RESUMEN
The amino acid cysteine and its oxidized dimeric form cystine are commonly believed to be synonymous in metabolic functions. Cyst(e)ine depletion not only induces amino acid response but also triggers ferroptosis, a non-apoptotic cell death. Here, we report that unlike general amino acid starvation, cyst(e)ine deprivation triggers ATF4 induction at the transcriptional level. Unexpectedly, it is the shortage of lysosomal cystine, but not the cytosolic cysteine, that elicits the adaptative ATF4 response. The lysosome-nucleus signaling pathway involves the aryl hydrocarbon receptor (AhR) that senses lysosomal cystine via the kynurenine pathway. A blockade of lysosomal cystine efflux attenuates ATF4 induction and sensitizes ferroptosis. To potentiate ferroptosis in cancer, we develop a synthetic mRNA reagent, CysRx, that converts cytosolic cysteine to lysosomal cystine. CysRx maximizes cancer cell ferroptosis and effectively suppresses tumor growth in vivo. Thus, intracellular nutrient reprogramming has the potential to induce selective ferroptosis in cancer without systematic starvation.
Asunto(s)
Quistes , Ferroptosis , Humanos , Cisteína , Cistina , Ferroptosis/genética , Aminoácidos , LisosomasRESUMEN
To survive, mammalian cells must adapt to environmental challenges. While the cellular response to mild stress has been widely studied, how cells respond to severe stress remains unclear. We show here that under severe hyperosmotic stress, cells enter a transient hibernation-like state in anticipation of recovery. We demonstrate this adaptive pausing response (APR) is a coordinated cellular response that limits ATP supply and consumption through mitochondrial fragmentation and widespread pausing of mRNA translation. This pausing is accomplished by ribosome stalling at translation initiation codons, which keeps mRNAs poised to resume translation upon recovery. We further show that recovery from severe stress involves ISR (integrated stress response) signaling that permits cell cycle progression, resumption of growth, and reversal of mitochondria fragmentation. Our findings indicate that cells can respond to severe stress via a hibernation-like mechanism that preserves vital elements of cellular function under harsh environmental conditions.
Asunto(s)
Proliferación Celular , Fibroblastos/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/biosíntesis , Presión Osmótica , Biosíntesis de Proteínas , Ribosomas/metabolismo , Adaptación Fisiológica , Adenosina Trifosfato/metabolismo , Animales , Codón Iniciador , Fibroblastos/patología , Células HEK293 , Humanos , Cinética , Ratones , Mitocondrias/genética , Mitocondrias/patología , Proteínas Mitocondriales/genética , Ribosomas/genética , Transducción de SeñalRESUMEN
The integrated stress response (ISR) facilitates cellular adaptation to stress conditions via the common target eIF2α. During ISR, the selective translation of stress-related mRNAs often relies on alternative mechanisms, such as leaky scanning or reinitiation, but the underlying mechanism remains incompletely understood. Here we report that, in response to amino acid starvation, the reinitiation of ATF4 is not only governed by the eIF2α signaling pathway, but is also subjected to regulation by mRNA methylation in the form of N6-methyladenosine (m6A). While depleting m6A demethylases represses ATF4 reinitiation, knocking down m6A methyltransferases promotes ATF4 translation. We demonstrate that m6A in the 5' UTR controls ribosome scanning and subsequent start codon selection. Global profiling of initiating ribosomes reveals widespread alternative translation events influenced by dynamic mRNA methylation. Consistently, Fto transgenic mice manifest enhanced ATF4 expression, highlighting the critical role of m6A in translational regulation of ISR at cellular and organismal levels.
Asunto(s)
Adenosina/análogos & derivados , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/fisiología , Factor 2 Eucariótico de Iniciación/metabolismo , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/genética , Ribosomas/fisiología , Estrés Fisiológico , Regiones no Traducidas 5' , Adenosina/farmacología , Animales , Células Cultivadas , Codón Iniciador , Factor 2 Eucariótico de Iniciación/genética , Fibroblastos , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Fosforilación , ARN Mensajero/metabolismoRESUMEN
N6-Methyladenosine (m6A) is the most abundant chemical modification occurring on eukaryotic mRNAs, and has been reported to be involved in almost all stages of mRNA metabolism. The distribution of m6A sites is notably asymmetric along mRNAs, with a strong preference toward the 3' terminus of the transcript. How m6A regional preference is shaped remains incompletely understood. In this study, by performing m6A-seq on chromatin-associated RNAs, we found that m6A regional preference arises during transcription. Nucleosome occupancy is remarkedly increased in the region downstream of m6A sites, suggesting an intricate interplay between m6A methylation and nucleosome-mediated transcriptional dynamics. Notably, we found a remarkable slowdown of Pol-II movement around m6A sites. In addition, inhibiting Pol-II movement increases nearby m6A methylation levels. By analyzing massively parallel assays for m6A, we found that RNA secondary structures inhibit m6A methylation. Remarkably, the m6A sites associated with Pol-II pausing tend to be embedded within RNA secondary structures. These results suggest that Pol-II pausing could affect the accessibility of m6A motifs to the methyltransferase complex and subsequent m6A methylation by mediating RNA secondary structure. Overall, our study reveals a crucial role of transcriptional dynamics in the formation of m6A regional preference.
Asunto(s)
Adenosina , Adenosina/análogos & derivados , ARN Polimerasa II , ARN Mensajero , Transcripción Genética , Adenosina/metabolismo , Metilación , ARN Mensajero/metabolismo , ARN Mensajero/genética , ARN Polimerasa II/metabolismo , Humanos , Conformación de Ácido Nucleico , Nucleosomas/metabolismo , Nucleosomas/genética , Metiltransferasas/metabolismo , Metiltransferasas/genética , Cromatina/metabolismo , Cromatina/genética , Cromatina/químicaRESUMEN
In eukaryotic cells, protein synthesis typically begins with the binding of eIF4F to the 7-methylguanylate (m7G) cap found on the 5' end of the majority of mRNAs. Surprisingly, overall translational output remains robust under eIF4F inhibition. The broad spectrum of eIF4F-resistant translatomes is incompatible with cap-independent translation mediated by internal ribosome entry sites (IRESs). Here, we report that N6-methyladenosine (m6A) facilitates mRNA translation that is resistant to eIF4F inactivation. Depletion of the methyltransferase METTL3 selectively inhibits translation of mRNAs bearing 5' UTR methylation, but not mRNAs with 5' terminal oligopyrimidine (TOP) elements. We identify ABCF1 as a critical mediator of m6A-promoted translation under both stress and physiological conditions. Supporting the role of ABCF1 in m6A-facilitated mRNA translation, ABCF1-sensitive transcripts largely overlap with METTL3-dependent mRNA targets. By illustrating the scope and mechanism of eIF4F-independent mRNA translation, these findings reshape our current perceptions of cellular translational pathways.
Asunto(s)
Adenosina/análogos & derivados , Factor 4F Eucariótico de Iniciación/metabolismo , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Caperuzas de ARN/genética , ARN Mensajero/metabolismo , Regiones no Traducidas 5'/genética , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina/farmacología , Factor 4F Eucariótico de Iniciación/genética , Células HeLa , Humanos , Sitios Internos de Entrada al Ribosoma , Metiltransferasas/genética , Metiltransferasas/metabolismo , Caperuzas de ARN/efectos de los fármacos , ARN Mensajero/genéticaRESUMEN
Gene expression can be post-transcriptionally regulated via dynamic and reversible RNA modifications. N1-methyladenosine (m1A) is a recently identified mRNA modification; however, little is known about its precise location and biogenesis. Here, we develop a base-resolution m1A profiling method, based on m1A-induced misincorporation during reverse transcription, and report distinct classes of m1A methylome in the human transcriptome. m1A in 5' UTR, particularly those at the mRNA cap, associate with increased translation efficiency. A different, small subset of m1A exhibit a GUUCRA tRNA-like motif, are evenly distributed in the transcriptome, and are dependent on the methyltransferase TRMT6/61A. Additionally, we show that m1A is prevalent in the mitochondrial-encoded transcripts. Manipulation of m1A level via TRMT61B, a mitochondria-localizing m1A methyltransferase, demonstrates that m1A in mitochondrial mRNA interferes with translation. Collectively, our approaches reveal distinct classes of m1A methylome and provide a resource for functional studies of m1A-mediated epitranscriptomic regulation.
Asunto(s)
Adenosina/análogos & derivados , Núcleo Celular/metabolismo , Mitocondrias/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , Imagen Individual de Molécula/métodos , Regiones no Traducidas 5' , Adenosina/metabolismo , Células HEK293 , Humanos , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Biosíntesis de Proteínas , Caperuzas de ARN , Interferencia de ARN , ARN Mensajero/genética , ARN de Transferencia/genética , Transfección , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismoRESUMEN
SAMD9 and SAMD9L (SAMD9/9L) are antiviral factors and tumor suppressors, playing a critical role in innate immune defense against poxviruses and the development of myeloid tumors. SAMD9/9L mutations with a gain-of-function (GoF) in inhibiting cell growth cause multisystem developmental disorders including many pediatric myelodysplastic syndromes. Predicted to be multidomain proteins with an architecture like that of the NOD-like receptors, SAMD9/9L molecular functions and domain structures are largely unknown. Here, we identified a SAMD9/9L effector domain that functions by binding to double-stranded nucleic acids (dsNA) and determined the crystal structure of the domain in complex with DNA. Aided with precise mutations that differentially perturb dsNA binding, we demonstrated that the antiviral and antiproliferative functions of the wild-type and GoF SAMD9/9L variants rely on dsNA binding by the effector domain. Furthermore, we showed that GoF variants inhibit global protein synthesis, reduce translation elongation, and induce proteotoxic stress response, which all require dsNA binding by the effector domain. The identification of the structure and function of a SAMD9/9L effector domain provides a therapeutic target for SAMD9/9L-associated human diseases.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Modelos Moleculares , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Supresoras de Tumor/química , Sitios de Unión , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación , Unión Proteica , Estrés Fisiológico , Relación Estructura-Actividad , Proteínas Supresoras de Tumor/metabolismoRESUMEN
In eukaryotic cells, many messenger RNAs (mRNAs) possess upstream open reading frames (uORFs) in addition to the main coding region. After uORF translation, the ribosome could either recycle at the stop codon or resume scanning for downstream start codons in a process known as reinitiation. Accumulating evidence suggests that some initiation factors, including eukaryotic initiation factor 3 (eIF3), linger on the early elongating ribosome, forming an eIF3-80S complex. Very little is known about how eIF3 is carried along with the 80S during elongation and whether the eIF3-80S association is subject to regulation. Here, we report that eIF3a undergoes dynamic O-linked N-acetylglucosamine (O-GlcNAc) modification in response to nutrient starvation. Stress-induced de-O-GlcNAcylation promotes eIF3 retention on the elongating ribosome and facilitates activating transcription factor 4 (ATF4) reinitiation. Eliminating the modification site from eIF3a via CRISPR genome editing induces ATF4 reinitiation even under the nutrient-rich condition. Our findings illustrate a mechanism in balancing ribosome recycling and reinitiation, thereby linking the nutrient stress response and translational reprogramming.
Asunto(s)
Factor 3 de Iniciación Eucariótica/metabolismo , Regulación de la Expresión Génica/fisiología , Proliferación Celular , Codón de Terminación , Medios de Cultivo/química , ADN Complementario , Factor 3 de Iniciación Eucariótica/genética , Células HEK293 , Células HeLa , Humanos , Iniciación de la Cadena Peptídica Traduccional , Estrés FisiológicoRESUMEN
Long noncoding RNAs (lncRNAs) are key regulators of gene expression in diverse cellular contexts and biological processes. Given the surprising range of shapes and sizes, how distinct lncRNAs achieve functional specificity remains incompletely understood. Here, we identified a heat shock-inducible lncRNA, Heat, in mouse cells that acts as a transcriptional brake to restrain stress gene expression. Functional characterization reveals that Heat directly binds to heat shock transcription factor 1 (HSF1), thereby targeting stress genes in a trans-acting manner. Intriguingly, Heat is heavily methylated in the form of m6A. Although dispensable for HSF1 binding, Heat methylation is required for silencing stress genes to attenuate heat shock response. Consistently, m6A depletion results in prolonged activation of stress genes. Furthermore, Heat mediates these effects via the nuclear m6A reader YTHDC1, forming a transcriptional silencing complex for stress genes. Our study reveals a crucial role of nuclear epitranscriptome in the transcriptional regulation of heat shock response.
Asunto(s)
Adenosina/análogos & derivados , Factores de Transcripción del Choque Térmico/metabolismo , Respuesta al Choque Térmico/genética , ARN Largo no Codificante/metabolismo , Transcripción Genética , Adenosina/metabolismo , Animales , Cromatina/metabolismo , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Células HeLa , Humanos , Metiltransferasas/metabolismo , Ratones , Unión Proteica , Factores de Empalme de ARN/metabolismo , ARN Largo no Codificante/genética , Estrés Fisiológico/genéticaRESUMEN
Transcriptional programming plays a key role in determining the cell state. Timely reconfiguration of chromatin structure and attenuation of pluripotent genes are required for efficient embryonic stem cell (ESC) differentiation. Here, we identify METTL3, a core N6-methyladenosine (m6A) catalyzing enzyme, as a crucial modulator of dynamic transcription and chromatin accessibility upon ESC-derived cardiac differentiation. Genome-wide analysis of chromatin-associated RNAs revealed that depletion of METTL3 failed to dramatically attenuate the transcription of pluripotent genes, as well as activate nascent cardiomyocyte-specific transcripts upon differentiation. Consistently, ATAC-seq analysis showed that loss of METTL3 markedly attenuated the dynamic alteration of chromatin accessibility at both promoters and gene bodies, resulting in reduced sensitivity of ESC chromatin structure to cardiac differentiation signal. Furthermore, we found that METTL3 negatively regulated the histone modifications H3K4me3 and H3K36me3, which are involved in METTL3-modulated dynamic chromatin architecture during cell state transition. Unexpectedly, using chromatin-associated m6A sequencing, we found that nuclear m6A underwent a dramatic increase upon differentiation, which correlates with the decrease of chromatin accessibility. Collectively, our findings reveal that METTL3 and nuclear m6A epitranscriptome couple with chromatin state to ensure transcriptional regulation of cell fate transition.
Asunto(s)
Cromatina , Células Madre Embrionarias , Cromatina/genética , Diferenciación Celular/genética , Células Madre Embrionarias/metabolismo , Código de Histonas , Regiones Promotoras Genéticas/genética , Metiltransferasas/genética , Metiltransferasas/metabolismoRESUMEN
Traditional annotation of protein-encoding genes relied on assumptions, such as one open reading frame (ORF) encodes one protein and minimal lengths for translated proteins. With the serendipitous discoveries of translated ORFs encoded upstream and downstream of annotated ORFs, from alternative start sites nested within annotated ORFs and from RNAs previously considered noncoding, it is becoming clear that these initial assumptions are incorrect. The findings have led to the realization that genetic information is more densely coded and that the proteome is more complex than previously anticipated. As such, interest in the identification and characterization of the previously ignored 'dark proteome' is increasing, though we note that research in eukaryotes and bacteria has largely progressed in isolation. To bridge this gap and illustrate exciting findings emerging from studies of the dark proteome, we highlight recent advances in both eukaryotic and bacterial cells. We discuss progress in the detection of alternative ORFs as well as in the understanding of functions and the regulation of their expression and posit questions for future work.
Asunto(s)
Regulación de la Expresión Génica , Sistemas de Lectura Abierta , Iniciación de la Cadena Peptídica Traduccional , Proteoma/genética , Enfermedad/genética , Regulación Bacteriana de la Expresión Génica , Humanos , Fusión de Membrana , Proteínas de la Membrana/metabolismo , Biosíntesis de Proteínas , Proteínas/fisiología , Transcripción GenéticaRESUMEN
RNA modification in the form of N6-methyladenosine (m6A) regulates nearly all the post-transcriptional processes. The asymmetric m6A deposition suggests that regional methylation may have distinct functional consequences. However, current RNA biology tools do not distinguish the contribution of individual m6A modifications. Here we report the development of 'm6A editing', a powerful approach that enables m6A installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single-chain m6A methyltransferase that can be programmed with a guide RNA. The resultant m6A 'writers' allow functional comparison of single site methylation in different messenger RNA regions. We further engineered m6A 'erasers' by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable m6A editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome.
Asunto(s)
Adenosina/análogos & derivados , Sistemas CRISPR-Cas , Edición Génica/métodos , Metiltransferasas/metabolismo , ARN Mensajero/metabolismo , Adenosina/química , Adenosina/metabolismo , Secuencia de Bases , Línea Celular , Humanos , Metiltransferasas/clasificación , ARN Mensajero/química , ARN Mensajero/genéticaRESUMEN
The structure of mRNA in vivo is unwound to some extent in response to multiple factors involved in the translation process, resulting in significant differences from the structure of the same mRNA in vitro. In this study, we have proposed a novel application of deep neural networks, named DeepDRU, to predict the degree of mRNA structure unwinding in vivo by fitting five quantifiable features that may affect mRNA folding: ribosome density (RD), minimum folding free energy (MFE), GC content, translation initiation ribosome density (INI) and mRNA structure position (POS). mRNA structures with adjustment of the simulated structural features were designed and then fed into the trained DeepDRU model. We found unique effect regions of these five features on mRNA structure in vivo. Strikingly, INI is the most critical factor affecting the structure of mRNA in vivo, and structural sequence features, including MFE and GC content, have relatively smaller effects. DeepDRU provides a new paradigm for predicting the unwinding capability of mRNA structure in vivo. This improved knowledge about the mechanisms of factors influencing the structural capability of mRNA to unwind will facilitate the design and functional analysis of mRNA structure in vivo.
Asunto(s)
Conformación de Ácido Nucleico , ARN Mensajero/química , Saccharomyces cerevisiae/química , Redes Neurales de la Computación , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genéticaRESUMEN
It is common wisdom that codon usage bias has evolved in the selection for efficient translation, in which highly expressed genes are encoded predominantly by optimal codons. However, a growing body of evidence suggests regulatory roles for non-optimal codons in translation dynamics. Here we report that in mammalian cells, non-optimal codons play a critical role in promoting selective mRNA translation during amino acid starvation. During starvation, in contrast to genes encoding ribosomal proteins whose translation is highly sensitive to amino acid deprivation, translation of genes involved in the cellular protein degradation pathways remains unaffected. We found that these two gene groups bear different codon composition, with non-optimal codons being highly enriched in genes encoding the ubiquitin-proteasome system. Supporting the selective tRNA charging model originally proposed in Escherichia coli, we demonstrated that tRNA isoacceptors decoding rare codons are maintained in translating ribosomes under amino acid starvation. Finally, using luciferase reporters fused with endogenous gene-derived sequences, we show that codon optimality contributes to differential mRNA translation in response to amino acid starvation. These results highlight the physiological significance of codon usage bias in cellular adaptation to stress.
Asunto(s)
Aminoácidos/metabolismo , Codón , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN de Transferencia/genética , Ribosomas/metabolismoRESUMEN
Codon bias at the 5' terminal of coding sequence (CDS) is known to be distinct from the rest of the CDS. A number of events occur in this short region to regulate early translation elongation and co-translational translocation. In the genes encoding secretory proteins, there is a special signal sequence which has a higher occurrence of rare codons. In this study, we analyzed codon bias of secretory genes in several eukaryotes. The results showed that secretory genes in the species except mammals had a higher occurrence of rare codons in the 5' terminal of CDS, and the bias was greater than the same region of non-secretory genes. GO analysis revealed that secretory genes containing rare codon clusters in different regions were responsible for various roles in gene functions. Moreover, codon bias in the region encoding the hydrophobic region of protein is similar in secretory and non-secretory genes, indicating that codon bias in secretory genes was partly influenced by amino acid bias. Rare codon clusters are found more frequently in specific regions, and continuous rare codons are not favoured probably because they will increase the probability of ribosome collision and drop-off. Based on ribosome profiling data, there is no significant difference in the average translation efficiencies between rare and optimal codons. Higher ribosomal density in the 5' terminal may result from ribosome pausing which could be involved in different translation events. These findings collectively provided rich information on codon bias in secretory genes, which may shed light on the co-effect of codon bias, mRNA structure and tRNA abundance in translational regulations.
Asunto(s)
Codón/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Animales , Biología Computacional/métodos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Sistemas de Lectura Abierta , Plantas/genética , ARN Mensajero/química , Ribosomas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Messenger RNA (mRNA) secondary structure decreases the elongation rate, as ribosomes must unwind every structure they encounter during translation. Therefore, the strength of mRNA secondary structure is assumed to be reduced in highly translated mRNAs. However, previous studies in vitro reported a positive correlation between mRNA folding strength and protein abundance. The counterintuitive finding suggests that mRNA secondary structure affects translation efficiency in an undetermined manner. Here, we analyzed the folding behavior of mRNA during translation and its effect on translation efficiency. We simulated translation process based on a novel computational model, taking into account the interactions among ribosomes, codon usage and mRNA secondary structures. We showed that mRNA secondary structure shortens ribosomal distance through the dynamics of folding strength. Notably, when adjacent ribosomes are close, mRNA secondary structures between them disappear, and codon usage determines the elongation rate. More importantly, our results showed that the combined effect of mRNA secondary structure and codon usage in highly translated mRNAs causes a short ribosomal distance in structural regions, which in turn eliminates the structures during translation, leading to a high elongation rate. Together, these findings reveal how the dynamics of mRNA secondary structure coupling with codon usage affect translation efficiency.
Asunto(s)
Biosíntesis de Proteínas , ARN Mensajero/química , Saccharomyces cerevisiae/genética , Codón , Conformación de Ácido Nucleico , Pliegue del ARN , Ribosomas/metabolismoRESUMEN
Amino acids show apparent propensities toward their neighbors. In addition to preferences of amino acids for their neighborhood context, amino acid substitutions are also considered to be context-dependent. However, context-dependence patterns of amino acid substitutions still remain poorly understood. Using relative entropy, we investigated the neighbor preferences of 20 amino acids and the context-dependent effects of amino acid substitutions with protein sequences in human, mouse, and dog. For 20 amino acids, the highest relative entropy was mostly observed at the nearest adjacent site of either N- or C-terminus except C and G. C showed the highest relative entropy at the third flanking site and periodic pattern was detected at G flanking sites. Furthermore, neighbor preference patterns of amino acids varied greatly in different secondary structures. We then comprehensively investigated the context-dependent effects of amino acid substitutions. Our results showed that nearly half of 380 substitution types were evidently context dependent, and the context-dependent patterns relied on protein secondary structures. Among 20 amino acids, P elicited the greatest effect on amino acid substitutions. The underlying mechanisms of context-dependent effects of amino acid substitutions were possibly mutation bias at a DNA level and natural selection. Our findings may improve secondary structure prediction algorithms and protein design; moreover, this study provided useful information to develop empirical models of protein evolution that consider dependence between residues.
Asunto(s)
Aminoácidos/metabolismo , Proteínas/química , Algoritmos , Sustitución de Aminoácidos , Aminoácidos/química , Animales , Perros , Entropía , Humanos , Ratones , Estructura Secundaria de Proteína , Proteínas/metabolismoRESUMEN
The long-term use of antibiotics can cause drug resistance. Natural polysaccharides are a novel means of treating bacterial infections, and the development and utilization of litchi pericarp polysaccharide (LPPs) as a bacteriostatic active substance offer a new research direction for the high-value utilization of litchi by-products. This study revealed that LPPs inhibited Staphylococcus aureus more than Escherichia coli, Listeria monocytogenes, and Salmonella typhimurium, with the minimum inhibitory concentrations of 145, 205, 325, and 445 µg/mL, respectively. The inhibitory activity of LPPs was insignificant for Bacillus subtilis at 505 µg/mL. The assessment of antibacterial mechanisms revealed that LPPs influenced the growth, conductivity, protein, and nucleic acid, reducing sugar, respiratory chain dehydrogenase activity, bacterial lipid peroxidation, intracellular adenosine triphosphate, and extracellular alkaline phosphatase levels of S. aureus. Of note, LPPs could modify the cell wall integrity and cell membrane permeability of S. aureus, resulting in the leakage of intracellular large and small molecules, inhibition of cellular respiratory metabolism, and oxidative losses. These processes exhibited an inhibitory effect and made the bacterium nonfunctional, thereby affecting its growth and metabolism or causing cell death. These findings provide support and insights into the potential application of LPPs as a natural antimicrobial agent.
RESUMEN
While lysine methylation is well-known for regulating gene expression transcriptionally, its implications in translation have been largely uncharted. Trimethylation at lysine 22 (K22me3) on RPL40, a core ribosomal protein located in the GTPase activation center, was first reported 27 years ago. Yet, its methyltransferase and role in translation remain unexplored. Here, we report that SMYD5 has robust in vitro activity toward RPL40 K22 and primarily catalyzes RPL40 K22me3 in cells. The loss of SMYD5 and RPL40 K22me3 leads to reduced translation output and disturbed elongation as evidenced by increased ribosome collisions. SMYD5 and RPL40 K22me3 are upregulated in hepatocellular carcinoma (HCC) and negatively correlated with patient prognosis. Depleting SMYD5 renders HCC cells hypersensitive to mTOR inhibition in both 2D and 3D cultures. Additionally, the loss of SMYD5 markedly inhibits HCC development and growth in both genetically engineered mouse and patient-derived xenograft (PDX) models, with the inhibitory effect in the PDX model further enhanced by concurrent mTOR suppression. Our findings reveal a novel role of the SMYD5 and RPL40 K22me3 axis in translation elongation and highlight the therapeutic potential of targeting SMYD5 in HCC, particularly with concurrent mTOR inhibition. This work also conceptually broadens the understanding of lysine methylation, extending its significance from transcriptional regulation to translational control.
Asunto(s)
Carcinoma Hepatocelular , N-Metiltransferasa de Histona-Lisina , Neoplasias Hepáticas , Lisina , Metiltransferasas , Proteínas Ribosómicas , Animales , Humanos , Ratones , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Lisina/metabolismo , Metilación , Ratones Desnudos , Biosíntesis de Proteínas , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genética , Metiltransferasas/genética , Metiltransferasas/metabolismoRESUMEN
A translating ribosome is typically thought to follow the reading frame defined by the selected start codon. Using super-resolution ribosome profiling, here we report pervasive out-of-frame translation immediately from the start codon. The start codon-associated ribosome frameshifting (SCARF) stems from the slippage of ribosomes during the transition from initiation to elongation. Using a massively paralleled reporter assay, we uncovered sequence elements acting as SCARF enhancers or repressors, implying that start codon recognition is coupled with reading frame fidelity. This finding explains thousands of mass spectrometry spectra unannotated from human proteome. Mechanistically, we find that the eukaryotic initiation factor 5B (eIF5B) maintains the reading frame fidelity by stabilizing initiating ribosomes. Intriguingly, amino acid starvation induces SCARF by proteasomal degradation of eIF5B. The stress-induced SCARF protects cells from starvation by enabling amino acid recycling and selective mRNA translation. Our findings illustrate a beneficial effect of translational "noise" in nutrient stress adaptation.