N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA. Very little is known of the function of m6A in the immune system or its role in host–pathogen interactions. Here we investigated the topology, dynamics, and bidirectional influences of the viral–host RNA methylomes during HIV-1 infection of human CD4 T cells. We show that viral infection triggers a massive increase in m6A in both host and viral mRNAs. In HIV-1 mRNA, we identified 14 methylation peaks in coding and noncoding regions, splicing junctions, and splicing regulatory sequences. We also identified a set of 56 human gene transcripts that were uniquely methylated in HIV-1-infected T cells and were enriched for functions in viral gene expression. The functional relevance of m6A for viral replication was demonstrated by silencing of the m6A writer or the eraser enzymes, which decreased or increased HIV-1 replication, respectively. Furthermore, methylation of two conserved adenosines in the stem loop II region of HIV-1 Rev Response Element (RRE) RNA enhanced binding of HIV-1 Rev protein to the RRE in vivo and influenced nuclear export of RNA. Our results identify a new mechanism for the control of HIV-1 replication and its interaction with the host immune system.

Although immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment, many patients do not respond or develop resistance to ICB. N6-methylation of adenosine (m6A) in RNA regulates many pathophysiological processes. Here, we show that deletion of the m6A demethylase Alkbh5 sensitized tumors to cancer immunotherapy. Alkbh5 has effects on m6A density and splicing events in tumors during ICB. Alkbh5 modulates Mct4/Slc16a3 expression and lactate content of the tumor microenvironment and the composition of tumor-infiltrating Treg and myeloid-derived suppressor cells. Importantly, a small-molecule Alkbh5 inhibitor enhanced the efficacy of cancer immunotherapy. Notably, the ALKBH5 gene mutation and expression status of melanoma patients correlate with their response to immunotherapy. Our results suggest that m6A demethylases in tumor cells contribute to the efficacy of immunotherapy and identify ALKBH5 as a potential therapeutic target to enhance immunotherapy outcome in melanoma, colorectal, and potentially other cancers.

N1-methyladenosine (mA) is an important post-transcriptional modification in RNA; however, the exact biological role of mA remains to be determined. By employing a quantitative proteomics method, we identified multiple putative protein readers of mA in RNA, including several YTH domain family proteins. We showed that YTHDF1-3 and YTHDC1, but not YTHDC2, could bind directly to mA in RNA. We also found that Trp in YTHDF2, a conserved residue in the hydrophobic pocket of the YTH domain that is necessary for its binding to N-methyladenosine (mA), is required for its recognition of mA. An analysis of previously published data revealed transcriptome-wide colocalization of YTH domain-containing proteins and mA sites in HeLa cells, suggesting that YTH domain-containing proteins can bind to mA in cells. Together, our results uncovered YTH domain-containing proteins as readers for mA in RNA and provided new insight into the functions of mA in RNA biology.

It is urgent and important to understand the relationship of the widespread severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) with host immune response and study the underlining molecular mechanism. N 6 -methylation of adenosine (m6A) in RNA regulates many physiological and disease processes. Here, we investigate m6A modification of SARS-CoV-2 gene in regulating host cell innate immune response. Our data show that SARS-CoV-2 virus has m6A modifications which are enriched in the 3’-end of the viral genome. We find that host cell m6A methyltransferase METTL3 depletion decreases m6A levels in SARS-CoV-2 and host genes, and m6A reduction in viral RNA increases RIG-I binding and subsequently enhances downstream innate immune signaling pathway and inflammatory gene expression. METTL3 expression is reduced and inflammatory genes are induced in severe COVID-19 patients. These findings will aid to understand the COVID-19 pathogenesis and help in designing future studies of regulating innate immunity for COVID-19 treatment.

N 6 -methyladenosine (m 6 A) has emerged as the most abundant mRNA modification that regulates gene expression in many physiological processes. m 6 A modification in RNA controls cellular proliferation and pluripotency and has been implicated in the progression of multiple disease states, including cancer. RNA m 6 A methylation is controlled by a multiprotein “writer” complex including the enzymatic factor methyltransferase-like protein 3 (METTL3) that regulates methylation and two “eraser” proteins, RNA demethylase ALKBH5 (ALKBH5) and fat mass- and obesity-associated protein (FTO), that demethylate m 6 A in transcripts. FTO can also demethylate N 6 ,2′- O -dimethyladenosine (m 6 A m ), which is found adjacent to the m 7 G cap structure in mRNA. FTO has recently gained interest as a potential cancer target, and small molecule FTO inhibitors such as meclofenamic acid have been shown to prevent tumor progression in both acute myeloid leukemia and glioblastoma in vivo models. However, current FTO inhibitors are unsuitable for clinical applications due to either poor target selectivity or poor pharmacokinetics. In this work, we describe the structure-based design, synthesis, and biochemical evaluation of a new class of FTO inhibitors. Rational design of 20 small molecules with low micromolar IC 50 ’s and specificity toward FTO over ALKBH5 identified two competitive inhibitors FTO-02 and FTO-04. Importantly, FTO-04 prevented neurosphere formation in patient-derived glioblastoma stem cells (GSCs) without inhibiting the growth of healthy neural stem cell-derived neurospheres. Finally, FTO-04 increased m 6 A and m 6 A m levels in GSCs consistent with FTO inhibition. These results support FTO-04 as a potential new lead for treatment of glioblastoma.