021/01/26

REPOSITORIES: GEO

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Project description:RNA epigenetics plays crucial roles in physiological and pathological processes. As the most abundant methylation of mRNA, 0.2-0.7% of the total adenosine, m6A is dynamically deposited co-transcriptionally by the methyltransferase complex, reversely removed by demethylases recognized and directly regulated by “reader” proteins. As the first identified m6A ‘reader’ protein, YTHDF2 recognizes and destabilizes m6A modified mRNAs by inducing mRNA decay or degradation in P-body and plays indispensable oncogenic or tumor suppressive roles in multiple malignant contexts by mediating distinct m6A modified mRNAs. Here, we report that YTHDF2 promotes the development of B cell malignancies by facilitating energy supplements and immune evasion. RNA-sequencing (RNA-seq) data reveal that a set of ATP production- and immune response-related genes are consistently and significantly dysregulated upon YTHDF2 knockdown (KD) in both B cell acute lymphoblastic leukemia (B-ALL) patient-derived xenotransplant (PDX) cells (IAH8R) and diffuse large B cell lymphoma (DLBCL) cells (SU-DHL-4). RNA immunoprecipitation sequencing (RIP-seq) of either wildtype or mutant YTHDF2 proteins identify target genes directly bind to YTHDF2 dependent on the RNA binding pockets. Methylation RIP-seq (MeRIP-seq) assays identify either m6A-moidfied genes or m5C-modified mRNAs, which is further confirmed by bisulfite-converted mRNA sequencing (BS-RNA-seq) in malignant B cells. Mechanically, we find that YTHDF2 enhances ATP synthesis by sustaining mRNA stabilities of target genes as an m5C reader protein by interacting with PABPC1, a well-know m5C-modified mRNA stabilizer. Further RNA-seq upon PABPC1 KD and RIP-seq assays for PABPC1 reveal the overlapped genes which are also regulated and bound with YTHDF2 via m5C modification. Strikingly, RNA-seq and m6A-MeRIP-seq data show that YTHDF2 promotes immune evasion by destabilizing CD19 and MHC-II molecules (e.g., HLA-DMA and HLA-DMB) in an m6A-dependent manner. Single cell RNA-seq (scRNA-seq) data further reveal the immune microenvironment and cell proportions in bone marrow of Ythdf2 conditional KO mouse models (Ythdf2fl/fl). Our proof-of-concept study discovers that a YTHDF2 selective inhibitor (e.g. CCI-38) significantly inhibits cell metabolism and immune evasion in malignant B cells (KOPN-8) by RNA-seq. Collectively, our data show that YTHDF2 promotes ATP synthesis and immune evasion and serves as a promising therapeutic target in B cell malignancies.