A primary hallmark of pathological cardiac hypertrophy is excess protein synthesis due to enhanced translational activity. However, regulatory mechanisms at the translational level under cardiac stress remain poorly understood. Here we examined the translational regulations in a mouse cardiac hypertrophy model induced by transaortic constriction (TAC) and explored the conservative networks versus the translatome pattern in human dilated cardiomyopathy (DCM). The results showed that the heart weight to body weight ratio was significantly elevated, and the ejection fraction and fractional shortening significantly decreased 8 weeks after TAC. Puromycin incorporation assay showed that TAC significantly increased protein synthesis rate in the left ventricle. RNA-seq revealed 1,632 differentially expressed genes showing functional enrichment in pathways including extracellular matrix remodeling, metabolic processes, and signaling cascades associated with pathological cardiomyocyte growth. When combined with ribosome profiling analysis, we revealed that translation efficiency (TE) of 1,495 genes was enhanced, while the TE of 933 genes was inhibited following TAC. In DCM patients, 1,354 genes were upregulated versus 1,213 genes were downregulated at the translation level. Although the majority of the genes were not shared between mouse and human, we identified 93 genes, including Nos3, Kcnj8, Adcy4, Itpr1, Fasn, Scd1, etc., with highly conserved translational regulations. These genes were remarkably associated with myocardial function, signal transduction, and energy metabolism, particularly related to cGMP-PKG signaling and fatty acid metabolism. Motif analysis revealed enriched regulatory elements in the 5' untranslated regions (5'UTRs) of transcripts with differential TE, which exhibited strong cross-species sequence conservation. Our study revealed novel regulatory mechanisms at the translational level in cardiac hypertrophy and identified conserved translation-sensitive targets with potential applications to treat cardiac hypertrophy and heart failure in the clinic.
Animals ; Humans ; Cardiomegaly/physiopathology* ; Ribosomes/physiology* ; Protein Biosynthesis/physiology* ; Mice ; Cardiomyopathy, Dilated/genetics* ; Ribosome Profiling

Loss of protein homeostasis is a hallmark of cellular senescence, and ribosome pausing plays a crucial role in the collapse of proteostasis. However, our understanding of ribosome pausing in senescent cells remains limited. In this study, we utilized ribosome profiling and G-quadruplex RNA immunoprecipitation sequencing techniques to explore the impact of RNA G-quadruplex (rG4) on the translation efficiency in senescent cells. Our results revealed a reduction in the translation efficiency of rG4-rich genes in senescent cells and demonstrated that rG4 structures within coding sequence can impede translation both in vivo and in vitro. Moreover, we observed a significant increase in the abundance of rG4 structures in senescent cells, and the stabilization of the rG4 structures further exacerbated cellular senescence. Mechanistically, the RNA helicase DHX9 functions as a key regulator of rG4 abundance, and its reduced expression in senescent cells contributing to increased ribosome pausing. Additionally, we also observed an increased abundance of rG4, an imbalance in protein homeostasis, and reduced DHX9 expression in aged mice. In summary, our findings reveal a novel biological role for rG4 and DHX9 in the regulation of translation and proteostasis, which may have implications for delaying cellular senescence and the aging process.
G-Quadruplexes ; Cellular Senescence ; Ribosomes/genetics* ; Humans ; Animals ; Mice ; DEAD-box RNA Helicases/genetics* ; Protein Biosynthesis ; RNA/chemistry* ; Neoplasm Proteins

Nucleolar complex associated 4 homolog (NOC4L) is a key factor in ribosome biogenesis, and this study aims to investigate its roles in activated T cells from the perspective of translation regulation. Firstly, flow cytometry was employed to determine the expression levels of NOC4L in the CD4⁺ T cells under different conditions in the transgenic reporter mice expressing Noc4l^mCherry. Subsequently, the expression of NOC4L along with cell proliferation was examined under Th1 and Th17 polarization conditions. Finally, in vitro experiments were conducted to identify the proteins interacting with NOC4L during the activation of Th1 and Th17 cells, on the basis of which the potential mechanisms of NOC4L were explored. The results showed that the expression level of NOC4L increased in activated CD4⁺ T cells, and the expression of NOC4L was closely associated with the proliferation and division of activated T cells. The in vitro experiments revealed interactions between NOC4L and proteins involved in ribosome assembly and cell proliferation during T cell activation. These findings lay a foundation for probing into the post-transcriptional regulation in helper T cells and hold profound significance for understanding the activation and regulatory mechanisms of T cells.
Animals ; Mice ; Lymphocyte Activation ; Cell Proliferation ; Mice, Transgenic ; Nuclear Proteins/genetics* ; Th1 Cells/immunology* ; Th17 Cells/metabolism* ; CD4-Positive T-Lymphocytes/immunology* ; Ribosomes/metabolism*

Ribosomal engineering is a technique that can improve the biosynthesis of secondary metabolites in the antibiotics-resistant mutants by attacking the bacterial RNA polymerase or ribosome units using the corresponding antibiotics. Ribosomal engineering can be used to discover and increase the production of valuable bioactive secondary metabolites from almost all actinomycetes strains regardless of their genetic accessibility. As a consequence, ribosomal engineering has been widely applied to genome mining and production optimization of secondary metabolites in actinomycetes. To date, more than a dozen of new molecules were discovered and production of approximately 30 secondary metabolites were enhanced using actinomycetes mutant strains generated by ribosomal engineering. This review summarized the mechanism, development, and protocol of ribosomal engineering, highlighting the application of ribosomal engineering in actinomycetes, with the aim to facilitate future development of ribosomal engineering and discovery of actinomycetes secondary metabolites.
Actinobacteria/metabolism* ; Actinomyces/genetics* ; Anti-Bacterial Agents/metabolism* ; Multigene Family ; Ribosomes/genetics*

In recent years, two novel proteins in the ribosomes of mycobacteria have been discovered by cryo-electron microscopy. The protein bS22 is located near the decoding center of the 30S subunit, and the protein bL37 is located near the peptidyl transferase center of the 50S subunit. Since these two proteins bind to conserved regions of the ribosome targeted by antibiotics, it is speculated that they might affect the binding of related drugs to these targets. Therefore, we knocked out the genes encoding these two proteins in wild-type Mycolicibacterium smegmatis mc²155 through homologous recombination, and then determined the growth curves of these mutants and their sensitivity to related antibiotics. The results showed that compared with the wild-type strain, the growth rate of these two mutants did not change significantly. However, mutant ΔbS22 showed increased sensitivity to capreomycin, kanamycin, amikacin, streptomycin, gentamicin, paromomycin, and hygromycin B, while mutant ΔbL37 showed increased sensitivity to linezolid. These changes in antibiotics sensitivity were restored by gene complementation. This study hints at the possibility of using ribosomal proteins bS22 and bL37 as targets for drug design.
Anti-Bacterial Agents/pharmacology* ; Cryoelectron Microscopy ; Mycobacterium/genetics* ; Ribosomal Proteins/genetics* ; Ribosomes/metabolism*

Carrimycin (CAM) is a new antibiotics with isovalerylspiramycins (ISP) as its major components. It is produced by Streptomyces spiramyceticus integrated with a heterogenous 4″-O-isovaleryltransferase gene (ist). However, the present CAM producing strain carries two resistant gene markers, which makes it difficult for further genetic manipulation. In addition, isovalerylation of spiramycin (SP) could be of low efficiency as the ist gene is located far from the SP biosynthesis gene cluster. In this study, ist and its positive regulatory gene acyB2 were inserted into the downstream of orf54 gene neighboring to SP biosynthetic gene cluster in Streptomyces spiramyceticus 1941 by using the CRISPR-Cas9 technique. Two new markerless CAM producing strains, 54IA-1 and 54IA-2, were obtained from the homologous recombination and plasmid drop-out. Interestingly, the yield of ISP in strain 54IA-2 was much higher than that in strain 54IA-1. Quantitative real-time PCR assay showed that the ist, acyB2 and some genes associated with SP biosynthesis exhibited higher expression levels in strain 54IA-2. Subsequently, strain 54IA-2 was subjected to rifampicin (RFP) resistance selection for obtaining high-yield CAM mutants by ribosome engineering. The yield of ISP in mutants resistant to 40 μg/mL RFP increased significantly, with the highest up to 842.9 μg/mL, which was about 6 times higher than that of strain 54IA-2. Analysis of the sequences of the rpoB gene of these 7 mutants revealed that the serine at position 576 was mutated to alanine existed in each sequenced mutant. Among the mutants carrying other missense mutations, strain RFP40-6-8 which carries a mutation of glutamine (424) to leucine showed the highest yield of ISP. In conclusion, two markerless novel CAM producing strains, 54IA-1 and 54IA-2, were successfully developed by using CRISPR-Cas9 technique. Furthermore, a novel CAM high-yielding strain RFP40-6-8 was obtained through ribosome engineering. This study thus demonstrated a useful combinatory approach for improving the production of CAM.
CRISPR-Cas Systems/genetics* ; Genetic Engineering ; Ribosomes ; Spiramycin ; Streptomyces/genetics*

Cryoelectron Microscopy ; Mycobacterium smegmatis ; genetics ; ultrastructure ; RNA, Ribosomal ; ultrastructure ; Ribosomal Proteins ; genetics ; isolation & purification ; Ribosomes ; genetics ; ultrastructure

Under different red (R):blue (B) photon flux ratios, the growth performance of rapeseed (Brassica napus L.) is significantly different. Rapeseed under high R ratios shows shade response, while under high B ratios it shows sun-type morphology. Rapeseed under monochromatic red or blue light is seriously stressed. Transcriptomic and proteomic methods were used to analyze the metabolic pathway change of rapeseed (cv. "Zhongshuang 11") leaves under different R:B photon flux ratios (including 100R:0B%, 75R:25B%, 25R:75B%, and 0R:100B%), based on digital gene expression (DGE) and two-dimensional gel electrophoresis (2-DE). For DGE analysis, 2054 differentially expressed transcripts (|log₂(fold change)|≥1, q<0.005) were detected among the treatments. High R ratios (100R:0B% and 75R:25B%) enhanced the expression of cellular structural components, mainly the cell wall and cell membrane. These components participated in plant epidermis development and anatomical structure morphogenesis. This might be related to the shade response induced by red light. High B ratios (25R:75B% and 0R:100B%) promoted the expression of chloroplast-related components, which might be involved in the formation of sun-type chloroplast induced by blue light. For 2-DE analysis, 37 protein spots showed more than a 2-fold difference in expression among the treatments. Monochromatic light (ML; 100R:0B% and 0R:100B%) stimulated accumulation of proteins associated with antioxidation, photosystem II (PSII), DNA and ribosome repairs, while compound light (CL; 75R:25B% and 25R:75B%) accelerated accumulation of proteins associated with carbohydrate, nucleic acid, amino acid, vitamin, and xanthophyll metabolisms. These findings can be useful in understanding the response mechanisms of rapeseed leaves to different R:B photon flux ratios.
Brassica napus/radiation effects* ; Brassica rapa/radiation effects* ; Carbon/chemistry* ; Chloroplasts/radiation effects* ; Computational Biology ; Electrophoresis, Gel, Two-Dimensional ; Gene Expression Regulation, Plant/radiation effects* ; Image Processing, Computer-Assisted ; Light ; Mass Spectrometry ; Metabolic Networks and Pathways ; Nitrogen/chemistry* ; Photons ; Photosystem II Protein Complex/genetics* ; Plant Leaves/radiation effects* ; Plant Proteins/genetics* ; Proteome ; Ribosomes ; Transcription, Genetic ; Transcriptome

Through introducing mutations into ribosomes by obtaining spontaneous drug resistance of microorganisms, ribosome engineering technology is an effective approach to develop mutant strains that overproduce secondary metabolites. In this study, ribosome engineering was used to improve the yield of butenyl-spinosyns produced by Saccharopolyspora pogona by screening streptomycin resistant mutants. The yields of butenyl-spinosyns were then analyzed and compared with the parent strain. Among the mutants, S13 displayed the greatest increase in the yield of butenyl-spinosyns, which was 1.79 fold higher than that in the parent strain. Further analysis of the metabolite profile of S13 by mass spectrometry lead to the discovery of Spinosyn α1, which was absent from the parent strain. DNA sequencing showed that there existed two point mutations in the conserved regions of rpsL gene which encodes ribosomal protein S12 in S13. The mutations occurred a C to A and a C to T transversion mutations occurred at nucleotide pair 314 and 320 respectively, which resulted in the mutations of Proline (105) to Gultamine and Alanine (107) to Valine. It also demonstrated that S13 exhibited genetic stability even after five passages.
Genetic Engineering ; Macrolides ; metabolism ; Point Mutation ; Ribosomal Proteins ; genetics ; Ribosomes ; metabolism ; Saccharopolyspora ; metabolism

OBJECTIVETo re-analyze the data published in order to explore plausible biological pathways that can be used to explain the anti-aging effect of curcumin.

METHODSMicroarray data generated from other study aiming to investigate effect of curcumin on extending lifespan of Drosophila melanogaster were further used for pathway prediction analysis. The differentially expressed genes were identified by using GeneSpring GX with a criterion of 3.0-fold change. Two Cytoscape plugins including BisoGenet and molecular complex detection (MCODE) were used to establish the protein-protein interaction (PPI) network based upon differential genes in order to detect highly connected regions. The function annotation clustering tool of Database for Annotation, Visualization and Integrated Discovery (DAVID) was used for pathway analysis.

RESULTSA total of 87 genes expressed differentially in D. melanogaster melanogaster treated with curcumin were identified, among which 50 were up-regulated significantly and 37 were remarkably down-regulated in D. melanogaster melanogaster treated with curcumin. Based upon these differential genes, PPI network was constructed with 1,082 nodes and 2,412 edges. Five highly connected regions in PPI networks were detected by MCODE algorithm, suggesting anti-aging effect of curcumin may be underlined through five different pathways including Notch signaling pathway, basal transcription factors, cell cycle regulation, ribosome, Wnt signaling pathway, and p53 pathway.

CONCLUSIONGenes and their associated pathways in D. melanogaster melanogaster treated with anti-aging agent curcumin were identified using PPI network and MCODE algorithm, suggesting that curcumin may be developed as an alternative therapeutic medicine for treating aging-associated diseases.

Aging ; drug effects ; genetics ; Animals ; Cell Cycle ; drug effects ; genetics ; Curcumin ; pharmacology ; Drosophila Proteins ; genetics ; metabolism ; Drosophila melanogaster ; drug effects ; genetics ; Gene Expression Regulation ; drug effects ; Gene Regulatory Networks ; drug effects ; Genes, Insect ; Protein Biosynthesis ; drug effects ; genetics ; Protein Interaction Maps ; drug effects ; genetics ; Receptors, Notch ; genetics ; metabolism ; Ribosomes ; drug effects ; metabolism ; Signal Transduction ; drug effects ; genetics ; Tumor Suppressor Protein p53 ; metabolism ; Wnt Signaling Pathway ; drug effects ; genetics