The RNA M6 A Reader YTHDF2 Controls NK Cell Antitumor And Antiviral Immunity Part 4

It is well known that YTHDF2 acts as an m6A reader, which recognizes and binds m6A-containing RNAs and regulates the degradation of m6A transcripts.
We then performed RNA immunoprecipitation (RIP)–seq using YTHDF2 antibody to map the target transcripts bound by YTHDF2 in NK cells. YTHDF2- binding sites were enriched in the protein-coding sequence region and 39UTR (Fig. S5, E, and F). 

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We identified 3,951 potential YTHDF2 binding peaks from 1,290 transcripts, 426 (33%) of which were detected with significant m6A enrichment. GO enrichment analysis showed that the 1,290 YTHDF2 target transcripts were enriched in protein translation, RNA binding, and RNA splicing (Fig. S5 G).

We then identified the potential target transcripts from overlapping transcripts through RNA-seq, m6A-seq, and RIP-seq and found that a set of 29 transcripts bound by YTHDF2 and marked with m6A (in both Ythdf2WT and Ythdf2ΔNK NK cells) were differentially expressed between NK cells from Ythdf2WT and Ythdf2ΔNK mice (Fig. 7 G). Among them, 12 transcripts were upregulated and 17 transcripts were downregulated in NK cells from Ythdf2ΔNK mice compared with those from Ythdf2WT NK cells (Fig. 7 G). 

Based on our functional characterization of YTHDF2 and the GO analysis from the RNA-seq data, Ythdf2 deficiency affects NK cell division and proliferation, possibly by facilitating mRNA degradation of cell-cycle checkpoints or negative regulators during cell division. 

Among the 12 upregulated transcripts, three have been reported to negatively regulate cell division or proliferation, including Mdm2 (murine double minute 2 [MDM2]; Frum et al., 2009; Giono and Manfredi, 2007; Giono et al., 2017), Tardbp (TAR DNA-binding protein 43 [TDP43]; Ayala et al., 2008; Sanna et al., 2020), and Crebzf (CREB/ATF BZIP transcription factor; Hu et al., 2020; López-Mateo et al., 2012), suggesting that they are the potential targets of YTHDF2 in NK cells. 

Of note, the m6A peaks fit well with the YTHDF2- binding sites at the 39UTR of Mdm2 and Tardbp and with the 59UTR of Crebzf genes, as shown by Integrative Genomics Viewer (Fig. 7 H). 

RIP using either m6A or YTHDF2 antibody following qPCR confirmed that Mdm2, Tardbp, and Crebzf were indeed m6A methylated and enriched predominately by YTHDF2 in NK cells (Fig. 7, I and J), supporting m6A-seq and RIP-seq data, which indicate that these three genes are potential targets of YTHDF2 in NK cells. 

To investigate whether YTHDF2 regulates Mdm2, Tardbp, and Crebzf expression through modulating the mRNA stability, we measured the mRNA degradation of the three targets by inhibition of transcription with actinomycin D in NK cells from Ythdf2ΔNK and Ythdf2WT mice. 

The results showed that Mdm2 and Tardbp, but not Crebzf, had longer half-lives in NK cells from Ythdf2ΔNK mice compared with those from Ythdf2WT mice (Fig. 7, K–M), suggesting that Mdm2 and Tardbp are directly regulated by YTHDF2 in NK cells. 

Using immunoblotting, we confirmed that the protein levels of MDM2 and TDP43 were upregulated in NK cells from Ythdf2ΔNK mice (Fig. S5 H). To further confirm that these two genes are functional targets of YTHDF2, we used Mdm2- or Tardbp-specific siRNA to knock down the expression of the two genes in vitro (Fig. S5, I, and J). 

We then compared cell proliferation and survival of Ythdf2ΔNK NK cells with versus without knockdown of Mdm2 or Tardbp in the presence of IL-15. The results showed that the knockdown of Tardbp could at least partially rescue the defect in cell proliferation and cell survival in NK cells from Ythdf2ΔNK mice (Fig. 7, N, and O). 

However, the knockdown of Mdm2 had no rescue effects (Fig. S5, K, and L). These results indicate that YTHDF2 regulates NK cell proliferation and division at least partially by inhibiting the mRNA stability of Tardbp.

Discussion

In this study, we report the multifaceted roles of YTHDF2-mediated m6A methylation in NK cell immunity. We found that YTHDF2, one of the most important readers of m6A modifications, is critical for maintaining NK cell homeostasis, maturation, IL–15–mediated survival, and antitumor and antiviral activity. 

We also identified a novel positive feedback loop between STAT5 and YTHDF2, downstream of IL-15, that contributes to effector functions and survival in mouse NK cells. Our study elucidates the biological roles of YTHDF2 or m6A methylation in general in NK cell innate immunity. It fills in the gap of knowledge as to how YTHDF2 regulates the innate immune response to malignant transformation and viral infection. 

Our findings provide a new direction to harness NK cell antitumor immunity and simultaneously advance our understanding of m6A modifications in shaping innate immunity. The m6A reader protein YTHDF2 regulates the stability of target mRNAs (Du et al., 2016; Wang et al., 2014). Numerous studies have supported the broad impact of YTHDF2 in various biological processes. 

YTHDF2 suppresses normal hematopoietic stem cell expansion and self-renewal but is also required for long-term hematopoietic stem cell maintenance (Li et al., 2018; Mapperley et al., 2021 Paris et al., 2019; Wang et al., 2018a). Recently, this gene is involved in restraining inflammation during bacterial infection (Wu et al., 2020a). The role and mechanism of YTHDF2 in tumor development have been well studied. 

YTHDF2 promotes leukemic stem cell development and acute myeloid leukemia initiation (Paris et al., 2019). Besides leukemia, YTHDF2 has been shown to promote the development of solid tumors, including prostate cancer (Li et al., 2020), glioblastoma (Dixit et al., 2021), and hepatocellular carcinoma (Chen et al., 2018; Hou et al., 2019; Zhang et al., 2020), by targeting diverse m6A-modified transcripts, such as tumor suppressors LHPP and NKX3-1 (Li et al., 2020), IGFBP3 (Dixit et al., 2021), OCT4 (Zhang et al., 2020), and SOCS2 (Chen et al., 2018). 

Although YTHDF2 plays a promoting role in tumor progression, our study reveals a beneficial role of YTHDF2 in the immune response to tumor cells, particularly in NK cells, which is a key component of innate immunity against viral infections and malignant transformation. 

Therefore, future development of YTHDF2 inhibitors to target tumor cells for cancer therapy should be pursued with caution as inhibition of YTHDF2 may impair the host antitumor response by NK cells. On the other hand, harnessing the antitumor activity of YTHDF2 in NK cells or other immune cells should consider its potential effect acting on tumor cells directly. Differential targeting of YTHDF2 in tumor cells and immune cells should maximize antitumor activity.

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