Extensive pre-mRNA back-splicing generates numerous circular RNAs (circRNAs) in human transcriptome. However, the biological functions of these circRNAs remain largely unclear. Here we report that N-6-methyladenosine (m(6)A), the most abundant base modification of RNA, promotes efficient initiation of protein translation from circRNAs in human cells. We discover that consensus m(6)A motifs are enriched in circRNAs and a single m(6)A site is sufficient to drive translation initiation. This m(6)A-driven translation requires initiation factor eIF4G2 and m(6)A reader YTHDF3, and is enhanced by methyltransferase METTL3/14, inhibited by demethylase FTO, and upregulated upon heat shock. Further analyses through polysome profiling, computational prediction and mass spectrometry reveal that m(6)A-driven translation of circRNAs is widespread, with hundreds of endogenous circRNAs having translation potential. Our study expands the coding landscape of human transcriptome, and suggests a role of circRNA-derived proteins in cellular responses to environmental stress.
FOXP3-expressing regulatory T （Treg） cells, which suppress aberrant immune response against self-antigens, also suppress anti-tumor immune response. Infiltration of a large number of Treg cells into tumor tissues is often associ- ated with poor prognosis. There is accumulating evidence that the removal of Treg cells is able to evoke and enhance anti-tumor immune response. However, systemic depletion of Treg cells may concurrently elicit deleterious autoim- munity. One strategy for evoking effective tumor immunity without antoimmunity is to specifically target terminally differentiated effector Treg cells rather than all FOXP3＋ T cells, because effector Treg cells are the predominant cell type in tumor tissues. Various cell surface molecules, including chemokine receptors such as CCR4, that are specifi- cally expressed by effector Treg cells can be the candidates for depleting effector Treg cells by specific cell-depleting monoclonal antibodies. In addition, other immunological characteristics of effector Treg cells, such as their high ex- pression of CTLA-4, active proliferation, and apoptosis-prone tendency, can be exploited to control specifically their functions. For example, anti-CTLA-4 antibody may kill effector Treg ceils or attenuate their suppressive activity. It is hoped that combination of Treg-cell targeting （e.g., by reducing Treg cells or attenuating their suppressive activity in tumor tissues） with the activation of tumor-specific effector T cells （e.g., by cancer vaccine or immune checkpoint blockade） will make the current cancer immunotherapy more effective.
N-6-methyladenosine (m(6)A) 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, m(6)A binding protein YTHDF1 facilitates translation of m(6)A-modified mRNAs, and YTHDF2 accelerates the decay of m(6)A-modified transcripts. The biological function of YTHDF3, another cytoplasmic m(6)A 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 m(6)A-modified transcripts. These results indicate that together with YTHDF1 and YTHDF2, YTHDF3 plays critical roles to accelerate metabolism of m(6)A-modified mRNAs in the cytoplasm. All three YTHDF proteins may act in an integrated and cooperative manner to impact fundamental biological processes related to m(6)A RNA methylation.
N6 -methyladenosine (m6 A) is the most common internal modification in eukaryotic mRNA. It is dynamically installed and removed, and acts as a new layer of mRNA metabolism, regulating biological processes including stem cell pluripotency, cell differentiation, and energy homeostasis. m6 A is recognized by selective binding proteins; YTHDF1 and YTHDF3 work in concert to affect the translation of m6 A-containing mRNAs, YTHDF2 expedites mRNA decay, and YTHDC1 affects the nuclear processing of its targets. The biological function of YTHDC2, the final member of the YTH protein family, remains unknown. We report that YTHDC2 selectively binds m6 A at its consensus motif. YTHDC2 enhances the translation efficiency of its targets and also decreases their mRNA abundance. Ythdc2 knockout mice are infertile; males have significantly smaller testes and females have significantly smaller ovaries compared to those of littermates. The germ cells of Ythdc2 knockout mice do not develop past the zygotene stage and accordingly, Ythdc2 is upregulated in the testes as meiosis begins. Thus, YTHDC2 is an m6 A-binding protein that plays critical roles during spermatogenesis.
N^6-methyladenosine （m^6A） is the most common internal modification in eukaryotic mRNA. It is dynamically in- stalled and removed, and acts as a new layer of mRNA metabolism, regulating biological processes including stem cell pluripotency, cell differentiation, and energy homeostasis, m^6A is recognized by selective binding proteins; YTHDF1 and YTHDF3 work in concert to affect the translation of m^6A-containing mRNAs, YTHDF2 expedites mRNA decay, and YTHDC1 affects the nuclear processing of its targets. The biological function of YTHDC2, the final member of the YTH protein family, remains unknown. We report that YTHDC2 selectively binds m6A at its consensus motif. YTHDC2 enhances the translation efficiency of its targets and also decreases their mRNA abundance. Ythdc2 knock- out mice are infertile; males have significantly smaller testes and females have significantly smaller ovaries compared to those oflittermates. The germ cells of Ythdc2 knockout mice do not develop past the zygotene stage and according- ly, Ythdc2 is upregulated in the testes as meiosis begins. Thus, YTHDC2 is an m6A-binding protein that plays critical roles during svermatogenesis.
Chimeric antigen receptor （CAR） gene-engineered T cell therapy holds the potential to make a meaningful differ- ence in the lives of patients with terminal cancers. For decades, cancer therapy was based on biophysical parameters, with surgical resection to debulk, followed by radiation and chemotherapy to target the rapidly growing tumor cells, while mostly sparing quiescent normal tissues. One breakthrough occurred with allogeneic bone-marrow transplant for patients with leukemia, which provided a sometimes curative therapy. The field of adoptive cell therapy for sol- id tumors was established with the discovery that tumor-infiltrating lymphocytes could be expanded and used to treat and even cure patients with metastatic melanoma. Tumor-specific T-cell receptors （TCRs） were identified and engineered into patient peripheral blood lymphocytes, which were also found to treat tumors. However, these were limited by patient HLA-restriction. Close behind came generation of CAR, combining the exquisite recognition of an antibody with the effector function of a T cell. The advent of CD19-targeted CARs for treating patients with multiple forms of advanced B-cell malignancies met with great success, with up to 95% response rates. Applying CAR treat- ment to solid tumors, however, has just begun, but already certain factors have been made clear： the tumor target is of utmost importance for clinicians to do no harm; and solid tumors respond differently to CAR therapy compared with hematologic ones. Here we review the state of clinical gene-engineered T cell immunotherapy, its successes, chal- lenges, and future.
5-methylcytosine (m C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m C modiﬁcation is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m C profiles of mammalian transcriptomes and suggests an essential role for m C modification in mRNA export and post-transcriptional regulation.
Immunotherapy using dendritic cell （DC）-based vaccination is an approved approach for harnessing the potential of a patient＇s own immune system to eliminate tumor cells in metastatic hormone-refractory cancer. Overall, al- though many DC vaccines have been tested in the clinic and proven to be immunogenic, and in some cases associated with clinical outcome, there remains no consensus on how to manufacture DC vaccines. In this review we will discuss what has been learned thus far about human DC biology from clinical studies, and how current approaches to apply DC vaccines in the clinic could be improved to enhance anti-tumor immunity.
5-methylcytosine (m(5)C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m(5)C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m(5)C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m(5)C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREF's nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of AL-YREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m(5)C profiles of mammalian transcriptomes and suggests an essential role for m(5)C modification in mRNA export and post-transcriptional regulation.
Glycolysis has long been considered as the major metabolic process for energy production and anabolic growth in cancer cells. Although such a view has been instrumental for the development of powerful imaging tools that are still used in the clinics, it is now clear that mitochondria play a key role in oncogenesis. Besides exerting central bioenergetic functions, mitochondria provide indeed building blocks for tumor anabolism, control redox and calcium homeostasis, participate in transcriptional regulation, and govern cell death. Thus, mitochondria constitute promising targets for the development of novel anticancer agents. However, tumors arise, progress, and respond to therapy in the context of an intimate crosstalk with the host immune system, and many immunological functions rely on intact mitochondrial metabolism. Here, we review the cancer cell-intrinsic and cell-extrinsic mechanisms through which mitochondria influence all steps of oncogenesis, with a focus on the therapeutic potential of targeting mitochondrial metabolism for cancer therapy.
Dear Editor, The clustered regularly interspaced short palindromic repeat （CRISPR）/CRISPR-associated protein （Cas） system has revolutionized biomedical research and facilitated the development of new therapies based on genome editing . A major roadblock to achieve the therapeutic potential of the CRISPR/Cas system is the lack of a safe and effectiving in vivo delivery method.
Certain low pathogenic avian influenza viruses can mutate to highly pathogenic viruses when they circulate in domestic poultry, at which point they can cause devastating poultry diseases and severe economic damage. The H7N9 influenza viruses that emerged in 2013 in China had caused severe human infections and deaths. However, these viruses were nonlethal in poultry. It is unknown whether the H7N9 viruses can acquire additional mutations during their circulation in nature and become lethal to poultry and more dangerous for humans. Here, we evaluated the evolution of H7N9 viruses isolated from avian species between 2013 and 2017 in China and found 23 different genotypes, 7 of which were detected only in ducks and were genetically distinct from the other 16 genotypes that evolved from the 2013 H7N9 viruses. Importantly, some H7N9 viruses obtained an insertion of four amino acids in their hemagglutinin （HA） cleavage site and were lethal in chickens. The index strain was not lethal in mice or ferrets, but readily obtained the 627K or 701N mutation in its PB2 segment upon replication in ferrets, causing it to become highly lethal in mice and ferrets and to be transmitted efficiently in ferrets by respiratory droplet. H7N9 viruses bearing the HA insertion and PB2 627K mutation have been detected in humans in China. Our study indicates that the new H7N9 mutants are lethal to chickens and pose an increased threat to human health, and thus highlights the need to control and eradicate the H7N9 viruses to prevent a possible pandemic.
Ruptured and intact plasma membranes are classically considered as hallmarks of necrotic and apoptotic cell death, respectively. As such, apoptosis is usually considered a non-inflammatory process while necrosis triggers in- flammation. Recent studies on necroptosis and pyroptosis, two types of programmed necrosis, revealed that plasma membrane rupture is mediated by MLKL channels during necroptosis but depends on non-selective gasdermin D （GSDMD） pores during pyroptosis. Importantly, the morphology of dying cells executed by MLKL channels can be distinguished from that executed by GSDMD pores. Interestingly, it was found recently that secondary necrosis of apoptotic cells, a previously believed non-regulated form of cell lysis that occurs after apoptosis, can be programmed and executed by plasma membrane pore formation like that of pyroptosis. In addition, pyroptosis is associated with pyroptotic bodies, which have some similarities to apoptotic bodies. Therefore, different cell death programs induce distinctive reshuffling processes of the plasma membrane. Given the fact that the nature of released intracellular contents plays a crucial role in dying/dead cell-induced immunogenicity, not only membrane rupture or integrity but also the nature of plasma membrane breakdown would determine the fate of a cell as well as its ability to elicit an immune response. In this review, we will discuss recent advances in the field of apoptosis, necroptosis and pyroptosis, with an emphasis on the mechanisms underlying plasma membrane changes observed on dying cells and their implication in cell death-elicited immunogenicity.
A pre-existing T cell-inflamed tumor microenvironment has prognostic utility and also can be predictive for re- sponse to contemporary cancer immunotherapies. The generation of a spontaneous T cell response against tumor-as- sociated antigens depends on innate immune activation, which drives type I interferon （IFN） production. Recent work has revealed a major role for the STING pathway of cytosolic DNA sensing in this process. This cascade of events contributes to the activation of Batf3-1ineage dendritic cells （DCs）, which appear to be central to anti-tumor immunity. Non-T cell-inflamed tumors lack chemokines for Batf3 DC recruitment, have few Batf3 DCs, and lack a type I IFN gene signature, suggesting that failed innate immune activation may be the ultimate cause for lack of spontaneous T cell activation and accumulation. With this information in hand, new strategies for triggering innate immune activation and Batf3 DC recruitment are being developed, including novel STING agonists for de novo im- mune priming. Ultimately, the successful development of effective innate immune activators should expand the frac- tion of patients that can respond to immunotherapies, such as with checkpoint blockade antibodies.
Harnessing the immune system to eradicate malignant cells is becoming a most powerful new approach to cancer therapy. FDA approval of the immunotherapy-based drugs, sipuleucel-T （Provenge）, ipilimumab （Yervoy, anti-CT- LA-4）, and more recently, the programmed cell death （PD）-I antibody （pembroliznmab, Keytruda）, for the treatment of multiple types of cancer has greatly advanced research and clinical studies in the field of cancer immunotherapy. Furthermore, recent clinical trials, using NY-ESO-l-specific T cell receptor （TCR） or CD19-chimeric antigen re- ceptor （CAR）, have shown promising clinical results for patients with metastatic cancer. Current success of cancer immunotherapy is built upon the work of cancer antigens and co-inhibitory signaling molecules identified 20 years ago. Among the large numbers of target antigens, CD19 is the best target for CAR T cell therapy for blood cancer, but CAR-engineered T cell immunotherapy does not yet work in solid cancer. NY-ESO-1 is one of the best targets for TCR-based immunotherapy in solid cancer. Despite the great success of checkpoint blockade therapy, more than 50% of cancer patients fail to respond to blockade therapy. The advent of new technologies such as next-generation sequencing has enhanced our ability to search for new immune targets in onco-immunology and accelerated the de- velopment of immunotherapy with potentially broader coverage of cancer patients. In this review, we will discuss the recent progresses of cancer immunotherapy and novel strategies in the identification of new immune targets and mu- tation-derived antigens （neoantigens） for cancer immunotherapy and immunoprecision medicine.
Targeted integration of transgenes can be achieved by strategies based on homologous recombination （HR）, mi- crohomology-mediated end joining （MMEJ） or non-homologous end joining （NHEJ）. The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining （HMEJ）-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and -800 bp of homology arms, and the targeted genome. We found no significant improve- ment of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblas- toma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock- in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies.
METTL3 catalyzes the formation of N-6-methyl-adenosine (m(6)A) which has important roles in regulating various biological processes. However, the in vivo function of Mettl3 remains largely unknown in mammals. Here we generated germ cell-specific Mettl3 knockout mice and demonstrated that Mettl3 was essential for male fertility and spermatogenesis. The ablation of Mettl3 in germ cells severely inhibited spermatogonial differentiation and blocked the initiation of meiosis. Transcriptome and m(6)A profiling analysis revealed that genes functioning in spermatogenesis had altered profiles of expression and alternative splicing. Our findings provide novel insights into the function and regulatory mechanisms of Mettl3-mediated m(6)A modification in spermatogenesis and reproduction in mammals.
METTL3 catalyzes the formation of N -methyl-adenosine (m A) which has important roles in regulating various biological processes. However, the in vivo function of Mettl3 remains largely unknown in mammals. Here we generated germ cell-specific Mettl3 knockout mice and demonstrated that Mettl3 was essential for male fertility and spermatogenesis. The ablation of Mettl3 in germ cells severely inhibited spermatogonial differentiation and blocked the initiation of meiosis. Transcriptome and m A profiling analysis revealed that genes functioning in spermatogenesis had altered profiles of expression and alternative splicing. Our findings provide novel insights into the function and regulatory mechanisms of Mettl3-mediated m A modification in spermatogenesis and reproduction in mammals.