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1.
Curr Protoc ; 4(9): e70007, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39240231

RESUMO

Translation of mRNA into functional proteins is a fundamental process underlying many aspects of plant growth and development. Yet, the role of translational regulation in plants across diverse tissue types, including seeds, is not well known due to the lack of methods targeting these processes. Studying the seed translatome could unveil seed-specific regulatory mechanisms, offering valuable insights for breeding efforts to enhance seed traits. Polysome profiling is a widely used technique for studying mRNAs being translated. However, the traditional method is time-consuming and has a low polysome recovery rate; therefore, it requires substantial starting material. This is particularly challenging for species or mutants with limited seed quantities. Additionally, seed polysome fractions often yield low quality RNA due to the abundance of various compounds that interfere with conventional RNA extraction protocols. Here we present a robust polysome extraction method incorporating a size-exclusion step for polysome concentration streamlined with a rapid RNA extraction method optimized for seeds. This protocol works across multiple plant species and offers increased speed and robustness, requiring less than half the amount of seed tissue and time compared to conventional methods while ensuring high polysome recovery and yield of high-quality RNA for downstream experiments. These features make this protocol an ideal tool for studying seed translation efficiency and hold broad applicability across various plant species and tissues. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Robust polysome extraction for seeds Basic Protocol 2: Rapid fraction total RNA extraction.


Assuntos
Polirribossomos , RNA de Plantas , Sementes , Sementes/genética , Polirribossomos/metabolismo , Polirribossomos/genética , RNA de Plantas/isolamento & purificação , RNA de Plantas/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação
2.
Nat Commun ; 15(1): 7989, 2024 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-39284811

RESUMO

There is a growing interest in the creation of engineered condensates formed via liquid-liquid phase separation (LLPS) to exert precise cellular control in prokaryotes. However, de novo design of cellular condensates to control metabolic flux or protein translation remains a challenge. Here, we present a synthetic condensate platform, generated through the incorporation of artificial, disordered proteins to realize specific functions in Bacillus subtilis. To achieve this, the "stacking blocks" strategy is developed to rationally design a series of LLPS-promoting proteins for programming condensates. Through the targeted recruitment of biomolecules, our investigation demonstrates that cellular condensates effectively sequester biosynthetic pathways. We successfully harness this capability to enhance the biosynthesis of 2'-fucosyllactose by 123.3%. Furthermore, we find that condensates can enhance the translation specificity of tailored enzyme fourfold, and can increase N-acetylmannosamine titer by 75.0%. Collectively, these results lay the foundation for the design of engineered condensates endowed with multifunctional capacities.


Assuntos
Bacillus subtilis , Proteínas de Bactérias , Hexosaminas , Engenharia Metabólica , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Engenharia Metabólica/métodos , Hexosaminas/biossíntese , Hexosaminas/metabolismo , Hexosaminas/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Vias Biossintéticas , Engenharia de Proteínas/métodos , Biossíntese de Proteínas , Trissacarídeos/metabolismo , Trissacarídeos/biossíntese , Trissacarídeos/química , Extração Líquido-Líquido/métodos
3.
Nat Commun ; 15(1): 8119, 2024 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-39284850

RESUMO

The ribosome utilizes hydrogen bonding between mRNA codons and aminoacyl-tRNAs to ensure rapid and accurate protein production. Chemical modification of mRNA nucleobases can adjust the strength and pattern of this hydrogen bonding to alter protein synthesis. We investigate how the N1-methylpseudouridine (m1Ψ) modification, commonly incorporated into therapeutic and vaccine mRNA sequences, influences the speed and fidelity of translation. We find that m1Ψ does not substantially change the rate constants for amino acid addition by cognate tRNAs or termination by release factors. However, we also find that m1Ψ can subtly modulate the fidelity of amino acid incorporation in a codon-position and tRNA dependent manner in vitro and in human cells. Our computational modeling shows that altered energetics of mRNA:tRNA interactions largely account for the context dependence of the low levels of miscoding we observe on Ψ and m1Ψ containing codons. The outcome of translation on modified mRNA bases is thus governed by the sequence context in which they occur.


Assuntos
Códon , Biossíntese de Proteínas , Pseudouridina , RNA Mensageiro , RNA de Transferência , Pseudouridina/metabolismo , Pseudouridina/análogos & derivados , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Humanos , Códon/genética , RNA de Transferência/metabolismo , RNA de Transferência/genética , Ribossomos/metabolismo , Ligação de Hidrogênio , Células HEK293
4.
Int J Mol Sci ; 25(17)2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-39273561

RESUMO

Amylin promoter and transcriptional factors are well-established, inducible factors in the production of the main amyloidogenic pancreatic hormone, human islet amyloid peptide (hIAPP) or amylin. However, posttranscriptional mechanisms driving hIAPP expression in pancreas remain enigmatic, and hence were explored here. The translational assay revealed that both 5' and 3' untranslated regions (UTRs) of hIAPP restricted expression of the luciferase constructs only in constructs driven by the hIAPP promoter. Bioinformatics analysis revealed several putative seed sequences for a dozen micro RNAs (miRNAs) in hIAPP's 3' UTR. miR-182, miR-335, and miR-495 were the most downregulated miRNAs in stressed human islets exposed to endoplasmic reticulum (ER) or metabolic stressors, thapsigargin (TG) or high glucose (HG). Correspondingly, miR-335 mimics alone or in combination with miR-495 and miR-182 mimics significantly and potently (>3-fold) reduced hIAPP protein expression in HG-treated cultured human islets. siRNA-mediated silencing of Ago2 but not Ago1 significantly stimulated hIAPP expression and secretion from transfected, HG-treated human islets. Conversely, ectopic expression of Ago2 in hIAPP-expressing RIN-m5F cell line driven by CMV promoter reduced hIAPP intracellular protein levels. Collectively, the results point to a novel and synergistic role for hIAPP promoter, 5/3' UTRs and Ago-2/miR-335 complex in post-transcriptional regulation of hIAPP gene expression in normal and metabolically active ß-cells.


Assuntos
Proteínas Argonautas , Células Secretoras de Insulina , Insulinoma , Polipeptídeo Amiloide das Ilhotas Pancreáticas , MicroRNAs , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Células Secretoras de Insulina/metabolismo , Insulinoma/metabolismo , Insulinoma/genética , Insulinoma/patologia , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Biossíntese de Proteínas , Regiões 3' não Traduzidas , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Linhagem Celular Tumoral , Animais , Glucose/metabolismo
5.
Nat Commun ; 15(1): 7418, 2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-39223140

RESUMO

Small-molecule compounds that elicit mRNA-selective translation repression have attracted interest due to their potential for expansion of druggable space. However, only a limited number of examples have been reported to date. Here, we show that desmethyl desamino pateamine A (DMDA-PatA) represses translation in an mRNA-selective manner by clamping eIF4A, a DEAD-box RNA-binding protein, onto GNG motifs. By systematically comparing multiple eIF4A inhibitors by ribosome profiling, we found that DMDA-PatA has unique mRNA selectivity for translation repression. Unbiased Bind-n-Seq reveals that DMDA-PatA-targeted eIF4A exhibits a preference for GNG motifs in an ATP-independent manner. This unusual RNA binding sterically hinders scanning by 40S ribosomes. A combination of classical molecular dynamics simulations and quantum chemical calculations, and the subsequent development of an inactive DMDA-PatA derivative reveals that the positive charge of the tertiary amine on the trienyl arm induces G selectivity. Moreover, we identified that DDX3, another DEAD-box protein, is an alternative DMDA-PatA target with the same effects on eIF4A. Our results provide an example of the sequence-selective anchoring of RNA-binding proteins and the mRNA-selective inhibition of protein synthesis by small-molecule compounds.


Assuntos
RNA Helicases DEAD-box , Fator de Iniciação 4A em Eucariotos , Biossíntese de Proteínas , RNA Mensageiro , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Simulação de Dinâmica Molecular , Ribossomos/metabolismo , Motivos de Nucleotídeos , Ligação Proteica , Células HEK293 , Compostos de Epóxi , Tiazóis , Macrolídeos
6.
Nat Commun ; 15(1): 7681, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-39227397

RESUMO

Nascent chains undergo co-translational enzymatic processing as soon as their N-terminus becomes accessible at the ribosomal polypeptide tunnel exit (PTE). In eukaryotes, N-terminal methionine excision (NME) by Methionine Aminopeptidases (MAP1 and MAP2), and N-terminal acetylation (NTA) by N-Acetyl-Transferase A (NatA), is the most common combination of subsequent modifications carried out on the 80S ribosome. How these enzymatic processes are coordinated in the context of a rapidly translating ribosome has remained elusive. Here, we report two cryo-EM structures of multi-enzyme complexes assembled on vacant human 80S ribosomes, indicating two routes for NME-NTA. Both assemblies form on the 80S independent of nascent chain substrates. Irrespective of the route, NatA occupies a non-intrusive 'distal' binding site on the ribosome which does not interfere with MAP1 or MAP2 binding nor with most other ribosome-associated factors (RAFs). NatA can partake in a coordinated, dynamic assembly with MAP1 through the hydra-like chaperoning function of the abundant Nascent Polypeptide-Associated Complex (NAC). In contrast to MAP1, MAP2 completely covers the PTE and is thus incompatible with NAC and MAP1 recruitment. Together, our data provide the structural framework for the coordinated orchestration of NME and NTA in protein biogenesis.


Assuntos
Microscopia Crioeletrônica , Ribossomos , Humanos , Ribossomos/metabolismo , Acetilação , Processamento de Proteína Pós-Traducional , Sítios de Ligação , Biossíntese de Proteínas , Ligação Proteica , Metionina/metabolismo , Modelos Moleculares
7.
Cell Death Dis ; 15(9): 656, 2024 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-39242581

RESUMO

Thyroid cancer is the most frequently observed endocrine-related malignancy among which anaplastic thyroid cancer (ATC) is the most fatal subtype. The synthesis of protein is active to satisfy the rapid growth of ATC tumor, but the mechanisms regulating protein synthesis are still unknown. Our research revealed that kinetochore protein NUF2 played an essential role in protein synthesis and drove the progression of ATC. The prognosis of patients with thyroid carcinoma was positively correlated with high NUF2 expression. Depletion of NUF2 in ATC cells notably inhibited the proliferation and induced apoptosis, while overexpression of NUF2 facilitated ATC cell viability and colony formation. Deletion of NUF2 significantly suppressed the growth and metastasis of ATC in vivo. Notably, knockdown of NUF2 epigenetically inhibited the expression of magnesium transporters through reducing the abundance of H3K4me3 at promoters, thereby reduced intracellular Mg2+ concentration. Furthermore, we found the deletion of NUF2 or magnesium transporters significantly inhibited the protein synthesis mediated by the PI3K/Akt/mTOR pathway. In conclusion, NUF2 functions as an emerging regulator for protein synthesis by maintaining the homeostasis of intracellular Mg2+, which finally drives ATC progression.


Assuntos
Progressão da Doença , Homeostase , Magnésio , Carcinoma Anaplásico da Tireoide , Animais , Feminino , Humanos , Camundongos , Apoptose , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Magnésio/metabolismo , Camundongos Nus , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Carcinoma Anaplásico da Tireoide/metabolismo , Carcinoma Anaplásico da Tireoide/patologia , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Serina-Treonina Quinases TOR/metabolismo
8.
Development ; 151(17)2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-39250531

RESUMO

miR-31 is a highly conserved microRNA that plays crucial roles in cell proliferation, migration and differentiation. We discovered that miR-31 and some of its validated targets are enriched on the mitotic spindle of the dividing sea urchin embryo and mammalian cells. Using the sea urchin embryo, we found that miR-31 inhibition led to developmental delay correlated with increased cytoskeletal and chromosomal defects. We identified miR-31 to directly suppress several actin remodeling transcripts, including ß-actin, Gelsolin, Rab35 and Fascin. De novo translation of Fascin occurs at the mitotic spindle of sea urchin embryos and mammalian cells. Importantly, miR-31 inhibition leads to a significant a increase of newly translated Fascin at the spindle of dividing sea urchin embryos. Forced ectopic localization of Fascin transcripts to the cell membrane and translation led to significant developmental and chromosomal segregation defects, highlighting the importance of the regulation of local translation by miR-31 at the mitotic spindle to ensure proper cell division. Furthermore, miR-31-mediated post-transcriptional regulation at the mitotic spindle may be an evolutionarily conserved regulatory paradigm of mitosis.


Assuntos
MicroRNAs , Biossíntese de Proteínas , Fuso Acromático , Animais , MicroRNAs/metabolismo , MicroRNAs/genética , Fuso Acromático/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Mitose/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Desenvolvimento Embrionário/genética , Embrião não Mamífero/metabolismo , Segregação de Cromossomos/genética , Actinas/metabolismo , Actinas/genética , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/genética , Ouriços-do-Mar/metabolismo
9.
Nat Commun ; 15(1): 8020, 2024 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-39271704

RESUMO

Most RNA-protein condensates are composed of heterogeneous immiscible phases. However, how this multiphase organization contributes to their biological functions remains largely unexplored. Drosophila germ granules, a class of RNA-protein condensates, are the site of mRNA storage and translational activation. Here, using super-resolution microscopy and single-molecule imaging approaches, we show that germ granules have a biphasic organization and that translation occurs in the outer phase and at the surface of the granules. The localization, directionality, and compaction of mRNAs within the granule depend on their translation status, translated mRNAs being enriched in the outer phase with their 5'end oriented towards the surface. Translation is strongly reduced when germ granule biphasic organization is lost. These findings reveal the intimate links between the architecture of RNA-protein condensates and the organization of their different functions, highlighting the functional compartmentalization of these condensates.


Assuntos
Grânulos Citoplasmáticos , Proteínas de Drosophila , Drosophila melanogaster , Biossíntese de Proteínas , RNA Mensageiro , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Grânulos Citoplasmáticos/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Células Germinativas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Imagem Individual de Molécula , Drosophila/metabolismo , Drosophila/genética , Condensados Biomoleculares/metabolismo
10.
BMC Biol ; 22(1): 206, 2024 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-39272107

RESUMO

BACKGROUND: Diapause, a pivotal phase in the insect life cycle, enables survival during harsh environmental conditions. Unraveling the gene expression profiles of the diapause process helps uncover the molecular mechanisms that underlying diapause, which is crucial for understanding physiological adaptations. In this study, we utilize RNA-seq and Ribo-seq data to examine differentially expressed genes (DEGs) and translational efficiency during diapause of Asian corn borer (Ostrinia furnacalis, ACB). RESULTS: Our results unveil genes classified as "forwarded", "exclusive", "intensified", or "buffered" during diapause, shedding light on their transcription and translation regulation patterns. Furthermore, we explore the landscape of lncRNAs (long non-coding RNAs) during diapause and identify differentially expressed lncRNAs, suggesting their roles in diapause regulation. Comparative analysis of different types of diapause in insects uncovers shared and unique KEGG pathways. While shared pathways highlight energy balance, exclusive pathways in the ACB larvae indicate insect-specific adaptations related to nutrient utilization and stress response. Interestingly, our study also reveals dynamic changes in the HSP70 gene family and proteasome pathway during diapause. Manipulating HSP protein levels and proteasome pathway by HSP activator or inhibitor and proteasome inhibitor affects diapause, indicating their vital role in the process. CONCLUSIONS: In summary, these findings enhance our knowledge of how insects navigate challenging conditions through intricate molecular mechanisms.


Assuntos
Diapausa de Inseto , Mariposas , Animais , Mariposas/fisiologia , Mariposas/genética , Diapausa de Inseto/fisiologia , Diapausa de Inseto/genética , Transcriptoma , Biossíntese de Proteínas , Larva/crescimento & desenvolvimento , Larva/fisiologia , Larva/genética , Diapausa/genética , Diapausa/fisiologia , Genoma de Inseto , Transcrição Gênica
11.
Int J Mol Sci ; 25(17)2024 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-39273490

RESUMO

Until now, research has not taken into consideration the physicochemical purine-pyrimidine symmetries of the genetic code in the transcription and translation processes of proteinogenesis. Our Supersymmetry Genetic Code table, developed in 2022, is common and unique for all RNA and DNA living species. Its basic structure is a purine-pyrimidine symmetry net with double mirror symmetry. Accordingly, the symmetry of the genetic code directly shows its organisation based on the principle of nucleotide Watson-Crick and codon-anticodon pairing. The maximal purine-pyrimidine symmetries of codons show that each codon has a strictly defined and unchangeable position within the genetic code. We discovered that the physicochemical symmetries of the genetic code play a fundamental role in recognising and differentiating codons from mRNA and the anticodon tRNA and aminoacyl-tRNA synthetases in the transcription and translation processes. These symmetries also support the wobble hypothesis with non-Watson-Crick pairing interactions between the translation process from mRNA to tRNA. The Supersymmetry Genetic Code table shows a specific arrangement of the second base of codons, according to which it is possible that an anticodon from tRNA recognises whether a codon from mRNA belongs to an amino acid with two or four codons, which is very important in the purposeful use of the wobble pairing process. Therefore, we show that canonical and wobble pairings essentially do not lead to misreading and errors during translation, and we point out the role of physicochemical purine-pyrimidine symmetries in decreasing disorder according to error minimisation and preserving the integrity of biological processes during proteinogenesis.


Assuntos
Códon , DNA , Código Genético , Biossíntese de Proteínas , Purinas , Transcrição Gênica , Purinas/metabolismo , DNA/genética , DNA/metabolismo , DNA/química , Códon/genética , Pirimidinas/química , Pirimidinas/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Anticódon/genética
12.
PLoS Pathog ; 20(9): e1012480, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39226332

RESUMO

Norovirus infection is characterised by a rapid onset of disease and the development of debilitating symptoms including projectile vomiting and diffuse diarrhoea. Vaccines and antivirals are sorely lacking and developments in these areas are hampered by the lack of an adequate cell culture system to investigate human norovirus replication and pathogenesis. Herein, we describe how the model norovirus, Mouse norovirus (MNV), produces a viral protein, NS3, with the functional capacity to attenuate host protein translation which invokes the activation of cell death via apoptosis. We show that this function of NS3 is conserved between human and mouse viruses and map the protein domain attributable to this function. Our study highlights a critical viral protein that mediates crucial activities during replication, potentially identifying NS3 as a worthy target for antiviral drug development.


Assuntos
Infecções por Caliciviridae , Macrófagos , Norovirus , Norovirus/fisiologia , Animais , Camundongos , Infecções por Caliciviridae/virologia , Macrófagos/virologia , Macrófagos/metabolismo , Humanos , Biossíntese de Proteínas , Replicação Viral/fisiologia , Morte Celular/fisiologia , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genética , Apoptose
14.
Life Sci Alliance ; 7(11)2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-39256052

RESUMO

Eukaryotic gene expression is regulated at the transcriptional and post-transcriptional levels, with disruption of regulation contributing significantly to human diseases. The 5' m7G mRNA cap is a central node in post-transcriptional regulation, participating in both mRNA stabilization and translation efficiency. In mammals, DCP1a and DCP1b are paralogous cofactor proteins of the mRNA cap hydrolase DCP2. As lower eukaryotes have a single DCP1 cofactor, the functional advantages gained by this evolutionary divergence remain unclear. We report the first functional dissection of DCP1a and DCP1b, demonstrating that they are non-redundant cofactors of DCP2 with unique roles in decapping complex integrity and specificity. DCP1a is essential for decapping complex assembly and interactions between the decapping complex and mRNA cap-binding proteins. DCP1b is essential for decapping complex interactions with protein degradation and translational machinery. DCP1a and DCP1b impact the turnover of distinct mRNAs. The observation that different ontological groups of mRNA molecules are regulated by DCP1a and DCP1b, along with their non-redundant roles in decapping complex integrity, provides the first evidence that these paralogs have qualitatively distinct functions.


Assuntos
Endorribonucleases , Capuzes de RNA , Estabilidade de RNA , RNA Mensageiro , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Endorribonucleases/metabolismo , Endorribonucleases/genética , Capuzes de RNA/metabolismo , Capuzes de RNA/genética , Estabilidade de RNA/genética , Proteínas de Ligação ao Cap de RNA/metabolismo , Proteínas de Ligação ao Cap de RNA/genética , Células HEK293 , Biossíntese de Proteínas , Ligação Proteica , Regulação da Expressão Gênica , Transativadores
15.
Curr Opin Chem Biol ; 82: 102523, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39226865

RESUMO

Localized protein translation occurs through trafficking of mRNAs and protein translation machineries to different compartments of the cell, leading to rapid on-site synthesis of proteins in response to signaling cues. The spatiotemporally precise nature of the local translation process necessitates continual developments of technologies reviewed herein to visualize and map biomolecular components and the translation process with better spatial and temporal resolution and with fewer artifacts. We also discuss approaches to control local translation, which can serve as a design paradigm for subcellular genetic devices for eukaryotic synthetic biology.


Assuntos
Biossíntese de Proteínas , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Imagem Molecular/métodos , Animais , Biologia Sintética/métodos , Proteínas/metabolismo
16.
Commun Biol ; 7(1): 1083, 2024 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-39232119

RESUMO

Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation (REI) at short upstream open reading frames (uORFs) harboring penultimate codons that confer heightened dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited REI at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on REI at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) REI. We found that the Tma proteins generally impede REI at native uORF4 and its variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on REI at native uORF1 and equipping it with Tma-hyperdependent penultimate codons generally did not confer Tma-dependent REI; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the REI potential of the uORF and penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica , Fases de Leitura Aberta , RNA Mensageiro , Subunidades Ribossômicas Menores de Eucariotos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/genética , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas
17.
RNA Biol ; 21(1): 8-18, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-39233564

RESUMO

In eukaryotes, the ribosomal small subunit (40S) is composed of 18S rRNA and 33 ribosomal proteins. 18S rRNA has a special secondary structure and is an indispensable part of the translation process. Herein, a special sequence located in mammalian 18S rRNA named Poly(G)7box, which is composed of seven guanines, was found. Poly(G)7 can form a special and stable secondary structure by binding to the translation elongation factor subunit eEF1D and the ribosomal protein RPL32. Poly(G)7box was transfected into cells, and the translation efficiency of cells was inhibited. We believe that Poly(G)7box is an important translation-related functional element located on mammalian 18S rRNA, meanwhile the Poly(G)7 located on mRNA 5' and 3' box does not affect mRNA translation.


Assuntos
Biossíntese de Proteínas , RNA Ribossômico 18S , RNA Ribossômico 18S/metabolismo , RNA Ribossômico 18S/genética , Humanos , Animais , Conformação de Ácido Nucleico , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequência de Bases , Guanina/metabolismo , Mamíferos/genética
18.
Nutrients ; 16(17)2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39275186

RESUMO

As older adults tend to reduce their intake of animal-source proteins, plant-source proteins may offer valuable resources for better protein intake. The aim of this study was to assess whether the pea proteins can be used to achieve blood amino acid levels that stimulate muscle protein synthesis. We measured variations in plasma amino acid concentrations in young and older adults given pea (NUTRALYS® S85 Plus) or whey proteins either alone or in a standardized meal. The effect of amino acid concentrations on protein synthesis in C2C12 myotubes was determined. In terms of results, plasma amino acid concentrations reflected the difference between the amino acid contents of whey and pea proteins. Blood leucine showed a greater increase of 91 to 130% with whey protein compared to pea protein, while the opposite was observed for arginine (A greater increase of 147 to 210% with pea compared to whey). Culture media prepared with plasmas from the human study induced age-dependent but not protein-type-dependent changes in myotube protein synthesis. In conclusion, pea and whey proteins have the same qualities in terms of their properties to maintain muscle protein synthesis. Pea proteins can be recommended for older people who do not consume enough animal-source proteins.


Assuntos
Aminoácidos , Fibras Musculares Esqueléticas , Proteínas de Ervilha , Proteínas do Soro do Leite , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Humanos , Masculino , Animais , Idoso , Aminoácidos/sangue , Camundongos , Feminino , Adulto , Adulto Jovem , Biossíntese de Proteínas/efeitos dos fármacos , Linhagem Celular , Proteínas Musculares/biossíntese , Proteínas Musculares/metabolismo , Pisum sativum/química
19.
Cell Metab ; 36(9): 1945-1962, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-39232280

RESUMO

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.


Assuntos
Biossíntese de Proteínas , RNA Mensageiro , Humanos , Animais , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Redes e Vias Metabólicas , Processamento Pós-Transcricional do RNA
20.
RNA Biol ; 21(1): 11-22, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-39190491

RESUMO

Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability and is caused by mutations in the gene encoding the Fragile X messenger ribonucleoprotein (FMRP). FMRP is an evolutionarily conserved and neuronally enriched RNA-binding protein (RBP) with functions in RNA editing, RNA transport, and protein translation. Specific target RNAs play critical roles in neurodevelopment, including the regulation of neurite morphogenesis, synaptic plasticity, and cognitive function. The different biological functions of FMRP are modulated by its cooperative interaction with distinct sets of neuronal RNA and protein-binding partners. Here, we focus on interactions between FMRP and components of the microRNA (miRNA) pathway. Using the Drosophila S2 cell model system, we show that the Drosophila ortholog of FMRP (dFMRP) can repress translation when directly tethered to a reporter mRNA. This repression requires the activity of AGO1, GW182, and MOV10/Armitage, conserved proteins associated with the miRNA-containing RNA-induced silencing complex (miRISC). Additionally, we find that untagged dFMRP can interact with a short stem-loop sequence in the translational reporter, a prerequisite for repression by exogenous miR-958. Finally, we demonstrate that dFmr1 interacts genetically with GW182 to control neurite morphogenesis. These data suggest that dFMRP may recruit the miRISC to nearby miRNA binding sites and repress translation via its cooperative interactions with evolutionarily conserved components of the miRNA pathway.


Assuntos
Proteínas de Drosophila , Proteína do X Frágil da Deficiência Intelectual , MicroRNAs , Neuritos , Biossíntese de Proteínas , Animais , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Neuritos/metabolismo , Morfogênese/genética , Complexo de Inativação Induzido por RNA/metabolismo , Complexo de Inativação Induzido por RNA/genética , Drosophila/metabolismo , Drosophila/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Ligação Proteica
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