Browsing by Subject "N6-methyladenosine"

The intracellular innate immune response to viral infection is among the first lines of defense against these pathogens. For the early establishment of an antiviral cellular state and initiation of inflammatory responses, type I interferons (IFNs) are particularly important, as they potently induce the production of hundreds of IFN-stimulated genes (ISGs), many of which have antiviral functions. The type I IFN response requires tight molecular coordination to achieve both efficient production of antiviral proteins and controlled shutoff of inflammatory responses to avoid tissue damage and autoimmunity. Despite the importance of regulation of this antiviral response, current knowledge of the molecular controls governing its activation and suppression remains incomplete. Further, although ISGs have diverse functions and are induced to differing potencies, our understanding of regulatory controls governing the expression of individual or subclasses of ISGs is limited. Current knowledge of type I IFN response regulation is predominantly centered on transcriptional and post-translational regulatory controls. However, post-transcriptional regulation of antiviral responses has begun to emerge as an important layer of control. An example of these post-transcriptional regulatory controls is the RNA base modification N6-methyladenosine (m6A), which regulates many aspects of mRNA metabolism through transcript-specific effects. m6A deposition is mediated by a cellular complex of proteins including METTL3 and METTL14 (METTL3/14) and other cofactors, and m6A can also be removed from RNA by the demethylase proteins FTO and ALKBH5. The presence of m6A on viral and host RNAs has been shown to influence the outcome of infection by diverse viruses. However, the role of m6A in the response to type I IFNs has not been explored. To investigate the role of m6A in the type I IFN response, we began by manipulating m6A levels in the transcriptome through perturbation of the expression of the cellular m6A machinery and measuring the induction of ISGs after IFN treatment. We found that depletion of the m6A methyltransferase proteins METTL3 and METTL14 (METTL3/14) resulted in less protein production of a subset of ISGs, including the antiviral genes IFITM1 and MX1, after IFN treatment. However, the expression of other ISGs and the overall activation of the IFN responses were unchanged. Using methyl RNA immunoprecipitation and sequencing (meRIP-seq), we found that the transcripts of many ISGs are modified by m6A, and these included the METTL3/14-regulated ISGs IFITM1 and MX1 that we had identified. Using polysome profiling and ribosome profiling, we determined that METTL3/14-regulated ISGs are translationally enhanced by METTL3/14. Additionally, ablation of putative m6A sites within the 3’UTR of IFITM1 decreased the translation of a reporter molecule. Overexpression of the m6A reader protein YTHDF1, which has known roles in promoting translation, enhanced the expression of IFITM1 in an m6A binding-dependent fashion. These experiments characterized METTL3/14 and m6A as novel enhancers of the type I IFN response. To determine whether m6A contributes to type I IFN-mediated viral restriction, we depleted or overexpressed METTL3/14 and pretreated cells with a low dose of IFN-β prior to infection with vesicular stomatitis virus (VSV). Interestingly, METTL3/14 depletion decreased the expression of ISGs and allowed increased VSV infection, while METTL3/14 overexpression had the opposite effect. Together, these studies demonstrate that METTL3/14 and m6A enhance the antiviral effect of type I IFN by promoting the translation of ISGs to support the establishment of an antiviral cellular state. Having discovered a role for m6A in the type I IFN response, we also investigated the role of an m6A demethylase protein, FTO. FTO polymorphisms can have profound effects on human health. Certain polymorphisms are associated with fat mass and obesity, cardiovascular disease, while others can cause growth retardation or embryonic lethality. However, the molecular functions of FTO and the cellular pathways that it affects are still not well characterized. We depleted FTO and measured the production of ISGs following IFN-β treatment and found that the production of m6A-regulated ISGs was increased, as expected. However, unexpectedly, we found that FTO depletion increased the mRNA levels of a subset of ISGs. Pulse labeling of nascent transcripts revealed that FTO suppresses the transcription of these ISGs and that FTO-depleted cells are primed for the production of certain ISGs in response to IFN. We then used cells lacking PCIF1, the writer of 2’-O-N6-dimethyladenosine (m6Am), an RNA modification that FTO can also remove, and found that FTO-mediated regulation of ISGs occurs independently of the m6Am modification. These results identify FTO as a transcriptional regulator of a subset of ISGs, which will add an important dimension to our understanding of the molecular functions of FTO and its contributions to inflammatory disease. Future research revealing the mechanisms by which FTO suppresses ISG transcription will be of great interest. Together, these data identify novel functions of m6A and its related cellular machinery in both positive and negative regulation of the type I IFN response and antiviral gene expression.

Hepatitis C virus (HCV) exploits nearly all aspects of cellular RNA biology to regulate its viral RNA genome during infection. However, the molecular mechanisms by which HCV exploits one aspect of RNA regulation, RNA modification with N6-methyladenosine (m6A), are still emerging. This is because the current understanding of how RNA becomes m6A modified involves a variety of nuclear localized mechanisms that are incompatible with the cytoplasmic lifecycle of HCV. Thus, we set out to bridge the gap between our current understanding of m6A biology and how HCV RNA becomes m6A modified.In this work, we find that m6A modification of HCV RNA by the m6A-methyltransferase proteins METTL3 and METTL14 is regulated by WTAP. WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m6A-methyltransferase complex and known to target METTL3 to mRNA. We found that HCV infection induces localization of WTAP to the cytoplasm. Importantly, we found that WTAP is required for both METTL3 interaction with HCV RNA and for m6A modification across the viral RNA genome. Further, we found that WTAP, like METTL3 and METTL14, negatively regulates the production of infectious HCV virions, a process that we have previously shown is regulated by m6A. Excitingly, WTAP regulation of both HCV RNA m6A modification and virion production were independent of its ability to localize to the nucleus. Together, these results reveal that WTAP is critical for HCV RNA m6A modification by METTL3 and METTL14 in the cytoplasm.

RNA is a versatile and tractable biomolecule that serves as a critical component of life, whether as a script for protein production, a carrier of genetic information, a scaffold, or an enzyme. The fate and function of RNA can be influenced by chemical modifications such as N6-methyladenosine (m6A). Here we sought to identify the role of m6A during infection by positive-sense RNA viruses in the Flaviviridae family.

First, we investigated the role of m6A on viral RNA. We mapped m6A on the viral RNA genomes of hepatitis C virus (HCV), dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), and yellow fever virus (YFV). We then studied HCV as a model RNA and virus to understand the function of m6A on Flaviviridae RNA genomes. We found that the m6A methyltransferases METTL3 and METTL14 reduced HCV infectious particle production without affection viral RNA replication, while the m6A demethylase FTO had the opposite effect. Similarly, the m6A-binding YTHDF1-3 proteins also inhibited HCV particle production. Furthermore, the YTHDF proteins relocalized to cytoplasmic lipid droplets, the sites of HCV particle assembly, during infection. We then identified that m6A in a specific region of viral RNA was responsible for the role of m6A in viral particle production. Abrogation of m6A modification increased viral RNA binding to the capsid protein Core, an important step of HCV assembly, and also increased HCV particle production. These data suggest that m6A inhibits HCV particle production and that m6A modification of viral RNA can have a functional consequence for infection.

We then investigated how m6A on cellular mRNA can impact Flaviviridae infection. Working in collaboration with Dr. Chris Mason’s lab, we developed stringent analytical tools for detecting m6A changes. When we applied these tools, we found that a subset of cellular transcripts had altered m6A modification following infection by DENV, ZIKV, WNV, and HCV. We identified that innate immune signaling and ER stress, cellular pathways which are activated during Flaviviridae infection, contribute to altered m6A modification of two model transcripts RIOK3 and CIRBP. The gain of m6A on RIOK3 promotes the translation of this transcript, while loss of m6A on CIRBP influences its alternative splicing. Importantly, the RIOK3, CIRBP, and other transcripts with altered m6A modification can promote or inhibit Flaviviridae infection. Taken together, these results highlight the important role of m6A on both viral and cellular RNA in regulating infection.