ABSTRACT: Interferon (IFN) stimulates a whole repertoire of cellular genes, collectively referred to as ISGs (Interferon-stimulated genes). ISG20, a 3´-5´ exonuclease enzyme, has been previously shown to bind and degrade hepatitis B Virus (HBV) transcripts. Here, we show that the N6-methyladenosine (m6A)-modified HBV transcripts are selectively recognized and processed for degradation by ISG20. Moreover, this effect of ISG20 is critically regulated by m6A reader protein, YTHDF2 (YTH-domain family 2). Previously, we identified a unique m6A site within HBV transcripts and confirmed that methylation at nucleotide A1907 regulates HBV lifecycle. In this report, we now show that the methylation at A1907 is a critical regulator of IFN-? mediated decay of HBV RNA. We observed that the HBV RNAs become less sensitive to ISG20 mediated degradation when methyltransferase enzymes or m6A reader protein YTHDF2 are silenced in HBV expressing cells. By using an enzymatically inactive form ISG20D94G, we further demonstrated that ISG20 forms a complex with m6A modified HBV RNA and YTHDF2 protein. Due to terminal redundancy, HBV genomic nucleotide A1907 position is acquired twice by pregenomic RNA (pgRNA) during transcription and therefore the sites of methylation are encoded within 5´ and 3´ epsilon stem loops. We generated HBV mutants that lack m6A site at either one (5´ or 3´) or both the termini (5´& 3´). Using these mutants, we demonstrated that m6A modified HBV RNAs are subjected to ISG20-mediated decay and propose sequence of events, in which ISG20 binds with YTHDF2 and recognizes m6A-modified HBV transcripts to carry out the ribonuclease activity. This is the first study, which identifies a hitherto unknown role of m6A modification of RNA in IFN-? induced viral RNA degradation and proposes a new role of YTHDF2 protein as a cofactor required for IFN-? mediated viral RNA degradation.
Project description:Post-transcriptional gene expression regulation of RNA has emerged as a key factor that controls mammalian protein production. RNA trafficking, translation efficiency, and stability are all controlled at the transcript level. For example, in addition to the commonly known processing steps of capping, splicing, and polyadenylation, RNA can be chemically modified. In eukaryotes, N6-methyladenosine (m6A) is the most prevalent mRNA modification. While the writers, erasers, and readers for m6A are rapidly being uncovered and studied at the whole-cell level, their competitive interplay to regulate methylated RNA transcripts has yet to be elucidated. To address this limitation, we report the development of programmable dPspCas13b-m6A reader proteins to investigate the regulatory effects of specific readers on single transcripts in live cells. We fused the two most well-characterized m6A reader proteins, YTHDF1 and YTHDF2, to a catalytically inactive PspCas13b protein, which can target the reader to a specific RNA of interest using guide RNA (gRNA) complementarity. We then demonstrate that the fused reader proteins each retain their reported functional role on a reporter construct: YTHDF2 induces degradation and YTHDF1 enhances translation. Finally, we show that the system can target endogenous mRNA transcripts within cells, using YTHDF2 as an exemplar, where we found tethering with YTHDF2 leads to decay of the target transcript. The development of dCas13b-based tools to study the regulation of endogenous RNAs will dramatically enhance our understanding of how RNA regulation occurs at the single RNA level. Additionally, our new tools, which permit transcript-specific mediated decay or enhanced protein production, will find utility in synthetic biology applications aimed at controlling genetic information flow at the RNA level.
Project description:The mRNA N6-methyladenosine (m6A) modification has emerged as an essential regulator of normal and malignant hematopoiesis. Inactivation of the m6A mRNA reader YTHDF2, which recognizes m6A-modified transcripts to promote m6A-mRNA degradation, results in hematopoietic stem cell (HSC) expansion and compromises acute myeloid leukemia. Here we investigate the long-term impact of YTHDF2 deletion on HSC maintenance and multilineage hematopoiesis. We demonstrate that Ythdf2-deficient HSCs from young mice fail upon serial transplantation, display increased abundance of multiple m6A-modified inflammation-related transcripts, and chronically activate proinflammatory pathways. Consistent with the detrimental consequences of chronic activation of inflammatory pathways in HSCs, hematopoiesis-specific Ythdf2 deficiency results in a progressive myeloid bias, loss of lymphoid potential, HSC expansion, and failure of aged Ythdf2-deficient HSCs to reconstitute multilineage hematopoiesis. Experimentally induced inflammation increases YTHDF2 expression, and YTHDF2 is required to protect HSCs from this insult. Thus, our study positions YTHDF2 as a repressor of inflammatory pathways in HSCs and highlights the significance of m6A in long-term HSC maintenance.
Project description:YTHDF2 binds and destabilizes N6-methyladenosine (m6A)-modified mRNA. The extent to which this branch of m6A RNA-regulatory pathway functions in vivo and contributes to mammalian development remains unknown. Here we find that YTHDF2 deficiency is partially permissive in mice and results in female-specific infertility. Using conditional mutagenesis, we demonstrate that YTHDF2 is autonomously required within the germline to produce MII oocytes that are competent to sustain early zygotic development. Oocyte maturation is associated with a wave of maternal RNA degradation, and the resulting relative changes to the MII transcriptome are integral to oocyte quality. The loss of YTHDF2 results in the failure to regulate transcript dosage of a cohort of genes during oocyte maturation, with enrichment observed for the YTHDF2-binding consensus and evidence of m6A in these upregulated genes. In summary, the m6A-reader YTHDF2 is an intrinsic determinant of mammalian oocyte competence and early zygotic development.
Project description:Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (?) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-? interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ? directly in absence of any other cellular proteins, indicating a direct ? RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ?. In addition, the lower stem portion of ? is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ? abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-? interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ?, as the deletion of ExoIII abolished in vitro ISG20-? binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general.
Project description:Epitranscriptomic RNA modifications can serve as recognition elements for the recruitment of effector proteins (i.e., "readers") to modified transcripts. While these interactions play an important role in mRNA regulation, there is a major gap in our understanding of the sequence determinants critical for the binding of readers to modified sequence motifs. Here, we develop a high-throughput platform, relying upon in vitro selection with a site-specifically modified random sequence RNA library and next-generation sequencing, to profile the binding specificity of RNA modification reader proteins. We apply our approach to interrogate the effect of sequence context on the interactions of YTH-domain proteins with N6-methyladenosine (m6A)-modified RNA. We find that while the in vitro binding preferences of YTHDC1 strongly overlap with the well-characterized DR(m6A)CH motif, the related YTH-domain proteins YTHDF1 and YTHDF2 can bind tightly to noncanonical m6A-containing sequences. Our results reveal the principles underlying substrate selection by m6A reader proteins and provide a powerful approach for investigating protein-modified RNA interactions in an unbiased manner.
Project description:Many viral RNAs are modified by methylation of the N6 position of adenosine (m6A). m6A is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) expresses m6A-modified RNAs, but the effects of m6A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m6A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m6A "reader" protein YTHDF2 increases IAV gene expression and replication. To address whether m6A residues modulate IAV RNA function in cis, we mapped m6A residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate m6A on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression while leaving other IAV mRNAs and proteins unaffected, and they also resulted in reduced IAV pathogenicity in mice. Thus, m6A residues in IAV transcripts enhance viral gene expression.
Project description:BACKGROUND:N6-methyladenosine (m6A) is the most abundant modification in mRNA of humans. Emerging evidence has supported the fact that m6A is comprehensively involved in various diseases especially cancers. As a crucial reader, YTHDF2 usually mediates the degradation of m6A-modified mRNAs in m6A-dependent way. However, the function and mechanisms of m6A especially YTHDF2 in prostate cancer (PCa) still remain elusive. METHODS:To investigate the functions and mechanisms of YTHDF2 in PCa, in vitro, in vivo biofunctional assays and epigenetics experiments were performed. Endogenous expression silencing of YTHDF2 and METTL3 was established with lentivirus-based shRNA technique. Colony formation, flow cytometry and trans-well assays were performed for cell function identifications. Subcutaneous xenografts and metastatic mice models were combined with in vivo imaging system to investigate the phenotypes when knocking down YTHDF2 and METTL3. m6A RNA immunoprecipitation (MeRIP) sequencing, mRNA sequencing, RIP-RT-qPCR and bioinformatics analysis were mainly used to screen and validate the direct common targets of YTHDF2 and METTL3. In addition, TCGA database was also used to analyze the expression pattern of YTHDF2, METTL3 and the common target LHPP in PCa, and their correlation with clinical prognosis. RESULTS:The upregulated YTHDF2 and METTL3 in PCa predicted a worse overall survival rate. Knocking down YTHDF2 or METTL3 markedly inhibited the proliferation and migration of PCa in vivo and in vitro. LHPP and NKX3-1 were identified as the direct targets of both YTHDF2 and METTL3. YTHDF2 directly bound to the m6A modification sites of LHPP and NKX3-1 to mediate the mRNA degradation. Knock-down of YTHDF2 or METTL3 significantly induced the expression of LHPP and NKX3-1 at both mRNA and protein level with inhibited phosphorylated AKT. Overexpression of LHPP and NKX3-1 presented the consistent phenotypes and AKT phosphorylation inhibition with knock-down of YTHDF2 or METTL3. Phosphorylated AKT was consequently confirmed as the downstream of METTL3/YTHDF2/LHPP/NKX3-1 to induce tumor proliferation and migration. CONCLUSION:We propose a novel regulatory mechanism in which YTHDF2 mediates the mRNA degradation of the tumor suppressors LHPP and NKX3-1 in m6A-dependent way to regulate AKT phosphorylation-induced tumor progression in prostate cancer. We hope our findings may provide new concepts of PCa biology.
Project description:The N6-methyladenosine (m6A) modification regulates mRNA stability and translation. Here, we show that transcriptomic m6A modification can be dynamic and the m6A reader protein YTH N6-methyladenosine RNA binding protein 2 (YTHDF2) promotes mRNA decay during cell cycle. Depletion of YTHDF2 in HeLa cells leads to the delay of mitotic entry due to overaccumulation of negative regulators of cell cycle such as Wee1-like protein kinase (WEE1). We demonstrate that WEE1 transcripts contain m6A modification, which promotes their decay through YTHDF2. Moreover, we found that YTHDF2 protein stability is dependent on cyclin-dependent kinase 1 (CDK1) activity. Thus, CDK1, YTHDF2, and WEE1 form a feedforward regulatory loop to promote mitotic entry. We further identified Cullin 1 (CUL1), Cullin 4A (CUL4A), damaged DNA-binding protein 1 (DDB1), and S-phase kinase-associated protein 2 (SKP2) as components of E3 ubiquitin ligase complexes that mediate YTHDF2 proteolysis. Our study provides insights into how cell cycle mediators modulate transcriptomic m6A modification, which in turn regulates the cell cycle.
Project description:N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNAs (mRNAs), and plays important roles in cell differentiation and tissue development. It regulates multiple steps throughout the RNA life cycle including RNA processing, translation, and decay, via the recognition by selective binding proteins. In the cytoplasm, m6A binding protein YTHDF1 facilitates translation of m6A-modified mRNAs, and YTHDF2 accelerates the decay of m6A-modified transcripts. The biological function of YTHDF3, another cytoplasmic m6A binder of the YTH (YT521-B homology) domain family, remains unknown. Here, we report that YTHDF3 promotes protein synthesis in synergy with YTHDF1, and affects methylated mRNA decay mediated through YTHDF2. Cells deficient in all three YTHDF proteins experience the most dramatic accumulation of m6A-modified transcripts. These results indicate that together with YTHDF1 and YTHDF2, YTHDF3 plays critical roles to accelerate metabolism of m6A-modified mRNAs in the cytoplasm. All three YTHDF proteins may act in an integrated and cooperative manner to impact fundamental biological processes related to m6A RNA methylation.
Project description:The RNA N6-methyladenosine (m6A) methylation is installed by the METTL3-METTL14 methyltransferase complex. This modification has critical regulatory roles in various biological processes. Here, we report that deletion of Mettl14 dramatically reduces mRNA m6A methylation in developing B cells and severely blocks B cell development in mice. Deletion of Mettl14 impairs interleukin-7 (IL-7)-induced pro-B cell proliferation and the large-pre-B-to-small-pre-B transition and causes dramatic abnormalities in gene expression programs important for B cell development. Suppression of a group of transcripts by cytoplasmic m6A reader YTHDF2 is critical to the IL-7-induced pro-B cell proliferation. In contrast, the block in the large-pre-B-to-small-pre-B transition is independent of YTHDF1 or YTHDF2 but is associated with a failure to properly upregulate key transcription factors regulating this transition. Our data highlight the important regulatory roles of the RNA m6A methylation and its reader proteins in early B cell development.