Browsing by Subject "m6A"

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.

N6-methyladenosine (m6A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m6A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m6A contributes to brain development are inco¬¬mpletely understood. Here, we discover serine-/arginine-rich splicing factor 7 (SRSF7) and RNA-binding motif-containing protein 45 (RBM45) as m6A-binding proteins in transformed hippocampal neurons. We find that SRSF7 binds to exon-intron junctions in methylated pre-mRNA targets and regulates the gene expression of thousands of cellular mRNAs, including the m6A RNA methyltransferase, METTL3. We find that RBM45 binds to thousands of cellular RNAs, predominantly within intronic regions. Rbm45 depletion disrupts the constitutive splicing of a subset of target pre-mRNAs, leading to altered mRNA and protein levels through both m6A-dependent and m6A-independent mechanisms. Finally, we find that RBM45 is highly expressed during embryonic neurodevelopment, demonstrating that expression of RBM45 is necessary for neuroblastoma cell differentiation and that its depletion impacts the expression of genes involved in several neurodevelopmental signaling pathways. Altogether, our findings identify roles for SRSF7 and RBM45 in gene expression regulation, and highlight a previously unknown function for RBM45 in the control of pre-mRNA processing and neuronal differentiation, mediated in part by the recognition of methylated RNA.

Ribonucleic acid (RNA) is a versatile and dynamic biomolecule that serves as an indispensable component in the central dogma of molecular biology. The realization that RNA plays a wide variety of roles in gene expression and regulation has been accompanied by the discovery that virtually all types of RNA are chemically modified. These modifications have profound effects on RNA metabolism. N6-Methyladenosine (m6A) is an abundant post-transcriptional RNA modification that influences multiple aspects of gene expression. While m6A is thought to mainly function by recruiting reader proteins to specific RNA sites, the modification can also reshape RNA-protein and RNA−RNA interactions by altering RNA structure mainly by destabilizing base pairing. Here we sought to provide a broad and deep description of how m6A reshapes the folding and recognition landscape of RNA, which provides detailed mechanisms via which m6A exerts its biological functions.First, we show that when neighbored by a 5ʹ bulge, m6A stabilizes m6A–U base pairs and global RNA structure by ~1 kcal/mol. The bulge most likely provides the flexibility needed to allow optimal stacking between the methyl group and 3ʹ neighbor through a conformation that is stabilized by Mg2+. A bias toward this motif can help explain the global impact of methylation on RNA structure in transcriptome-wide studies. While m6A embedded in duplex RNA is poorly recognized by the YTH domain reader protein and m6A antibodies, both readily recognize m6A in this newly identified motif. The results uncover potentially abundant and functional m6A motifs that can modulate the epitranscriptomic structure landscape with important implications for the interpretation of transcriptome-wide data. In addition to altering RNA stability, m6A has also been shown to slow the kinetics of biochemical processes involving RNA-RNA interactions. However, little is known about how m6A affects the kinetic rates of RNA folding and conformational transitions that are important for RNA functions. We developed an NMR relaxation dispersion (RD) method to non-invasively and site-specifically measure nucleic acid hybridization kinetics. Using this method, we discovered that m6A selectively slows annealing rate while has minimal impact on melting rate in different sequence contexts and buffer conditions. To understand the mechanism of the m6A-induced slowdown of hybridization, we used NMR RD to dissect the kinetic pathways of duplex hybridization. We show that m6A pairs with uridine with the methylamino group in the anti conformation to form a Watson-Crick base pair that transiently exchanges on the millisecond timescale with a singly hydrogen-bonded low-populated (1%) mismatch-like conformation in which the methylamino group is syn. This ability to rapidly interchange between Watson-Crick or mismatch-like forms, combined with different syn:anti isomer preferences when paired (~1:100) versus unpaired (~10:1), explains how m6A robustly slows duplex annealing without affecting melting via two pathways in which isomerization occurs before or after duplex annealing. Our model quantitatively predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions and provides an explanation for why the modification robustly slows diverse cellular processes. Taken together, these results uncover the important role of m6A on modulating RNA-RNA and RNA-protein interactions through altering RNA structure and dynamics, highlighting the structural-dynamics-function relationship.

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.