RESUMO
Chemical modification of transcripts with 5' caps occurs in all organisms. Here, we report a systems-level mass spectrometry-based technique, CapQuant, for quantitative analysis of an organism's cap epitranscriptome. The method was piloted with 21 canonical caps-m7GpppN, m7GpppNm, GpppN, GpppNm, and m2,2,7GpppG-and 5 'metabolite' caps-NAD, FAD, UDP-Glc, UDP-GlcNAc, and dpCoA. Applying CapQuant to RNA from purified dengue virus, Escherichia coli, yeast, mouse tissues, and human cells, we discovered new cap structures in humans and mice (FAD, UDP-Glc, UDP-GlcNAc, and m7Gpppm6A), cell- and tissue-specific variations in cap methylation, and high proportions of caps lacking 2'-O-methylation (m7Gpppm6A in mammals, m7GpppA in dengue virus). While substantial Dimroth-induced loss of m1A and m1Am arose with specific RNA processing conditions, human lymphoblast cells showed no detectable m1A or m1Am in caps. CapQuant accurately captured the preference for purine nucleotides at eukaryotic transcription start sites and the correlation between metabolite levels and metabolite caps.
Assuntos
Epigênese Genética , Capuzes de RNA/química , Processamento Pós-Transcricional do RNA , Análise de Sequência de RNA/métodos , Transcriptoma , Animais , Células Cultivadas , Vírus da Dengue , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Capuzes de RNA/genética , RNA Viral/química , RNA Viral/genética , Saccharomyces cerevisiaeRESUMO
Regulation of cellular iron homeostasis is crucial as both iron excess and deficiency cause hematological and neurodegenerative diseases. Here we show that mice lacking iron-regulatory protein 2 (Irp2), a regulator of cellular iron homeostasis, develop diabetes. Irp2 post-transcriptionally regulates the iron-uptake protein transferrin receptor 1 (TfR1) and the iron-storage protein ferritin, and dysregulation of these proteins due to Irp2 loss causes functional iron deficiency in ß cells. This impairs Fe-S cluster biosynthesis, reducing the function of Cdkal1, an Fe-S cluster enzyme that catalyzes methylthiolation of t6A37 in tRNALysUUU to ms2t6A37. As a consequence, lysine codons in proinsulin are misread and proinsulin processing is impaired, reducing insulin content and secretion. Iron normalizes ms2t6A37 and proinsulin lysine incorporation, restoring insulin content and secretion in Irp2-/- ß cells. These studies reveal a previously unidentified link between insulin processing and cellular iron deficiency that may have relevance to type 2 diabetes in humans.
Assuntos
Insulina/metabolismo , Proteína 2 Reguladora do Ferro/metabolismo , Ferro/metabolismo , RNA de Transferência de Lisina/metabolismo , tRNA Metiltransferases/metabolismo , Animais , Linhagem Celular Tumoral , Intolerância à Glucose/genética , Homeostase , Células Secretoras de Insulina/metabolismo , Insulinoma/genética , Insulinoma/metabolismo , Proteína 2 Reguladora do Ferro/genética , Proteínas Ferro-Enxofre/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proinsulina/genética , Proinsulina/metabolismo , RNA de Transferência de Lisina/genética , Ratos , Resposta a Proteínas não Dobradas/genética , tRNA Metiltransferases/genéticaRESUMO
N(6)-Methyladenosine (m(6)A) represents the most prevalent internal modification on mRNA and requires a multicomponent m(6)A methyltransferase complex in mammals. How their plant counterparts determine the global m(6)A modification landscape and its molecular link to plant development remain unknown. Here we show that FKBP12 INTERACTING PROTEIN 37 KD (FIP37) is a core component of the m(6)A methyltransferase complex, which underlies control of shoot stem cell fate in Arabidopsis. The mutants lacking FIP37 exhibit massive overproliferation of shoot meristems and a transcriptome-wide loss of m(6)A RNA modifications. We further demonstrate that FIP37 mediates m(6)A RNA modification on key shoot meristem genes inversely correlated with their mRNA stability, thus confining their transcript levels to prevent shoot meristem overproliferation. Our results suggest an indispensable role of FIP37 in mediating m(6)A mRNA modification, which is required for maintaining the shoot meristem as a renewable source for continuously producing all aerial organs in plants.
Assuntos
Adenosina/análogos & derivados , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Transporte/metabolismo , Meristema/citologia , Brotos de Planta/citologia , RNA de Plantas/química , Células-Tronco/citologia , Adenosina/química , Adenosina/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Meristema/metabolismo , Mutação/genética , Fenótipo , Brotos de Planta/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Plantas/genética , Proteínas de Ligação a RNA , Células-Tronco/metabolismoRESUMO
Here we describe an analytical platform for systems-level quantitative analysis of modified ribonucleosides in any RNA species, with a focus on stress-induced reprogramming of tRNA as part of a system of translational control of cell stress response. This chapter emphasizes strategies and caveats for each of the seven steps of the platform workflow: (1) RNA isolation, (2) RNA purification, (3) RNA hydrolysis to individual ribonucleosides, (4) chromatographic resolution of ribonucleosides, (5) identification of the full set of modified ribonucleosides, (6) mass spectrometric quantification of ribonucleosides, (6) interrogation of ribonucleoside datasets, and (7) mapping the location of stress-sensitive modifications in individual tRNA molecules. We have focused on the critical determinants of analytical sensitivity, specificity, precision, and accuracy in an effort to ensure the most biologically meaningful data on mechanisms of translational control of cell stress response. The methods described here should find wide use in virtually any analysis involving RNA modifications.