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      mRNA circularization by METTL3-eIF3h enhances translation and promotes oncogenesis


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          N 6-Methyladenosine (m 6A) modification of messenger RNA (mRNA) is emerging as an important regulator of gene expression that impacts different developmental and biological processes, and altered m 6A homeostasis is linked to cancer 1- 5 . m 6A is catalyzed by METTL3 and enriched in the 3’ untranslated region (3’ UTR) of a large subset of mRNAs at sites close to the stop codon 5 . METTL3 can promote translation but the mechanism and widespread relevance remain unknown 1 . Here we show that METTL3 enhances translation only when tethered to reporter mRNA at sites close to the stop codon supporting a mRNA looping mechanism for ribosome recycling and translational control. Electron microscopy reveals the topology of individual polyribosomes with single METTL3 foci found in close proximity to 5’ cap-binding proteins. We identify a direct physical and functional interaction between METTL3 and the eukaryotic translation initiation factor 3 subunit h (eIF3h). METTL3 promotes translation of a large subset of oncogenic mRNAs, including Bromodomain-containing protein 4 (BRD4) that are also m 6A-modified in human primary lung tumors. The METTL3-eIF3h interaction is required for enhanced translation, formation of densely packed polyribosomes, and oncogenic transformation. METTL3 depletion inhibits tumorigenicity and sensitizes lung cancer cells to BRD4 inhibition. These findings uncover a mRNA looping mechanism of translation control and identify METTL3-eIF3h as a potential cancer therapeutic target.

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          Circularization of mRNA by eukaryotic translation initiation factors.

          Communication between the 5' cap structure and 3' poly(A) tail of eukaryotic mRNA results in the synergistic enhancement of translation. The cap and poly(A) tail binding proteins, eIF4E and Pab1p, mediate this effect in the yeast S. cerevisiae through their interactions with different parts of the translation factor eIF4G. Here, we demonstrate the reconstitution of an eIF4E/eIF4G/Pab1p complex with recombinant proteins, and show by atomic force microscopy that the complex can circularize capped, polyadenylated RNA. Our results suggest that formation of circular mRNA by translation factors could contribute to the control of mRNA expression in the eukaryotic cell.
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            eIF3d is an mRNA cap-binding protein required for specialized translation initiation

            Eukaryotic mRNAs contain a 5' cap structure critical for recruitment of the translation machinery and initiation of protein synthesis. mRNA recognition is thought to require direct interactions between eukaryotic initiation factor 4E (eIF4E) and the mRNA cap. However, translation of numerous capped mRNAs remains robust during cellular stress, early development, and cell cycle progression 1 despite eIF4E inactivation. Here we describe a new cellular cap-dependent pathway of translation initiation that relies on a previously unknown cap-binding activity of eIF3d, a subunit of the 800-kilodalton eukaryotic initiation factor 3 (eIF3) complex. A 1.4 Å crystal structure of the eIF3d cap-binding domain reveals unexpected homology to endonucleases involved in RNA turnover, and allows modeling of cap recognition by eIF3d. eIF3d makes specific contacts to the cap, as exemplified by cap analog competition, and these interactions are essential for assembly of translation initiation complexes on eIF3-specialized mRNAs 2 such as the cell proliferation regulator c-Jun. The c-Jun mRNA further encodes an inhibitory RNA element that blocks eIF4E recruitment, thus enforcing alternative cap recognition by eIF3d. Our results reveal a new mechanism of cap-dependent translation independent of eIF4E, and illustrate how modular RNA elements work in concert to direct specialized forms of translation initiation.
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              Structure of mammalian eIF3 in the context of the 43S preinitiation complex.

              During eukaryotic translation initiation, 43S complexes, comprising a 40S ribosomal subunit, initiator transfer RNA and initiation factors (eIF) 2, 3, 1 and 1A, attach to the 5'-terminal region of messenger RNA and scan along it to the initiation codon. Scanning on structured mRNAs also requires the DExH-box protein DHX29. Mammalian eIF3 contains 13 subunits and participates in nearly all steps of translation initiation. Eight subunits having PCI (proteasome, COP9 signalosome, eIF3) or MPN (Mpr1, Pad1, amino-terminal) domains constitute the structural core of eIF3, to which five peripheral subunits are flexibly linked. Here we present a cryo-electron microscopy structure of eIF3 in the context of the DHX29-bound 43S complex, showing the PCI/MPN core at ∼6 Å resolution. It reveals the organization of the individual subunits and their interactions with components of the 43S complex. We were able to build near-complete polyalanine-level models of the eIF3 PCI/MPN core and of two peripheral subunits. The implications for understanding mRNA ribosomal attachment and scanning are discussed.

                Author and article information

                15 August 2018
                19 September 2018
                September 2018
                19 March 2019
                : 561
                : 7724
                : 556-560
                [1 ]Stem Cell Program, Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA 02115, USA
                [2 ]Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
                [3 ]Center for Translational Medicine, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510080, China
                [4 ]Department of Pathology, Cancer Center, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA
                [5 ]Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain
                [6 ]Harvard School of Dental Medicine, Boston MA 02115, USA
                [7 ]Innovation Center for Biomedical Informatics, Georgetown University Medical Center, Washington, DC20057, USA
                [8 ]Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA
                [9 ]Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
                [10 ]Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
                [11 ]Department of Surgery, Brigham and Women’s Hospital, Boston, MA 02115, USA
                [12 ]Division of Hematology and Medical Oncology, NYU School of Medicine, New York, NY 10016
                [13 ]School of Biosciences and Medicine, University of Surrey, Guildford, United Kingdom
                [14 ]Harvard Initiative for RNA Medicine, Boston MA 02115, USA
                [15 ]Harvard Stem Cell Institute, Cambridge, MA 02138, USA
                Author notes

                Author contributions

                J.C., S.L. and R.I.G. designed the research; J.C. and S.L. contributed equally; J.C., S.L., W.Z., L.W., J.R. and W.K. performed all the experiments; J.C. performed in vitro translation assay, tethering assay, polysome fractionation and RNA-seq, co-IPs, EM, Cap-association assay and in situ PLA; S.L. performed GST Pull-Down Assay, Far-Western blotting, IHC staining, cell invasion assay and m 6A meRIP-Seq; W.Z. performed soft agar colony formation assays and in vivo tumor xenograft; L.W. performed EM; J.R. performed cell proliferation and apoptosis assays; W.K. performed in situ PLA.S.L., Q.L., P.D. and S.T. performed all bioinformatics analysis; W.R. and K.W. provided human lung cancer patient samples. N.L. provided eIF3 complex. J.C., S.L., and R.I.G. analyzed data and wrote the paper with input from other authors.

                Author information

                Reprints and permissions information is available at www.nature.com/reprints. Authors declare no competing financial interests. Correspondence and requests for materials should be addressed to rgregory@ 123456enders.tch.harvard.edu


                Present address: Biomedical Translational Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.

                [17 ]Corresponding author: Richard I. Gregory, Phone: (617)919-2273, rgregory@ 123456enders.tch.harvard.edu

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                n6-methyladenosine,m6a,mettl3,polysome,translation,closed loop,eif3h,lung adenocarcinoma,oncogene,brd4,jq1
                n6-methyladenosine, m6a, mettl3, polysome, translation, closed loop, eif3h, lung adenocarcinoma, oncogene, brd4, jq1


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