RESUMEN
The 7-methylguanosine cap added to the 5' end of mRNA is required for efficient gene expression in eukaryotes. In mammals, methylation of the guanosine cap is catalyzed by RNMT (RNA guanine-7 methyltransferase), an enzyme previously thought to function as a monomer. We have identified an obligate component of the mammalian cap methyltransferase, RAM (RNMT-Activating Mini protein)/Fam103a1, a previously uncharacterized protein. RAM consists of an N-terminal RNMT-activating domain and a C-terminal RNA-binding domain. As monomers RNMT and RAM have a relatively weak affinity for RNA; however, together their RNA affinity is significantly increased. RAM is required for efficient cap methylation in vitro and in vivo, and is indirectly required to maintain mRNA expression levels, for mRNA translation and for cell viability. Our findings demonstrate that RAM is an essential component of the core gene expression machinery.
Asunto(s)
Regulación de la Expresión Génica , Metiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , Biosíntesis de Proteínas/genética , Caperuzas de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Western Blotting , Supervivencia Celular , Secuencia Conservada , Guanosina/análogos & derivados , Guanosina/genética , Guanosina/metabolismo , Células HEK293 , Humanos , Inmunoprecipitación , Metilación , Metiltransferasas/genética , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estructura Terciaria de Proteína , Caperuzas de ARN/genética , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , S-Adenosilmetionina/genética , S-Adenosilmetionina/metabolismo , Alineación de SecuenciaRESUMEN
The mRNA cap is a structure added to RNA pol II transcripts in eukaryotes, which recruits factors involved in RNA processing, nuclear export and translation initiation. RNA guanine-7 methyltransferase (RNMT)-RNA-activating miniprotein (RAM), the mRNA cap methyltransferase complex, completes the basic functional mRNA cap structure, cap 0, by methylating the cap guanosine. Here, we report that RNMT-RAM co-ordinates mRNA processing with ribosome production. Suppression of RNMT-RAM reduces synthesis of the 45S ribosomal RNA (rRNA) precursor. RNMT-RAM is required for c-Myc expression, a major regulator of RNA pol I, which synthesises 45S rRNA. Constitutive expression of c-Myc restores rRNA synthesis when RNMT-RAM is suppressed, indicating that RNMT-RAM controls rRNA production predominantly by controlling c-Myc expression. We report that RNMT-RAM is recruited to the ribosomal DNA locus, which may contribute to rRNA synthesis in certain contexts.
Asunto(s)
Metiltransferasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Caperuzas de ARN/química , ARN Mensajero/metabolismo , ARN Ribosómico/metabolismo , Proteínas de Unión al ARN/metabolismo , Núcleo Celular , Cromatina/química , Cromatina/metabolismo , Células HeLa , Humanos , Metilación , Metiltransferasas/genética , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-myc/genética , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Ribosómico/genética , Proteínas de Unión al ARN/genética , Ribosomas/química , Ribosomas/metabolismo , Transcripción GenéticaRESUMEN
c-Myc is upregulated in response to growth factors and transmits the signal to proliferate by altering the gene expression landscape. When genetic alterations result in growth factor-independent c-Myc expression, it can become an oncogene. The majority of human tumour types exhibit a degree of c-Myc deregulation, resulting in unrestrained cell proliferation. c-Myc binds proximal to the promoter region of genes and recruits co-factors including histone acetyltransferases and RNA pol II kinases, which promote transcription. c-Myc also promotes formation of the cap structure at the 5' end of mRNA. The cap is 7-methylguanosine linked to the first transcribed nucleotide of RNA pol II transcripts via a 5' to 5' triphosphate bridge. The cap is added to the first transcribed nucleotide by the capping enzymes, RNGTT and RNMT-RAM. During the early stages of transcription, the capping enzymes are recruited to RNA pol II phosphorylated on Serine-5 of the C-terminal domain. The mRNA cap protects transcripts from degradation during transcription and recruits factors which promote RNA processing including, splicing, export and translation initiation. The proportion of transcripts with a cap structure is increased by elevating c-Myc expression, resulting in increased rates of translation. c-Myc promotes capping by promoting RNA pol II phosphorylation and by upregulating the enzyme SAHH which neutralises the inhibitory bi-product of methylation reactions, SAH. c-Myc-induced capping is required for c-Myc-dependent gene expression and cell proliferation. Targeting capping may represent a new therapeutic opportunity to inhibit c-Myc function in tumours. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology.
Asunto(s)
Neoplasias/genética , Biosíntesis de Proteínas , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Guanosina/análogos & derivados , Guanosina/genética , Humanos , Neoplasias/patología , Proteínas Proto-Oncogénicas c-myc/biosíntesis , ARN Polimerasa II/genéticaRESUMEN
Basic mechanisms in gene expression are currently being investigated as targets in cancer therapeutics. One such fundamental process is the addition of the cap to pre-mRNA, which recruits mediators of mRNA processing and translation initiation. Maturation of the cap involves mRNA cap guanosine N-7 methylation, catalysed by RNMT (RNA guanine-7 methyltransferase). In a panel of breast cancer cell lines, we investigated whether all are equivalently dependent on RNMT for proliferation. When cellular RNMT activity was experimentally reduced by 50%, the proliferation rate of non-transformed mammary epithelial cells was unchanged, whereas a subset of breast cancer cell lines exhibited reduced proliferation and increased apoptosis. Most of the cell lines which exhibited enhanced dependency on RNMT harboured oncogenic mutations in PIK3CA, which encodes the p110α subunit of PI3Kα. Conversely, all cell lines insensitive to RNMT depletion expressed wild-type PIK3CA. Expression of oncogenic PIK3CA mutants, which increase PI3K p110α activity, was sufficient to increase dependency on RNMT. Conversely, inhibition of PI3Kα reversed dependency on RNMT, suggesting that PI3Kα signalling is required. Collectively, these findings provide evidence to support RNMT as a therapeutic target in breast cancer and suggest that therapies targeting RNMT would be most valuable in a PIK3CA mutant background.
Asunto(s)
Neoplasias de la Mama/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Metiltransferasas/genética , Mutación , Caperuzas de ARN , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinogénesis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Humanos , Células MCF-7 , Metiltransferasas/metabolismo , ARN Mensajero/genética , Transducción de Señal/genética , Transcripción GenéticaRESUMEN
mRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription.