RESUMEN
Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.
Asunto(s)
Microscopía por Crioelectrón , Hidroliasas , Transferasas Intramoleculares , Seudouridina , ARN de Transferencia , Humanos , Dominio Catalítico , Células HEK293 , Hidroliasas/metabolismo , Hidroliasas/genética , Hidroliasas/química , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/enzimología , Modelos Moleculares , Mutación , Unión Proteica , Seudouridina/metabolismo , Seudouridina/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Especificidad por SustratoRESUMEN
Proteins begin to fold as they emerge from translating ribosomes. The kinetics of ribosome transit along a given mRNA can influence nascent chain folding, but the extent to which individual codon translation rates impact proteome integrity remains unknown. Here, we show that slower decoding of discrete codons elicits widespread protein aggregation in vivo. Using ribosome profiling, we find that loss of anticodon wobble uridine (U34) modifications in a subset of tRNAs leads to ribosome pausing at their cognate codons in S. cerevisiae and C. elegans. Cells lacking U34 modifications exhibit gene expression hallmarks of proteotoxic stress, accumulate aggregates of endogenous proteins, and are severely compromised in clearing stress-induced protein aggregates. Overexpression of hypomodified tRNAs alleviates ribosome pausing, concomitantly restoring protein homeostasis. Our findings demonstrate that modified U34 is an evolutionarily conserved accelerator of decoding and reveal an unanticipated role for tRNA modifications in maintaining proteome integrity.
Asunto(s)
Caenorhabditis elegans/metabolismo , Biosíntesis de Proteínas , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Codón , Agregado de Proteínas , Ribosomas/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Estrés Fisiológico , Uridina/genéticaRESUMEN
Post-translational modifications by ubiquitin-like proteins (UBLs) are essential for nearly all cellular processes. Ubiquitin-related modifier 1 (Urm1) is a unique UBL, which plays a key role in tRNA anticodon thiolation as a sulfur carrier protein (SCP) and is linked to the noncanonical E1 enzyme Uba4 (ubiquitin-like protein activator 4). While Urm1 has also been observed to conjugate to target proteins like other UBLs, the molecular mechanism of its attachment remains unknown. Here, we reconstitute the covalent attachment of thiocarboxylated Urm1 to various cellular target proteins in vitro, revealing that, unlike other known UBLs, this process is E2/E3-independent and requires oxidative stress. Furthermore, we present the crystal structures of the peroxiredoxin Ahp1 before and after the covalent attachment of Urm1. Surprisingly, we show that urmylation is accompanied by the transfer of sulfur to cysteine residues in the target proteins, also known as cysteine persulfidation. Our results illustrate the role of the Uba4-Urm1 system as a key evolutionary link between prokaryotic SCPs and the UBL modifications observed in modern eukaryotes.
Asunto(s)
Ubiquitina , Ubiquitinas , Anticodón , Proteínas Portadoras/metabolismo , Cisteína , Peroxirredoxinas , Azufre/metabolismo , Ubiquitina/metabolismo , Ubiquitinas/metabolismoRESUMEN
Ribosome-enhanced translational miscoding of the genetic code causes protein dysfunction and loss of cellular fitness. During evolution, open reading frame length increased, necessitating mechanisms for enhanced translation fidelity. Indeed, eukaryal ribosomes are more accurate than bacterial counterparts, despite their virtually identical, conserved active centers. During the evolution of eukaryotic organisms ribosome expansions at the rRNA and protein level occurred, which potentially increases the options for translation regulation and cotranslational events. Here we tested the hypothesis that ribosomal RNA expansions can modulate the core function of the ribosome, faithful protein synthesis. We demonstrate that a short expansion segment present in all eukaryotes' small subunit, ES7S, is crucial for accurate protein synthesis as its presence adjusts codon-specific velocities and guarantees high levels of cognate tRNA selection. Deletion of ES7S in yeast enhances mistranslation and causes protein destabilization and aggregation, dramatically reducing cellular fitness. Removal of ES7S did not alter ribosome architecture but altered the structural dynamics of inter-subunit bridges thus affecting A-tRNA selection. Exchanging the yeast ES7S sequence with the human ES7S increases accuracy whereas shortening causes the opposite effect. Our study demonstrates that ES7S provided eukaryal ribosomes with higher accuracy without perturbing the structurally conserved decoding center.
Asunto(s)
Biosíntesis de Proteínas , ARN Ribosómico , Ribosomas , Saccharomyces cerevisiae , Biosíntesis de Proteínas/genética , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ribosomas/metabolismo , Ribosomas/genética , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Codón/genéticaRESUMEN
Fungal pathogens threaten ecosystems and human health. Understanding the molecular basis of their virulence is key to develop new treatment strategies. Here, we characterize NCS2*, a point mutation identified in a clinical baker's yeast isolate. Ncs2 is essential for 2-thiolation of tRNA and the NCS2* mutation leads to increased thiolation at body temperature. NCS2* yeast exhibits enhanced fitness when grown at elevated temperatures or when exposed to oxidative stress, inhibition of nutrient signalling, and cell-wall stress. Importantly, Ncs2* alters the interaction and stability of the thiolase complex likely mediated by nucleotide binding. The absence of 2-thiolation abrogates the in vivo virulence of pathogenic baker's yeast in infected mice. Finally, hypomodification triggers changes in colony morphology and hyphae formation in the common commensal pathogen Candida albicans resulting in decreased virulence in a human cell culture model. These findings demonstrate that 2-thiolation of tRNA acts as a key mediator of fungal virulence and reveal new mechanistic insights into the function of the highly conserved tRNA-thiolase complex.
Asunto(s)
ARN de Transferencia , Saccharomyces cerevisiae , Animales , Humanos , Ratones , Candida albicans/metabolismo , Ecosistema , Proteínas Fúngicas/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/patogenicidad , Azufre/metabolismo , Virulencia/genéticaRESUMEN
Ribosome profiling provides quantitative, comprehensive, and high-resolution snapshots of cellular translation by the high-throughput sequencing of short mRNA fragments that are protected by ribosomes from nucleolytic digestion. While the overall principle is simple, the workflow of ribosome profiling experiments is complex and challenging, and typically requires large amounts of sample, limiting its broad applicability. Here, we present a new protocol for ultra-rapid ribosome profiling from low-input samples. It features a robust strategy for sequencing library preparation within one day that employs solid phase purification of reaction intermediates, allowing to reduce the input to as little as 0.1 pmol of â¼30 nt RNA fragments. Hence, it is particularly suited for the analyses of small samples or targeted ribosome profiling. Its high sensitivity and its ease of implementation will foster the generation of higher quality data from small samples, which opens new opportunities in applying ribosome profiling.
Asunto(s)
Perfilado de Ribosomas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Biosíntesis de Proteínas , Perfilado de Ribosomas/métodos , Ribosomas/genética , Ribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin-related modifier 1 (Urm1) pathway is responsible for the synthesis of 2-thiolated wobble uridine (U34 ). During the key step of the modification cascade, the E1-like activating enzyme ubiquitin-like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C-terminus of its substrate Urm1. Subsequently, activated thiocarboxylated Urm1 (Urm1-COSH) can serve as a sulfur donor for specific tRNA thiolases or participate in ubiquitin-like conjugation reactions. Structural and mechanistic details of Uba4 and Urm1 have remained elusive but are key to understand the evolutionary branch point between ubiquitin-like proteins (UBL) and sulfur-relay systems. Here, we report the crystal structures of full-length Uba4 and its heterodimeric complex with its substrate Urm1. We show how the two domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the C-terminus of Urm1. Finally, we uncover how the catalytic domains of Uba4 communicate efficiently during the reaction cycle and identify a mechanism that enables Uba4 to protect itself against self-conjugation with its own product, namely activated Urm1-COSH.
Asunto(s)
Nucleotidiltransferasas/química , ARN de Transferencia/química , Azufre/química , Sulfurtransferasas/química , Ubiquitinas/química , Humanos , Nucleotidiltransferasas/metabolismo , ARN de Transferencia/metabolismo , Azufre/metabolismo , Sulfurtransferasas/metabolismo , Ubiquitinas/metabolismoRESUMEN
Reprogramming of mRNA translation has a key role in cancer development and drug resistance 1 . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation2,3. Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U34) tRNA (U34 enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF V600E oncogene and by resistance to targeted therapy in melanoma. We show that BRAF V600E -expressing melanoma cells are dependent on U34 enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U34 enzymes. Mechanistically, U34 enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U34 enzymes and HIF1α. Together, these results demonstrate that U34 enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.
Asunto(s)
Codón/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Biosíntesis de Proteínas , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Codón/efectos de los fármacos , Femenino , Humanos , Masculino , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Melanoma/patología , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fosforilación , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN de Transferencia/química , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Transducción de Señal , Factores de Elongación Transcripcional , Uridina/química , Uridina/genética , Uridina/metabolismo , Vemurafenib/farmacología , Vemurafenib/uso terapéutico , Pez Cebra/genéticaRESUMEN
Estrogen receptor alpha (ERα) activity is associated with increased cancer cell proliferation. Studies aiming to understand the impact of ERα on cancer-associated phenotypes have largely been limited to its transcriptional activity. Herein, we demonstrate that ERα coordinates its transcriptional output with selective modulation of mRNA translation. Importantly, translational perturbations caused by depletion of ERα largely manifest as "translational offsetting" of the transcriptome, whereby amounts of translated mRNAs and corresponding protein levels are maintained constant despite changes in mRNA abundance. Transcripts whose levels, but not polysome association, are reduced following ERα depletion lack features which limit translation efficiency including structured 5'UTRs and miRNA target sites. In contrast, mRNAs induced upon ERα depletion whose polysome association remains unaltered are enriched in codons requiring U34-modified tRNAs for efficient decoding. Consistently, ERα regulates levels of U34-modifying enzymes and thereby controls levels of U34-modified tRNAs. These findings unravel a hitherto unprecedented mechanism of ERα-dependent orchestration of transcriptional and translational programs that may be a pervasive mechanism of proteome maintenance in hormone-dependent cancers.
Asunto(s)
Neoplasias de la Mama/genética , Receptor alfa de Estrógeno/genética , Regulación Neoplásica de la Expresión Génica , Polirribosomas/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proliferación Celular , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Células MCF-7 , Polirribosomas/metabolismo , ARN Mensajero/metabolismo , Transducción de Señal , Activación TranscripcionalRESUMEN
Protein methylation occurs primarily on lysine and arginine, but also on some other residues, such as histidine. METTL18 is the last uncharacterized member of a group of human methyltransferases (MTases) that mainly exert lysine methylation, and here we set out to elucidate its function. We found METTL18 to be a nuclear protein that contains a functional nuclear localization signal and accumulates in nucleoli. Recombinant METTL18 methylated a single protein in nuclear extracts and in isolated ribosomes from METTL18 knockout (KO) cells, identified as 60S ribosomal protein L3 (RPL3). We also performed an RPL3 interactomics screen and identified METTL18 as the most significantly enriched MTase. We found that His-245 in RPL3 carries a 3-methylhistidine (3MH; τ-methylhistidine) modification, which was absent in METTL18 KO cells. In addition, both recombinant and endogenous METTL18 were found to be automethylated at His-154, thus further corroborating METTL18 as a histidine-specific MTase. Finally, METTL18 KO cells displayed altered pre-rRNA processing, decreased polysome formation and codon-specific changes in mRNA translation, indicating that METTL18-mediated methylation of RPL3 is important for optimal ribosome biogenesis and function. In conclusion, we have here established METTL18 as the second human histidine-specific protein MTase, and demonstrated its functional relevance.
Asunto(s)
Biosíntesis de Proteínas , Proteína Metiltransferasas/metabolismo , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Secuencias de Aminoácidos , Nucléolo Celular/enzimología , Células HEK293 , Células HeLa , Histidina/metabolismo , Humanos , Señales de Localización Nuclear , Proteína Metiltransferasas/química , Procesamiento Postranscripcional del ARN , Proteína Ribosomal L3 , Ribosomas/metabolismoRESUMEN
RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem-loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.
Asunto(s)
Adenosina/análogos & derivados , Metiltransferasas/genética , ARN Mensajero/genética , ARN Ribosómico 28S/genética , Adenosina/química , Adenosina/genética , Catálisis , Humanos , Metilación , Metiltransferasas/química , Unión Proteica/genética , ARN Ribosómico 28S/químicaRESUMEN
The identification of peptide sequences and their post-translational modifications (PTMs) is a crucial step in the analysis of bottom-up proteomics data. The recent development of open modification search (OMS) engines allows virtually all PTMs to be searched for. This not only increases the number of spectra that can be matched to peptides but also greatly advances the understanding of the biological roles of PTMs through the identification, and the thereby facilitated quantification, of peptidoforms (peptide sequences and their potential PTMs). Whereas the benefits of combining results from multiple protein database search engines have been previously established, similar approaches for OMS results have been missing so far. Here we compare and combine results from three different OMS engines, demonstrating an increase in peptide spectrum matches of 8-18%. The unification of search results furthermore allows for the combined downstream processing of search results, including the mapping to potential PTMs. Finally, we test for the ability of OMS engines to identify glycosylated peptides. The implementation of these engines in the Python framework Ursgal facilitates the straightforward application of the OMS with unified parameters and results files, thereby enabling yet unmatched high-throughput, large-scale data analysis.
Asunto(s)
Algoritmos , Programas Informáticos , Bases de Datos de Proteínas , Procesamiento Proteico-Postraduccional , Proteómica , Motor de BúsquedaRESUMEN
tRNA are post-transcriptionally modified by chemical modifications that affect all aspects of tRNA biology. An increasing number of mutations underlying human genetic diseases map to genes encoding for tRNA modification enzymes. However, our knowledge on human tRNA-modification genes remains fragmentary and the most comprehensive RNA modification database currently contains information on approximately 20% of human cytosolic tRNAs, primarily based on biochemical studies. Recent high-throughput methods such as DM-tRNA-seq now allow annotation of a majority of tRNAs for six specific base modifications. Furthermore, we identified large gaps in knowledge when we predicted all cytosolic and mitochondrial human tRNA modification genes. Only 48% of the candidate cytosolic tRNA modification enzymes have been experimentally validated in mammals (either directly or in a heterologous system). Approximately 23% of the modification genes (cytosolic and mitochondrial combined) remain unknown. We discuss these 'unidentified enzymes' cases in detail and propose candidates whenever possible. Finally, tissue-specific expression analysis shows that modification genes are highly expressed in proliferative tissues like testis and transformed cells, but scarcely in differentiated tissues, with the exception of the cerebellum. Our work provides a comprehensive up to date compilation of human tRNA modifications and their enzymes that can be used as a resource for further studies.
Asunto(s)
Enzimas/análisis , Enzimas/genética , ARN de Transferencia/metabolismo , Citosol/metabolismo , Humanos , Especificidad de Órganos/genética , Proteómica , ARN de Transferencia/química , ARN de Transferencia/genéticaRESUMEN
The impact of RNA structures in coding sequences (CDS) within mRNAs is poorly understood. Here, we identify a novel and highly conserved mechanism of translational control involving RNA structures within coding sequences and the DEAD-box helicase Dhh1. Using yeast genetics and genome-wide ribosome profiling analyses, we show that this mechanism, initially derived from studies of the Brome Mosaic virus RNA genome, extends to yeast and human mRNAs highly enriched in membrane and secreted proteins. All Dhh1-dependent mRNAs, viral and cellular, share key common features. First, they contain long and highly structured CDSs, including a region located around nucleotide 70 after the translation initiation site; second, they are directly bound by Dhh1 with a specific binding distribution; and third, complementary experimental approaches suggest that they are activated by Dhh1 at the translation initiation step. Our results show that ribosome translocation is not the only unwinding force of CDS and uncover a novel layer of translational control that involves RNA helicases and RNA folding within CDS providing novel opportunities for regulation of membrane and secretome proteins.
Asunto(s)
ARN Helicasas DEAD-box/genética , Iniciación de la Cadena Peptídica Traduccional , Biosíntesis de Proteínas , ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Bromovirus/genética , Exones/genética , Regulación de la Expresión Génica/genética , Humanos , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta/genética , ARN Mensajero/genética , Ribosomas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Post-transcriptional chemical modifications of (t)RNA molecules are crucial in fundamental biological processes, such as translation. Despite their biological importance and accumulating evidence linking them to various human diseases, technical challenges have limited their detection and accurate quantification. Here, we present a sensitive capillary nanoflow liquid chromatography mass spectrometry (nLC-MS) pipeline for quantitative high-resolution analysis of ribonucleoside modifications from complex biological samples. We evaluated two porous graphitic carbon (PGC) materials and one end-capped C18 reference material as stationary phases for reversed-phase separation. We found that these matrices have complementing retention and separation characteristics, including the capability to separate structural isomers. PGC and C18 matrices yielded excellent signal-to-noise ratios in nLC-MS while differing in the separation capability and sensitivity for various nucleosides. This emphasizes the need for tailored LC-MS setups for optimally detecting as many nucleoside modifications as possible. Detection ranges spanning up to six orders of magnitude enable the analysis of individual ribonucleosides down to femtomol concentrations. Furthermore, normalizing the obtained signal intensities to a stable isotope labeled spike-in enabled direct comparison of ribonucleoside levels between different samples. In conclusion, capillary columns coupled to nLC-MS constitute a powerful and sensitive tool for quantitative analysis of modified ribonucleosides in complex biological samples. This setup will be invaluable for further unraveling the intriguing and multifaceted biological roles of RNA modifications.
Asunto(s)
Cromatografía Liquida , Espectrometría de Masas , Ribonucleósidos/análisis , Ribonucleósidos/química , Cromatografía Liquida/métodos , Grafito/química , Humanos , Espectrometría de Masas/métodos , ARN Bacteriano , ARN de Hongos , ARN de Transferencia/química , Ribonucleósidos/aislamiento & purificación , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Eukaryotic ubiquitin-like proteins (UBLs) have evolved from prokaryotic sulfur-carrier proteins (SCPs). Ubiquitin related modifier 1 (Urm1) shares biochemical and structural features of UBLs and SCPs and is essential for 2-thiolation of cytoplasmic tRNA. This chemical modification of wobble uridine is highly conserved amongst species and is achieved via Urm1 thiocarboxylation by the non-canonical ubiquitin activating 4 enzyme (Uba4), which contains an E1- and a Rhodanese (RHD) domain. While the RHD catalyzes the last step in Urm1-thiocarboxylate formation, the previous steps in Urm1 activation and the interplay between the two domains have remained elusive. To define the underlying mechanism, we established an Urm1 in vitro thiocarboxylation assay, which combined with structure-function and chemical profiling analyses revealed a critical thioester linkage between Urm1 and Uba4 residue Cys225. This linkage is indispensable for the Urm1 intramolecular transfer between the two domains of Uba4 and it is thus, essential for tRNA thiolation in vivo. These findings contribute to a deeper understanding of UBL evolution.
Asunto(s)
ARN de Transferencia/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Cisteína/química , Cisteína/metabolismo , Mutación , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Quantitative mass spectrometry (MS) is a key technique in many research areas (1), including proteomics, metabolomics, glycomics, and lipidomics. Because all of the corresponding molecules can be described by chemical formulas, universal quantification tools are highly desirable. Here, we present pyQms, an open-source software for accurate quantification of all types of molecules measurable by MS. pyQms uses isotope pattern matching that offers an accurate quality assessment of all quantifications and the ability to directly incorporate mass spectrometer accuracy. pyQms is, due to its universal design, applicable to every research field, labeling strategy, and acquisition technique. This opens ultimate flexibility for researchers to design experiments employing innovative and hitherto unexplored labeling strategies. Importantly, pyQms performs very well to accurately quantify partially labeled proteomes in large scale and high throughput, the most challenging task for a quantification algorithm.
Asunto(s)
Marcaje Isotópico/métodos , Proteoma/análisis , Proteómica/métodos , Programas Informáticos , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Algoritmos , Cromatografía Liquida , Glicómica , MetabolómicaRESUMEN
Many cellular proteins are methylated on lysine residues and this has been most intensively studied for histone proteins. Lysine methylations on non-histone proteins are also frequent, but in most cases the functional significance of the methylation event, as well as the identity of the responsible lysine (K) specific methyltransferase (KMT), remain unknown. Several recently discovered KMTs belong to the so-called seven-ß-strand (7BS) class of MTases and we have here investigated an uncharacterized human 7BS MTase currently annotated as part of the endothelin converting enzyme 2, but which should be considered a separate enzyme. Combining in vitro enzymology and analyzes of knockout cells, we demonstrate that this MTase efficiently methylates K36 in eukaryotic translation elongation factor 1 alpha (eEF1A) in vitro and in vivo. We suggest that this novel KMT is named eEF1A-KMT4 (gene name EEF1AKMT4), in agreement with the recently established nomenclature. Furthermore, by ribosome profiling we show that the absence of K36 methylation affects translation dynamics and changes translation speed of distinct codons. Finally, we show that eEF1A-KMT4 is part of a novel family of human KMTs, defined by a shared sequence motif in the active site and we demonstrate the importance of this motif for catalytic activity.
Asunto(s)
Factor 1 Eucariótico de Iniciación/metabolismo , Metiltransferasas/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Electroforesis en Gel de Poliacrilamida , Factor 1 Eucariótico de Iniciación/genética , Técnicas de Inactivación de Genes , N-Metiltransferasa de Histona-Lisina , Humanos , Lisina/genética , Lisina/metabolismo , Metilación , Metiltransferasas/genética , Filogenia , ARN Mensajero/genética , Homología de Secuencia de AminoácidoRESUMEN
Lysine methylation is abundant on histone proteins, representing a dynamic regulator of chromatin state and gene activity, but is also frequent on many non-histone proteins, including eukaryotic elongation factor 1 alpha (eEF1A). However, the functional significance of eEF1A methylation remains obscure and it has remained unclear whether eEF1A methylation is dynamic and subject to active regulation. We here demonstrate, using a wide range of in vitro and in vivo approaches, that the previously uncharacterized human methyltransferase METTL21B specifically targets Lys-165 in eEF1A in an aminoacyl-tRNA- and GTP-dependent manner. Interestingly, METTL21B-mediated eEF1A methylation showed strong variation across different tissues and cell lines, and was induced by altering growth conditions or by treatment with certain ER-stress-inducing drugs, concomitant with an increase in METTL21B gene expression. Moreover, genetic ablation of METTL21B function in mammalian cells caused substantial alterations in mRNA translation, as measured by ribosomal profiling. A non-canonical function for eEF1A in organization of the cellular cytoskeleton has been reported, and interestingly, METTL21B accumulated in centrosomes, in addition to the expected cytosolic localization. In summary, the present study identifies METTL21B as the enzyme responsible for methylation of eEF1A on Lys-165 and shows that this modification is dynamic, inducible and likely of regulatory importance.