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      Nuclear m 6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development

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          Abstract

          The N 6-methyladenosine (m 6A) modification is the most prevalent internal RNA modification in eukaryotes. The majority of m 6A sites are found in the last exon and 3’ UTRs. Here we show that the nuclear m 6A reader YTHDC1 is essential for embryo viability and germline development in mouse. Specifically, YTHDC1 is required for spermatogonial development in males and for oocyte growth and maturation in females; Ythdc1-deficient oocytes are blocked at the primary follicle stage. Strikingly, loss of YTHDC1 leads to extensive alternative polyadenylation in oocytes, altering 3’ UTR length. Furthermore, YTHDC1 deficiency causes massive alternative splicing defects in oocytes. The majority of splicing defects in mutant oocytes are rescued by introducing wild-type, but not m 6A-binding-deficient, YTHDC1. YTHDC1 is associated with the pre-mRNA 3’ end processing factors CPSF6, SRSF3, and SRSF7. Thus, YTHDC1 plays a critical role in processing of pre-mRNA transcripts in the oocyte nucleus and may have similar non-redundant roles throughout fetal development.

          Author summary

          The N 6-methyladenosine (m 6A) modification, one type of RNA methylation, is the most abundant internal RNA modification in eukaryote messenger RNAs. m 6A is specifically recognized by RNA-binding reader proteins. Here we report an essential role of the nuclear m 6A reader, YTHDC1, in embryo development and fertility. In particular, YTHDC1 is required for oocyte growth and maturation. YTHDC1-deficient oocytes exhibit massive defects in alternative splicing, which can be rescued by introducing into mutant oocytes wild-type, but not m 6A-binding-deficient, YTHDC1. Strikingly, loss of YTHDC1 causes extensive alternative polyadenylation in oocytes, resulting in altered 3’ UTR length. YTHDC1 interacts with the pre-mRNA 3’end processing factors CPSF6, SRSF3, and SRSF7. Thus, YTHDC1 is a key nuclear factor in the processing of pre-mRNA transcripts.

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          Ythdc2 is an N6-methyladenosine binding protein that regulates mammalian spermatogenesis

          N 6 -methyladenosine (m 6 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. m 6 A is recognized by selective binding proteins; YTHDF1 and YTHDF3 work in concert to affect the translation of m 6 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 m 6 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 m 6 A-binding protein that plays critical roles during spermatogenesis.
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            Progressive lengthening of 3' untranslated regions of mRNAs by alternative polyadenylation during mouse embryonic development.

            The 3' untranslated regions (3' UTRs) of mRNAs contain cis-acting elements for posttranscriptional regulation of gene expression. Here, we report that mouse genes tend to express mRNAs with longer 3' UTRs as embryonic development progresses. This global regulation is controlled by alternative polyadenylation and coordinates with initiation of organogenesis and aspects of embryonic development, including morphogenesis, differentiation, and proliferation. Using myogenesis of C2C12 myoblast cells as a model, we recapitulated this process in vitro and found that 3' UTR lengthening is likely caused by weakening of mRNA polyadenylation activity. Because alternative 3' UTR sequences are typically longer and have higher AU content than constitutive ones, our results suggest that lengthening of 3' UTR can significantly augment posttranscriptional control of gene expression during embryonic development, such as microRNA-mediated regulation.
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              m(6)A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency.

              N(6)-methyladenosine (m(6)A) has been recently identified as a conserved epitranscriptomic modification of eukaryotic mRNAs, but its features, regulatory mechanisms, and functions in cell reprogramming are largely unknown. Here, we report m(6)A modification profiles in the mRNA transcriptomes of four cell types with different degrees of pluripotency. Comparative analysis reveals several features of m(6)A, especially gene- and cell-type-specific m(6)A mRNA modifications. We also show that microRNAs (miRNAs) regulate m(6)A modification via a sequence pairing mechanism. Manipulation of miRNA expression or sequences alters m(6)A modification levels through modulating the binding of METTL3 methyltransferase to mRNAs containing miRNA targeting sites. Increased m(6)A abundance promotes the reprogramming of mouse embryonic fibroblasts (MEFs) to pluripotent stem cells; conversely, reduced m(6)A levels impede reprogramming. Our results therefore uncover a role for miRNAs in regulating m(6)A formation of mRNAs and provide a foundation for future functional studies of m(6)A modification in cell reprogramming.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole: Writing – original draftRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: ResourcesRole: Writing – review & editing
                Role: InvestigationRole: MethodologyRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: ResourcesRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: SupervisionRole: Writing – review & editing
                Role: ConceptualizationRole: Formal analysisRole: Funding acquisitionRole: InvestigationRole: MethodologyRole: ResourcesRole: SupervisionRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS Genet
                PLoS Genet
                plos
                plosgen
                PLoS Genetics
                Public Library of Science (San Francisco, CA USA )
                1553-7390
                1553-7404
                25 May 2018
                May 2018
                : 14
                : 5
                : e1007412
                Affiliations
                [1 ] Department of Biomedical Sciences, University of Pennsylvania, Philadelphia, United States of America
                [2 ] Department of Biology, University of Pennsylvania, Philadelphia, United States of America
                [3 ] Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, Davis, United States of America
                University of Nevada School of Medicine, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist.

                Author information
                http://orcid.org/0000-0002-5782-7303
                http://orcid.org/0000-0001-7532-0138
                http://orcid.org/0000-0003-2311-4089
                Article
                PGENETICS-D-18-00373
                10.1371/journal.pgen.1007412
                5991768
                29799838
                6ec0b5b4-8b95-44df-9836-9a637e72cbe0
                © 2018 Kasowitz et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 22 February 2018
                : 14 May 2018
                Page count
                Figures: 9, Tables: 0, Pages: 28
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/100000057, National Institute of General Medical Sciences;
                Award ID: R35GM118052
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/100009633, Eunice Kennedy Shriver National Institute of Child Health and Human Development;
                Award ID: HD022681
                Award Recipient :
                Funded by: Lalor foundation
                Award ID: Postdoctoral fellowship
                Award Recipient :
                This work was supported by National Institutes of Health/National Institute of General Medical Sciences grant R35GM118052 (to PJW), NIH/National Institute of Child Health and Human Development grant R01HD022681 (to RMS), and National Science Foundation (NSF) grant MCB-1623887 (to BDG). YX is supported by the Lalor foundation postdoctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Cell Biology
                Cellular Types
                Animal Cells
                Germ Cells
                OVA
                Oocytes
                Biology and Life Sciences
                Genetics
                Gene Expression
                Polyadenylation
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Messenger RNA
                Biology and Life Sciences
                Molecular Biology
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Research and Analysis Methods
                Molecular Biology Techniques
                Artificial Gene Amplification and Extension
                Polymerase Chain Reaction
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                Messenger RNA
                Untranslated Regions
                3' Utr
                Biology and life sciences
                Genetics
                Gene expression
                RNA processing
                Alternative Splicing
                Biology and life sciences
                Biochemistry
                Nucleic acids
                RNA
                RNA processing
                Alternative Splicing
                Biology and Life Sciences
                Developmental Biology
                Embryology
                Embryos
                Biology and Life Sciences
                Anatomy
                Reproductive System
                Ovaries
                Medicine and Health Sciences
                Anatomy
                Reproductive System
                Ovaries
                Custom metadata
                vor-update-to-uncorrected-proof
                2018-06-07
                Oocyte RNA-seq data are available under the NCBI/SRA number: SRP116737.

                Genetics
                Genetics

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