RESUMEN

Dinoflagellates, a class of unicellular eukaryotic phytoplankton, exhibit minimal transcriptional regulation, representing a unique model for exploring gene expression. The biosynthesis, distribution, regulation, and function of mRNA N1-methyladenosine (m1A) remain controversial due to its limited presence in typical eukaryotic mRNA. This study provides a comprehensive map of m1A in dinoflagellate mRNA and shows that m1A, rather than N6-methyladenosine (m6A), is the most prevalent internal mRNA modification in various dinoflagellate species, with an asymmetric distribution along mature transcripts. In Amphidinium carterae, we identify 6549 m1A sites characterized by a non-tRNA T-loop-like sequence motif within the transcripts of 3196 genes, many of which are involved in regulating carbon and nitrogen metabolism. Enriched within 3'UTRs, dinoflagellate mRNA m1A levels negatively correlate with translation efficiency. Nitrogen depletion further decreases mRNA m1A levels. Our data suggest that distinctive patterns of m1A modification might influence the expression of metabolism-related genes through translational control.

RESUMEN

Metabolism and mRNA translation represent critical steps involved in modulating gene expression and cellular physiology. Being the most energy-consuming process in the cell, mRNA translation is strictly linked to cellular metabolism and in synchrony with it. Indeed, several mRNAs for metabolic pathways are regulated at the translational level, resulting in translation being a coordinator of metabolism. On the other hand, there is a growing appreciation for how metabolism impacts several aspects of RNA biology. For example, metabolic pathways and metabolites directly control the selectivity and efficiency of the translational machinery, as well as post-transcriptional modifications of RNA to fine-tune protein synthesis. Consistently, alterations in the intricate interplay between translational control and cellular metabolism have emerged as a critical axis underlying human diseases. A better understanding of such events will foresee innovative therapeutic strategies in human disease states.

Asunto(s)

Biosíntesis de Proteínas , ARN Mensajero , Humanos , Animales , ARN Mensajero/metabolismo , ARN Mensajero/genética , Redes y Vías Metabólicas , Procesamiento Postranscripcional del ARN

RESUMEN

Viral infection often triggers eukaryotic initiator factor 2α (eIF2α) phosphorylation, leading to global 5'-cap-dependent translation inhibition. RSV encodes messenger RNAs (mRNAs) mimicking 5'-cap structures of host mRNAs and thus inhibition of cap-dependent translation initiation would likely also reduce viral translation. We confirmed that RSV limits widespread translation initiation inhibition and unexpectedly found that the fraction of ribosomes within polysomes increases during infection, indicating higher ribosome loading on mRNAs during infection. We found that AU-rich host transcripts that are less efficiently translated under normal conditions become more efficient at recruiting ribosomes, similar to RSV transcripts. Viral transcripts are transcribed in cytoplasmic inclusion bodies, where the viral AU-rich binding protein M2-1 has been shown to bind viral transcripts and shuttle them into the cytoplasm. We further demonstrated that M2-1 is found on polysomes, and that M2-1 might deliver host AU-rich transcripts for translation.

RESUMEN

Although messenger RNA translation is tightly regulated to preserve protein synthesis and cellular homeostasis, chronic exposure to interferon-γ (IFN-γ) in several cancers can lead to tryptophan (Trp) shortage via the indoleamine-2,3-dioxygenase (IDO)- kynurenine pathway and therefore promotes the production of aberrant peptides by ribosomal frameshifting and tryptophan-to-phenylalanine (W>F) codon reassignment events (substitutants) specifically at Trp codons. However, the effect of Trp depletion on the generation of aberrant peptides by ribosomal mistranslation in gastric cancer (GC) is still obscure. Here, it is shows that the abundant infiltrating lymphocytes in EBV-positive GC continuously secreted IFN-γ, upregulated IDO1 expression, leading to Trp shortage and the induction of W>F substitutants. Intriguingly, the production of W>F substitutants in EBV-positive GC is linked to antigen presentation and the activation of the mTOR/eIF4E signaling pathway. Inhibiting either the mTOR/eIF4E pathway or EIF4E expression counteracted the production and antigen presentation of W>F substitutants. Thus, the mTOR/eIF4E pathway exposed the vulnerability of gastric cancer by accelerating the production of aberrant peptides and boosting immune activation through W>F substitutant events. This work proposes that EBV-positive GC patients with mTOR/eIF4E hyperactivation may benefit from anti-tumor immunotherapy.

RESUMEN

BACKGROUND: N4-acetylcytidine (ac4C) represents a novel messenger RNA (mRNA) modification, and its associated acetyltransferase N-acetyltransferase 10 (NAT10) plays a crucial role in the initiation and progression of tumors by regulating mRNA functionality. However, its role in hepatocellular carcinoma (HCC) development and prognosis is largely unknown. This study aimed to elucidate the role of NAT10-mediated ac4C in HCC progression and provide a promising therapeutic approach. METHODS: The ac4C levels were evaluated by dot blot and ultra-performance liquid chromatography-tandem mass spectrometry with harvested HCC tissues. The expression of NAT10 was investigated using quantitative real-time polymerase chain reaction, western blotting, and immunohistochemical staining across 91 cohorts of HCC patients. To explore the underlying mechanisms of NAT10-ac4C in HCC, we employed a comprehensive approach integrating acetylated RNA immunoprecipitation and sequencing, RNA sequencing and ribosome profiling analyses, along with RNA immunoprecipitation, RNA pull-down, mass spectrometry, and site-specific mutation analyses. The drug affinity responsive targets stability, cellular thermal shift assay, and surface plasmon resonance assays were performed to assess the specific binding of NAT10 and Panobinostat. Furthermore, the efficacy of targeting NAT10-ac4C for HCC treatment was elucidated through in vitro experiments using HCC cells and in vivo HCC mouse models. RESULTS: Our investigation revealed a significant increase in both the ac4C RNA level and NAT10 expression in HCC. Notably, elevated NAT10 expression was associated with poor outcomes in HCC patients. Functionally, silencing NAT10 suppressed HCC proliferation and metastasis in vitro and in vivo. Mechanistically, NAT10 stimulates the ac4C modification within the coding sequence (CDS) of high mobility group protein B2 (HMGB2), which subsequently enhances HMGB2 translation by facilitating eukaryotic elongation factor 2 (eEF2) binding to the ac4C sites on HMGB2 mRNA's CDS. Additionally, high-throughput compound library screening revealed Panobinostat as a potent inhibitor of NAT10-mediated ac4C modification. This inhibition significantly attenuated HCC growth and metastasis in both in vitro experiments using HCC cells and in vivo HCC mouse models. CONCLUSIONS: Our study identified a novel oncogenic epi-transcriptome axis involving NAT10-ac4C/eEF2-HMGB2, which plays a pivotal role in regulating HCC growth and metastasis. The drug Panobinostat validates the therapeutic potential of targeting this axis for HCC treatment.

RESUMEN

Sensitization of spinal nociceptive circuits plays a crucial role in neuropathic pain. This sensitization depends on new gene expression that is primarily regulated via transcriptional and translational control mechanisms. The relative roles of these mechanisms in regulating gene expression in the clinically relevant chronic phase of neuropathic pain are not well understood. Here, we show that changes in gene expression in the spinal cord during the chronic phase of neuropathic pain are substantially regulated at the translational level. Downregulating spinal translation at the chronic phase alleviated pain hypersensitivity. Cell-type-specific profiling revealed that spinal inhibitory neurons exhibited greater changes in translation after peripheral nerve injury compared to excitatory neurons. Notably, increasing translation selectively in all inhibitory neurons or parvalbumin-positive (PV+) interneurons, but not excitatory neurons, promoted mechanical pain hypersensitivity. Furthermore, increasing translation in PV+ neurons decreased their intrinsic excitability and spiking activity, whereas reducing translation in spinal PV+ neurons prevented the nerve injury-induced decrease in excitability. Thus, translational control mechanisms in the spinal cord, particularly in inhibitory neurons, play a role in mediating neuropathic pain hypersensitivity.

RESUMEN

In male reproductive system, proteins containing the coiled-coil domain (CCDC) are predominantly expressed in specific regions including the testis, epididymis, seminal vesicle, and prostate. They play a vital role in centriole formation, sperm motility and flagellar development in male gametes. Despite being highly expressed in the testis, the exact physiological function of the coiled-coil domain-containing 189 (Ccdc189) gene remain largely unclear. Our research provides a comprehensive and detailed investigation into the localization of CCDC189 protein within the testis seminiferous tubules. CCDC189 specifically expressed in spermatocytes, round spermatids and elongating spermatids in mouse testis. The deletion of Ccdc189 in mouse leads to male infertility, characterized by significantly reduced sperm counts and motility. Abnormally shaped spermatozoa with irregular tails, exhibiting shortened and twisted morphology, were observed in the seminiferous tubules. Electron microscopy revealed disordered and missing peripheral microtubule doublets (MTD) and outer dense fibers (ODF) in the sperm flagella, accompanied by a consistent absence of central pairs (CP). The knockout of Ccdc189 resulted in oligo-astheno-teratozoospermia, which is characterized by low sperm count and reduced sperm motility and abnormal morphology. Furthermore, we identified poly(A)-binding protein cytoplasmic 1 (PABPC1) and PABPC2 as interacting proteins with CCDC189. These proteins belong to the poly(A)-binding protein (PABP) family and are involved in regulating mRNA translational activity in spermatogenic cells by specifically binding to poly(A) tails at the 3' ends of mRNAs.

RESUMEN

INTRODUCTION: Impaired brain protein synthesis, synaptic plasticity, and memory are major hallmarks of Alzheimer's disease (AD). The ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) has been shown to modulate protein synthesis, but its effects on memory in AD models remain elusive. METHODS: We investigated the effects of HNK on hippocampal protein synthesis, long-term potentiation (LTP), and memory in AD mouse models. RESULTS: HNK activated extracellular signal-regulated kinase 1/2 (ERK1/2), mechanistic target of rapamycin (mTOR), and p70S6 kinase 1 (S6K1)/ribosomal protein S6 signaling pathways. Treatment with HNK rescued hippocampal LTP and memory deficits in amyloid-ß oligomers (AßO)-infused mice in an ERK1/2-dependent manner. Treatment with HNK further corrected aberrant transcription, LTP and memory in aged APP/PS1 mice. DISCUSSION: Our findings demonstrate that HNK induces signaling and transcriptional responses that correct synaptic and memory deficits in AD mice. These results raise the prospect that HNK could serve as a therapeutic approach in AD. HIGHLIGHTS: The ketamine metabolite HNK activates hippocampal ERK/mTOR/S6 signaling pathways. HNK corrects hippocampal synaptic and memory defects in two mouse models of AD. Rescue of synaptic and memory impairments by HNK depends on ERK signaling. HNK corrects aberrant transcriptional signatures in APP/PS1 mice.

Asunto(s)

Enfermedad de Alzheimer , Modelos Animales de Enfermedad , Hipocampo , Ketamina , Ratones Transgénicos , Plasticidad Neuronal , Animales , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Ketamina/análogos & derivados , Ketamina/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Ratones , Potenciación a Largo Plazo/efectos de los fármacos , Péptidos beta-Amiloides/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , ARN Mensajero/metabolismo , Memoria/efectos de los fármacos , Masculino , Trastornos de la Memoria/tratamiento farmacológico , Ratones Endogámicos C57BL , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Presenilina-1/genética , Humanos

RESUMEN

Some chemotherapy drugs modulate the formation of stress granules (SGs), which are RNA-containing cytoplasmic foci contributing to stress response pathways. How SGs mechanistically contribute to pro-survival or pro-apoptotic functions must be better defined. The chemotherapy drug lomustine promotes SG formation by activating the stress-sensing eIF2α kinase HRI (encoded by the EIF2AK1 gene). Here, we applied a DNA microarray-based transcriptome analysis to determine the genes modulated by lomustine-induced stress and suggest roles for SGs in this process. We found that the expression of the pro-apoptotic EGR1 gene was specifically regulated in cells upon lomustine treatment. The appearance of EGR1-encoding mRNA in SGs correlated with a decrease in EGR1 mRNA translation. Specifically, EGR1 mRNA was sequestered to SGs upon lomustine treatment, probably preventing its ribosome translation and consequently limiting the degree of apoptosis. Our data support the model where SGs can selectively sequester specific mRNAs in a stress-specific manner, modulate their availability for translation, and thus determine the fate of a stressed cell.

Asunto(s)

Proteína 1 de la Respuesta de Crecimiento Precoz , Lomustina , ARN Mensajero , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Lomustina/farmacología , Gránulos de Estrés/metabolismo , Gránulos de Estrés/genética , Apoptosis/efectos de los fármacos , Antineoplásicos Alquilantes/farmacología

RESUMEN

Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing ß cells renders these cells susceptible to autoimmunity. We show that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reverses the mRNA translation block in stressed human islets and delays the onset of diabetes, reduces islet inflammation, and preserves ß cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice shows reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in ß cells. Spatial proteomics of islets from these mice shows an increase in the immune checkpoint protein PD-L1 in ß cells. Golgi membrane protein 1, whose levels increase following PERK inhibition in human islets and EndoC-ßH1 human ß cells, interacts with and stabilizes PD-L1. Collectively, our studies show that PERK activity enhances ß cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.

RESUMEN

Many types of human cancers suppress the expression of argininosuccinate synthase 1 (ASS1), a rate-limiting enzyme for arginine production. Although dependency on exogenous arginine can be harnessed by arginine-deprivation therapies, the impact of ASS1 suppression on the quality of the tumor proteome is unknown. We therefore interrogated proteomes of cancer patients for arginine codon reassignments (substitutants) and surprisingly identified a strong enrichment for cysteine (R>C) in lung tumors specifically. Most R>C events did not coincide with genetically encoded R>C mutations but were likely products of tRNA misalignments. The expression of R>C substitutants was highly associated with oncogenic kelch-like epichlorohydrin (ECH)-associated protein 1 (KEAP1)-pathway mutations and suppressed by intact-KEAP1 in KEAP1-mutated cancer cells. Finally, functional interrogation indicated a key role for R>C substitutants in cell survival to cisplatin, suggesting that regulatory codon reassignments endow cancer cells with more resilience to stress. Thus, we present a mechanism for enriching lung cancer proteomes with cysteines that may affect therapeutic decisions.

Asunto(s)

Arginina , Cisteína , Proteína 1 Asociada A ECH Tipo Kelch , Neoplasias Pulmonares , Proteoma , Humanos , Cisteína/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteoma/metabolismo , Arginina/metabolismo , Mutación , Argininosuccinato Sintasa/metabolismo , Argininosuccinato Sintasa/genética , Cisplatino/farmacología , Línea Celular Tumoral , Proteómica/métodos , Regulación Neoplásica de la Expresión Génica , Supervivencia Celular/efectos de los fármacos , ARN de Transferencia/metabolismo , ARN de Transferencia/genética

RESUMEN

Multiple myeloma (MM) is an incurable plasma cell cancer in the bone marrow. Immunomodulatory drugs, such as lenalidomide (LEN) and pomalidomide, are backbone agents in MM treatment, and LEN resistance is commonly seen in the MM clinic. In this study, we presented that heterogeneous nuclear ribonucleoprotein U (hnRNPU) affected MM resistance to LEN via the regulation of target mRNA translation. hnRNPULow MM cells exhibited upregulated CRBN and IKZF1 proteins, stringent IKZF1/3 protein degradation upon LEN addition and increased sensitivity to LEN. RNA pulldown assays and RNA electrophoretic mobility shift assays revealed that hnRNPU bound to the 3'-untranslated region of CRBN and IKZF1 mRNA. A sucrose gradient assay suggested that hnRNPU specifically regulated CRBN and IKZF1 mRNA translation. The competition of hnRNPU binding to its target mRNAs by small RNAs with hnRNPU-binding sites restored MM sensitivity to LEN. hnRNPU function in vivo was confirmed in an immunocompetent MM mouse model constructed by the inoculation of Crbn-humanized murine 5TGM1 cells into CrbnI391V/+ mice. Overall, this study suggests a novel mechanism of LEN sensitivity in which hnRNPU represses CRBN and IKZF1 mRNA translation.

Asunto(s)

Lenalidomida , Mieloma Múltiple , Lenalidomida/farmacología , Lenalidomida/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Humanos , Ratones , Animales , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Factor de Transcripción Ikaros/genética , Factor de Transcripción Ikaros/metabolismo

RESUMEN

Post-transcriptional mRNA regulation shapes gene expression, yet how cis-elements and mRNA translation interface to regulate mRNA stability is poorly understood. We find that the strength of translation initiation, upstream open reading frame (uORF) content, codon optimality, AU-rich elements, microRNA binding sites, and open reading frame (ORF) length function combinatorially to regulate mRNA stability. Machine-learning analysis identifies ORF length as the most important conserved feature regulating mRNA decay. We find that Upf1 binds poorly translated and untranslated ORFs, which are associated with a higher decay rate, including mRNAs with uORFs and those with exposed ORFs after stop codons. Our study emphasizes Upf1's converging role in surveilling mRNAs with exposed ORFs that are poorly translated, such as mRNAs with long ORFs, ORF-like 3' UTRs, and mRNAs containing uORFs. We propose that Upf1 regulation of poorly/untranslated ORFs provides a unifying mechanism of surveillance in regulating mRNA stability and homeostasis in an exon-junction complex (EJC)-independent nonsense-mediated decay (NMD) pathway that we term ORF-mediated decay (OMD).

Asunto(s)

ARN Helicasas , Estabilidad del ARN , Transactivadores , Humanos , Regiones no Traducidas 3'/genética , Degradación de ARNm Mediada por Codón sin Sentido , Sistemas de Lectura Abierta/genética , Biosíntesis de Proteínas , ARN Helicasas/metabolismo , ARN Helicasas/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Transactivadores/metabolismo , Transactivadores/genética , Células HEK293

RESUMEN

Bacteria have evolved diverse defense mechanisms to counter bacteriophage attacks. Genetic programs activated upon infection characterize phage-host molecular interactions and ultimately determine the outcome of the infection. In this study, we applied ribosome profiling to monitor protein synthesis during the early stages of sk1 bacteriophage infection in Lactococcus cremoris. Our analysis revealed major changes in gene expression within 5 minutes of sk1 infection. Notably, we observed a specific and severe downregulation of several pyr operons which encode enzymes required for uridine monophosphate biosynthesis. Consistent with previous findings, this is likely an attempt of the host to starve the phage of nucleotides it requires for propagation. We also observed a gene expression response that we expect to benefit the phage. This included the upregulation of 40 ribosome proteins that likely increased the host's translational capacity, concurrent with a downregulation of genes that promote translational fidelity (lepA and raiA). In addition to the characterization of host-phage gene expression responses, the obtained ribosome profiling data enabled us to identify two putative recoding events as well as dozens of loci currently annotated as pseudogenes that are actively translated. Furthermore, our study elucidated alterations in the dynamics of the translation process, as indicated by time-dependent changes in the metagene profile, suggesting global shifts in translation rates upon infection. Additionally, we observed consistent modifications in the ribosome profiles of individual genes, which were apparent as early as 2 minutes post-infection. The study emphasizes our ability to capture rapid alterations of gene expression during phage infection through ribosome profiling. IMPORTANCE: The ribosome profiling technology has provided invaluable insights for understanding cellular translation and eukaryotic viral infections. However, its potential for investigating host-phage interactions remains largely untapped. Here, we applied ribosome profiling to Lactococcus cremoris cultures infected with sk1, a major infectious agent in dairy fermentation processes. This revealed a profound downregulation of genes involved in pyrimidine nucleotide synthesis at an early stage of phage infection, suggesting an anti-phage program aimed at restricting nucleotide availability and, consequently, phage propagation. This is consistent with recent findings and contributes to our growing appreciation for the role of nucleotide limitation as an anti-viral strategy. In addition to capturing rapid alterations in gene expression levels, we identified translation occurring outside annotated regions, as well as signatures of non-standard translation mechanisms. The gene profiles revealed specific changes in ribosomal densities upon infection, reflecting alterations in the dynamics of the translation process.

Asunto(s)

Bacteriófagos , Lactococcus , Biosíntesis de Proteínas , Perfilado de Ribosomas , Regulación hacia Abajo , Bacteriófagos/genética , Bacteriófagos/metabolismo , ARN Mensajero/metabolismo , Nucleótidos/metabolismo , Uridina Monofosfato/metabolismo

RESUMEN

eEF2 post-translational modifications (PTMs) can profoundly affect mRNA translation dynamics. However, the physiologic function of eEF2K525 trimethylation (eEF2K525me3), a PTM catalyzed by the enzyme FAM86A, is unknown. Here, we find that FAM86A methylation of eEF2 regulates nascent elongation to promote protein synthesis and lung adenocarcinoma (LUAD) pathogenesis. The principal physiologic substrate of FAM86A is eEF2, with K525me3 modeled to facilitate productive eEF2-ribosome engagement during translocation. FAM86A depletion in LUAD cells causes 80S monosome accumulation and mRNA translation inhibition. FAM86A is overexpressed in LUAD and eEF2K525me3 levels increase through advancing LUAD disease stages. FAM86A knockdown attenuates LUAD cell proliferation and suppression of the FAM86A-eEF2K525me3 axis inhibits cancer cell and patient-derived LUAD xenograft growth in vivo. Finally, FAM86A ablation strongly attenuates tumor growth and extends survival in KRASG12C-driven LUAD mouse models. Thus, our work uncovers an eEF2 methylation-mediated mRNA translation elongation regulatory node and nominates FAM86A as an etiologic agent in LUAD.

Asunto(s)

Adenocarcinoma del Pulmón , Carcinogénesis , Neoplasias Pulmonares , Factor 2 de Elongación Peptídica , ARN Mensajero , Humanos , Animales , Metilación , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Factor 2 de Elongación Peptídica/metabolismo , Factor 2 de Elongación Peptídica/genética , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Carcinogénesis/genética , Carcinogénesis/metabolismo , Proliferación Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Extensión de la Cadena Peptídica de Translación , Ratones Desnudos , Procesamiento Proteico-Postraduccional , Femenino

RESUMEN

Huntington disease (HD) is caused by an expanded polyglutamine mutation in huntingtin (mHTT) that promotes prominent atrophy in the striatum and subsequent psychiatric, cognitive deficits, and choreiform movements. Multiple lines of evidence point to an association between HD and aberrant striatal mitochondrial functions; however, the present knowledge about whether (or how) mitochondrial mRNA translation is differentially regulated in HD remains unclear. We found that protein synthesis is diminished in HD mitochondria compared to healthy control striatal cell models. We utilized ribosome profiling (Ribo-Seq) to analyze detailed snapshots of ribosome occupancy of the mitochondrial mRNA transcripts in control and HD striatal cell models. The Ribo-Seq data revealed almost unaltered ribosome occupancy on the nuclear-encoded mitochondrial transcripts involved in oxidative phosphorylation (SDHA, Ndufv1, Timm23, Tomm5, Mrps22) in HD cells. By contrast, ribosome occupancy was dramatically increased for mitochondrially encoded oxidative phosphorylation mRNAs (mt-Nd1, mt-Nd2, mt-Nd4, mt-Nd4l, mt-Nd5, mt-Nd6, mt-Co1, mt-Cytb, and mt-ATP8). We also applied tandem mass tag-based mass spectrometry identification of mitochondrial proteins to derive correlations between ribosome occupancy and actual mature mitochondrial protein products. We found many mitochondrial transcripts with comparable or higher ribosome occupancy, but diminished mitochondrial protein products, in HD. Thus, our study provides the first evidence of a widespread dichotomous effect on ribosome occupancy and protein abundance of mitochondria-related genes in HD.

Asunto(s)

Enfermedad de Huntington , Mitocondrias , Biosíntesis de Proteínas , Perfilado de Ribosomas , Humanos , Línea Celular , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Espectrometría de Masas , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Fosforilación Oxidativa , ARN Mensajero/metabolismo , ARN Mensajero/genética , ARN Mitocondrial/metabolismo , ARN Mitocondrial/genética

RESUMEN

BACKGROUND INFORMATION: The dual-specificity phosphatase 3 (DUSP3) regulates cell cycle progression, proliferation, senescence, and DNA repair pathways under genotoxic stress. This phosphatase interacts with HNRNPC protein suggesting an involvement in the regulation of HNRNPC-ribonucleoprotein complex stability. In this work, we investigate the impact of DUSP3 depletion on functions of HNRNPC aiming to suggest new roles for this enzyme. RESULTS: The DUSP3 knockdown results in the tyrosine hyperphosphorylation state of HNRNPC increasing its RNA binding ability. HNRNPC is present in the cytoplasm where it interacts with IRES trans-acting factors (ITAF) complex, which recruits the 40S ribosome on mRNA during protein synthesis, thus facilitating the translation of mRNAs containing IRES sequence in response to specific stimuli. In accordance with that, we found that DUSP3 is present in the 40S, monosomes and polysomes interacting with HNRNPC, just like other previously identified DUSP3 substrates/interacting partners such as PABP and NCL proteins. By downregulating DUSP3, Tyr-phosphorylated HNRNPC preferentially binds to IRES-containing mRNAs within ITAF complexes preferentially in synchronized or stressed cells, as evidenced by the higher levels of proteins such as c-MYC and XIAP, but not their mRNAs such as measured by qPCR. Under DUSP3 absence, this increased phosphorylated-HNRNPC/RNA interaction reduces HNRNPC-p53 binding in presence of RNAs releasing p53 for specialized cellular responses. Similarly, to HNRNPC, PABP physically interacts with DUSP3 in an RNA-dependent manner. CONCLUSIONS AND SIGNIFICANCE: Overall, DUSP3 can modulate cellular responses to genotoxic stimuli at the translational level by maintaining the stability of HNRNPC-ITAF complexes and regulating the intensity and specificity of RNA interactions with RRM-domain proteins.

Asunto(s)

Daño del ADN , Fosfatasa 3 de Especificidad Dual , Ribonucleoproteína Heterogénea-Nuclear Grupo C , ARN Mensajero , Humanos , Fosfatasa 3 de Especificidad Dual/metabolismo , Fosfatasa 3 de Especificidad Dual/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Fosforilación , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo C/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo C/metabolismo

RESUMEN

BACKGROUND: While liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism underlying liver CSC self-renewal remains elusive. We aim to characterize the role of Methyltransferase 16 (METTL16), a recently identified RNA N6-methyladenosine (m6A) methyltransferase, in HCC development/maintenance, CSC stemness, as well as normal hepatogenesis. METHODS: Liver-specific Mettl16 conditional KO (cKO) mice were generated to assess its role in HCC pathogenesis and normal hepatogenesis. Hydrodynamic tail-vein injection (HDTVi)-induced de novo hepatocarcinogenesis and xenograft models were utilized to determine the role of METTL16 in HCC initiation and progression. A limiting dilution assay was utilized to evaluate CSC frequency. Functionally essential targets were revealed via integrative analysis of multi-omics data, including RNA-seq, RNA immunoprecipitation (RIP)-seq, and ribosome profiling. RESULTS: METTL16 is highly expressed in liver CSCs and its depletion dramatically decreased CSC frequency in vitro and in vivo. Mettl16 KO significantly attenuated HCC initiation and progression, yet only slightly influenced normal hepatogenesis. Mechanistic studies, including high-throughput sequencing, unveiled METTL16 as a key regulator of ribosomal RNA (rRNA) maturation and mRNA translation and identified eukaryotic translation initiation factor 3 subunit a (eIF3a) transcript as a bona-fide target of METTL16 in HCC. In addition, the functionally essential regions of METTL16 were revealed by CRISPR gene tiling scan, which will pave the way for the development of potential inhibitor(s). CONCLUSIONS: Our findings highlight the crucial oncogenic role of METTL16 in promoting HCC pathogenesis and enhancing liver CSC self-renewal through augmenting mRNA translation efficiency.

Asunto(s)

Carcinoma Hepatocelular , Neoplasias Hepáticas , Células Madre Neoplásicas , Animales , Humanos , Ratones , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Autorrenovación de las Células/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Metiltransferasas/genética , Metiltransferasas/metabolismo , Células Madre Neoplásicas/patología , Biosíntesis de Proteínas , Ribosomas/metabolismo , ARN

RESUMEN

The CRISPR-Cas system, initially for DNA-level gene editing and transcription regulation, has expanded to RNA targeting with the Cas13d family, notably the RfxCas13d. This advancement allows for mRNA targeting with high specificity, particularly after catalytic inactivation, broadening the exploration of translation regulation. This study introduces a CRISPR-dCas13d-eIF4G fusion module, combining dCas13d with the eIF4G translation regulatory element, enhancing target mRNA translation levels. This module, using specially designed sgRNAs, selectively boosts protein translation in targeted tissue cells without altering transcription, leading to notable protein expression upregulation. This system is applied to a kidney stone disease model, focusing on ferroptosis-linked GPX4 gene regulation. By targeting GPX4 with sgRNAs, its protein expression is upregulated in human renal cells and mouse kidney tissue, countering ferroptosis and resisting calcium oxalate-induced cell damage, hence mitigating stone formation. This study evidences the CRISPR-dCas13d-eIF4G system's efficacy in eukaryotic cells, presenting a novel protein translation research approach and potential kidney stone disease treatment advancements.

Asunto(s)

Sistemas CRISPR-Cas , Oxalato de Calcio , Modelos Animales de Enfermedad , Factor 4G Eucariótico de Iniciación , Ferroptosis , Ferroptosis/genética , Ratones , Animales , Oxalato de Calcio/metabolismo , Sistemas CRISPR-Cas/genética , Humanos , Factor 4G Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/metabolismo , Cálculos Renales/genética , Cálculos Renales/metabolismo , Biosíntesis de Proteínas/genética , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo

RESUMEN

In recent years, research into Parkinson's disease and similar neurodegenerative disorders has increasingly suggested that these conditions are synonymous with failures in proteostasis. However, the spotlight of this research has remained firmly focused on the tail end of proteostasis, primarily aggregation, misfolding, and degradation, with protein translation being comparatively overlooked. Now, there is an increasing body of evidence supporting a potential role for translation in the pathogenesis of PD, and its dysregulation is already established in other similar neurodegenerative conditions. In this paper, we consider how altered protein translation fits into the broader picture of PD pathogenesis, working hand in hand to compound the stress placed on neurons, until this becomes irrecoverable. We will also consider molecular players of interest, recent evidence that suggests that aggregates may directly influence translation in PD progression, and the implications for the role of protein translation in our development of clinically useful diagnostics and therapeutics.

Asunto(s)

Enfermedad de Parkinson , Humanos , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/metabolismo , Neuronas/metabolismo , Proteostasis , Biosíntesis de Proteínas , alfa-Sinucleína/metabolismo