RESUMEN
The N6-methyladenosine (m6A) RNA modification is used widely to alter the fate of mRNAs. Here we demonstrate that the C. elegans writer METT-10 (the ortholog of mouse METTL16) deposits an m6A mark on the 3' splice site (AG) of the S-adenosylmethionine (SAM) synthetase pre-mRNA, which inhibits its proper splicing and protein production. The mechanism is triggered by a rich diet and acts as an m6A-mediated switch to stop SAM production and regulate its homeostasis. Although the mammalian SAM synthetase pre-mRNA is not regulated via this mechanism, we show that splicing inhibition by 3' splice site m6A is conserved in mammals. The modification functions by physically preventing the essential splicing factor U2AF35 from recognizing the 3' splice site. We propose that use of splice-site m6A is an ancient mechanism for splicing regulation.
Asunto(s)
Adenosina/análogos & derivados , Sitios de Empalme de ARN/genética , Empalme del ARN/genética , Factor de Empalme U2AF/metabolismo , Adenosina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/genética , Secuencia Conservada/genética , Dieta , Células HeLa , Humanos , Intrones/genética , Metionina Adenosiltransferasa , Metilación , Metiltransferasas/química , Ratones , Mutación/genética , Conformación de Ácido Nucleico , Unión Proteica , Precursores del ARN/química , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Nuclear Pequeño , S-Adenosilmetionina , Transcriptoma/genéticaRESUMEN
The functional consequence of N6-methyladenosine (m6A) RNA modification is mediated by "reader" proteins of the YTH family. YTH domain-containing 2 (YTHDC2) is essential for mammalian fertility, but its molecular function is poorly understood. Here, we identify U-rich motifs as binding sites of YTHDC2 on 3' UTRs of mouse testicular RNA targets. Although its YTH domain is an m6A-binder in vitro, the YTH point mutant mice are fertile. Significantly, the loss of its 3'â5' RNA helicase activity causes mouse infertility, with the catalytic-dead mutation being dominant negative. Biochemical studies reveal that the weak helicase activity of YTHDC2 is enhanced by its interaction with the 5'â3' exoribonuclease XRN1. Single-cell transcriptomics indicate that Ythdc2 mutant mitotic germ cells transition into meiosis but accumulate a transcriptome with mixed mitotic/meiotic identity that fail to progress further into meiosis. Finally, our demonstration that ythdc2 mutant zebrafish are infertile highlights its conserved role in animal germ cell development.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Exorribonucleasas/metabolismo , ARN Helicasas , Pez Cebra , Animales , Fertilidad/genética , Mamíferos/metabolismo , Meiosis , Ratones , ARN/genética , ARN Helicasas/genética , ARN Helicasas/metabolismo , Pez Cebra/genéticaRESUMEN
RNA export is tightly coupled to splicing in metazoans. In the Drosophila germline, precursors for the majority of Piwi-interacting RNAs (piRNAs) are unspliced. In this issue of Genes & Development, Kneuss and colleagues (pp. 1208-1220) identify Nxf3 as a novel germline-specific export adapter for such unspliced transcripts. Their findings reveal the sequence of events leading from its role at the site of transcription to delivery of the cargo to cytoplasmic piRNA biogenesis sites.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Transporte Activo de Núcleo Celular , Animales , Elementos Transponibles de ADN , Drosophila/genética , ARN Interferente PequeñoRESUMEN
Internal modification of RNAs with N6-methyladenosine (m6A) is a highly conserved means of gene expression control. While the METTL3/METTL14 heterodimer adds this mark on thousands of transcripts in a single-stranded context, the substrate requirements and physiological roles of the second m6A writer METTL16 remain unknown. Here we describe the crystal structure of human METTL16 to reveal a methyltransferase domain furnished with an extra N-terminal module, which together form a deep-cut groove that is essential for RNA binding. When presented with a random pool of RNAs, METTL16 selects for methylation-structured RNAs where the critical adenosine is present in a bulge. Mouse 16-cell embryos lacking Mettl16 display reduced mRNA levels of its methylation target, the SAM synthetase Mat2a. The consequence is massive transcriptome dysregulation in â¼64-cell blastocysts that are unfit for further development. This highlights the role of an m6A RNA methyltransferase in facilitating early development via regulation of SAM availability.
Asunto(s)
Adenosina/análogos & derivados , Metiltransferasas/metabolismo , Metiltransferasas/ultraestructura , Adenosina/metabolismo , Animales , Desmetilación , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Expresión Génica/genética , Células HEK293 , Humanos , Metionina Adenosiltransferasa , Metilación , Metiltransferasas/fisiología , Ratones/embriología , Ratones Noqueados , ARN , Procesamiento Postranscripcional del ARN/fisiología , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/metabolismoRESUMEN
N6-methyladenosine (m6A) is an essential internal RNA modification that is critical for gene expression control in most organisms. Proteins with a YTH domain recognize m6A marks and are mediators of molecular functions like RNA splicing, mRNA decay, and translation control. Here we demonstrate that YTH domain-containing 2 (YTHDC2) is an m6A reader that is essential for male and female fertility in mice. High-throughput mapping of the m6A transcriptome and expression analysis in the Yhtdc2 mutant testes reveal an upregulation of m6A-enriched transcripts. Our biochemical studies indicate that YTHDC2 is an RNA-induced ATPase with a 3'â5' RNA helicase activity. Furthermore, YTHDC2 recruits the 5'â3' exoribonuclease XRN1 via Ankyrin repeats that are inserted in between the RecA modules of the RNA helicase domain. Our studies reveal a role for YTHDC2 in modulating the levels of m6A-modified germline transcripts to maintain a gene expression program that is conducive for progression through meiosis.
Asunto(s)
Adenosina/análogos & derivados , Regulación de la Expresión Génica/fisiología , Meiosis/fisiología , ARN Helicasas/metabolismo , ARN Mensajero/metabolismo , Adenosina/genética , Adenosina/metabolismo , Animales , Repetición de Anquirina , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Masculino , Ratones , Ratones Mutantes , Dominios Proteicos , ARN Helicasas/genética , ARN Mensajero/genéticaRESUMEN
Eukaryotic RNA pol II transcripts are capped at the 5' end by the methylated guanosine (m7G) moiety. In higher eukaryotes, CMTR1 and CMTR2 catalyze cap-proximal ribose methylations on the first (cap1) and second (cap2) nucleotides, respectively. These modifications mark RNAs as "self," blocking the activation of the innate immune response pathway. Here, we show that loss of mouse Cmtr1 or Cmtr2 leads to embryonic lethality, with non-overlapping sets of transcripts being misregulated, but without activation of the interferon pathway. In contrast, Cmtr1 mutant adult mouse livers exhibit chronic activation of the interferon pathway, with multiple interferon-stimulated genes being expressed. Conditional deletion of Cmtr1 in the germline leads to infertility, while global translation is unaffected in the Cmtr1 mutant mouse liver and human cells. Thus, mammalian cap1 and cap2 modifications have essential roles in gene regulation beyond their role in helping cellular transcripts to evade the innate immune system.
Asunto(s)
Caperuzas de ARN , Ribosa , Humanos , Animales , Ratones , Metilación , Caperuzas de ARN/metabolismo , Metiltransferasas/metabolismo , Interferones/metabolismo , Fertilidad , Mamíferos/metabolismoRESUMEN
AIMS: MicroRNA-378a, highly expressed in skeletal muscles, was demonstrated to affect myoblasts differentiation and to promote tumour angiogenesis. We hypothesized that miR-378a could play a pro-angiogenic role in skeletal muscle and may be involved in regeneration after ischaemic injury in mice. METHODS AND RESULTS: Silencing of miR-378a in murine C2C12 myoblasts did not affect differentiation but impaired their secretory angiogenic potential towards endothelial cells. miR-378a knockout (miR-378a-/-) in mice resulted in a decreased number of CD31-positive blood vessels and arterioles in gastrocnemius muscle. In addition, diminished endothelial sprouting from miR-378a-/- aortic rings was shown. Interestingly, although fibroblast growth factor 1 (Fgf1) expression was decreased in miR-378a-/- muscles, this growth factor did not mediate the angiogenic effects exerted by miR-378a. In vivo, miR-378a knockout did not affect the revascularization of the ischaemic muscles in both normo- and hyperglycaemic mice subjected to femoral artery ligation (FAL). No difference in regenerating muscle fibres was detected between miR-378a-/- and miR-378+/+ mice. miR-378a expression temporarily declined in ischaemic skeletal muscles of miR-378+/+ mice already on Day 3 after FAL. At the same time, in the plasma, the level of miR-378a-3p was enhanced. Similar elevation of miR-378a-3p was reported in the plasma of patients with intermittent claudication in comparison to healthy donors. Local adeno-associated viral vectors-based miR-378a overexpression was enough to improve the revascularization of the ischaemic limb of wild-type mice on Day 7 after FAL, what was not reported after systemic delivery of vectors. In addition, the number of infiltrating CD45+ cells and macrophages (CD45+ CD11b+ F4/80+ Ly6G-) was higher in the ischaemic muscles of miR-378a-/- mice, suggesting an anti-inflammatory action of miR-378a. CONCLUSIONS: Data indicate miR-378a role in the pro-angiogenic effect of myoblasts and vascularization of skeletal muscle. After the ischaemic insult, the anti-angiogenic effect of miR-378a deficiency might be compensated by enhanced inflammation.
Asunto(s)
Isquemia/metabolismo , MicroARNs/metabolismo , Músculo Esquelético/irrigación sanguínea , Mioblastos Esqueléticos/metabolismo , Neovascularización Fisiológica , Regeneración , Anciano , Animales , Estudios de Casos y Controles , Línea Celular , Modelos Animales de Enfermedad , Femenino , Terapia Genética , Humanos , Claudicación Intermitente/sangre , Claudicación Intermitente/genética , Isquemia/genética , Isquemia/fisiopatología , Isquemia/terapia , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/sangre , MicroARNs/genética , Persona de Mediana EdadRESUMEN
Heme oxygenase-1 (HO-1; encoded by Hmox1), a downstream target of the Nrf2 transcription factor, has been postulated to be a negative regulator of osteoclasts (OCLs) differentiation. Here, we further explored such a hypothesis by examining HO-1 effects in different stages of osteoclastogenesis. We confirmed the inhibition of the expression of OCLs markers by Nrf2. In contrast, both the lack of the active Hmox1 gene or HO-1 silencing in OCLs precursor cells, bone marrow macrophages (BMMs), decreased their differentiation towards OCLs, as indicated by the analysis of OCLs markers such as TRAP. However, no effect of HO-1 deficiency was observed when HO-1 expression was silenced in BMMs or RAW264.7 macrophage cell line pre-stimulated with RANKL (considered as early-stage OCLs). Moreover, cobalt protoporphyrin IX (CoPPIX) or hemin, the known HO-1 inducers, inhibited OCLs markers both in RANKL-stimulated RAW264.7 cells and BMMs. Strikingly, a similar effect occurred in HO-1-/- cells, indicating HO-1-independent activity of CoPPIX and hemin. Interestingly, plasma of HO-1-/- mice contained higher TRAP levels, which suggests an increased number of bone-resorbing OCLs in the absence of HO-1 in vivo. In conclusion, our data indicate that HO-1 is involved in the response of bone marrow macrophages to RANKL and the induction of OCLs markers, but it is dispensable in early-stage OCLs. However, in vivo HO-1 appears to inhibit OCLs formation.
Asunto(s)
Hemo-Oxigenasa 1/fisiología , Osteogénesis , Animales , Células de la Médula Ósea , Diferenciación Celular , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoclastos/citología , Osteoclastos/metabolismo , Ligando RANK , Células RAW 264.7 , Proteínas RecombinantesRESUMEN
AIMS: Nuclear factor E2-related factor 2 (Nrf2), a key cytoprotective transcription factor, regulates also proangiogenic mediators, interleukin-8 and heme oxygenase-1 (HO-1). However, hitherto its role in blood vessel formation was modestly examined. Particularly, although Nrf2 was shown to affect hematopoietic stem cells, it was not tested in bone marrow-derived proangiogenic cells (PACs). Here we investigated angiogenic properties of Nrf2 in PACs, endothelial cells, and inflammation-related revascularization. RESULTS: Treatment of endothelial cells with angiogenic cytokines increased nuclear localization of Nrf2 and induced expression of HO-1. Nrf2 activation stimulated a tube network formation, while its inhibition decreased angiogenic response of human endothelial cells, the latter effect reversed by overexpression of HO-1. Moreover, lack of Nrf2 attenuated survival, proliferation, migration, and angiogenic potential of murine PACs and affected angiogenic transcriptome in vitro. Additionally, angiogenic capacity of PAC Nrf2(-/-) in in vivo Matrigel assay and PAC mobilization in response to hind limb ischemia of Nrf2(-/-) mice were impaired. Despite that, restoration of blood flow in Nrf2-deficient ischemic muscles was better and accompanied by increased oxidative stress and inflammatory response. Accordingly, the anti-inflammatory agent etodolac tended to diminish blood flow in the Nrf2(-/-) mice. INNOVATION: Identification of a novel role of Nrf2 in angiogenic signaling of endothelial cells and PACs. CONCLUSION: Nrf2 contributes to angiogenic potential of both endothelial cells and PACs; however, its deficiency increases muscle blood flow under tissue ischemia. This might suggest a proangiogenic role of inflammation in the absence of Nrf2 in vivo, concomitantly undermining the role of PACs in such conditions.