Yuxiang Chen

All Publications

• RAPIDASH: Tag-free enrichment of ribosome-associated proteins reveals composition dynamics in embryonic tissue, cancer cells, and macrophages. Molecular cell Susanto, T. T., Hung, V., Levine, A. G., Chen, Y., Kerr, C. H., Yoo, Y., Oses-Prieto, J. A., Fromm, L., Zhang, Z., Lantz, T. C., Fujii, K., Wernig, M., Burlingame, A. L., Ruggero, D., Barna, M. 2024

  Abstract

  Ribosomes are emerging as direct regulators of gene expression, with ribosome-associated proteins (RAPs) allowing ribosomes to modulate translation. Nevertheless, a lack of technologies to enrich RAPs across sample types has prevented systematic analysis of RAP identities, dynamics, and functions. We have developed a label-free methodology called RAPIDASH to enrich ribosomes and RAPs from any sample. We applied RAPIDASH to mouse embryonic tissues and identified hundreds of potential RAPs, including Dhx30 and Llph, two forebrain RAPs important for neurodevelopment. We identified a critical role of LLPH in neural development linked to the translation of genes with long coding sequences. In addition, we showed that RAPIDASH can identify ribosome changes in cancer cells. Finally, we characterized ribosome composition remodeling during immune cell activation and observed extensive changes post-stimulation. RAPIDASH has therefore enabled the discovery of RAPs in multiple cell types, tissues, and stimuli and is adaptable to characterize ribosome remodeling in several contexts.

  View details for DOI 10.1016/j.molcel.2024.08.023

  View details for PubMedID 39260367

• Mechanical morphotype switching as an adaptive response in mycobacteria. Science advances Eskandarian, H. A., Chen, Y., Toniolo, C., Belardinelli, J. M., Palcekova, Z., Hom, L., Ashby, P. D., Fantner, G. E., Jackson, M., McKinney, J. D., Javid, B. 2024; 10 (1): eadh7957

  Abstract

  Invading microbes face a myriad of cidal mechanisms of phagocytes that inflict physical damage to microbial structures. How intracellular bacterial pathogens adapt to these stresses is not fully understood. Here, we report the discovery of a virulence mechanism by which changes to the mechanical stiffness of the mycobacterial cell surface confer refraction to killing during infection. Long-term time-lapse atomic force microscopy was used to reveal a process of "mechanical morphotype switching" in mycobacteria exposed to host intracellular stress. A "soft" mechanical morphotype switch enhances tolerance to intracellular macrophage stress, including cathelicidin. Both pharmacologic treatment, with bedaquiline, and a genetic mutant lacking uvrA modified the basal mechanical state of mycobacteria into a soft mechanical morphotype, enhancing survival in macrophages. Our study proposes microbial cell mechanical adaptation as a critical axis for surviving host-mediated stressors.

  View details for DOI 10.1126/sciadv.adh7957

  View details for PubMedID 38170768

• Selective translation by alternative bacterial ribosomes. Proceedings of the National Academy of Sciences of the United States of America Chen, Y. X., Xu, Z. Y., Ge, X., Sanyal, S., Lu, Z. J., Javid, B. 2020; 117 (32): 19487-19496

  Abstract

  Alternative ribosome subunit proteins are prevalent in the genomes of diverse bacterial species, but their functional significance is controversial. Attempts to study microbial ribosomal heterogeneity have mostly relied on comparing wild-type strains with mutants in which subunits have been deleted, but this approach does not allow direct comparison of alternate ribosome isoforms isolated from identical cellular contexts. Here, by simultaneously purifying canonical and alternative RpsR ribosomes from Mycobacterium smegmatis, we show that alternative ribosomes have distinct translational features compared with their canonical counterparts. Both alternative and canonical ribosomes actively take part in protein synthesis, although they translate a subset of genes with differential efficiency as measured by ribosome profiling. We also show that alternative ribosomes have a relative defect in initiation complex formation. Furthermore, a strain of M. smegmatis in which the alternative ribosome protein operon is deleted grows poorly in iron-depleted medium, uncovering a role for alternative ribosomes in iron homeostasis. Our work confirms the distinct and nonredundant contribution of alternative bacterial ribosomes for adaptation to hostile environments.

  View details for DOI 10.1073/pnas.2009607117

  View details for PubMedID 32723820

  View details for PubMedCentralID PMC7431078

• Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems. Journal of visualized experiments : JoVE Chen, Y. X., Pan, M., Chen, Y. M., Javid, B. 2019

  Abstract

  The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

  View details for DOI 10.3791/59453

  View details for PubMedID 31081825

• More than merely drug resistance. Nature microbiology Chen, Y. X., Javid, B. 2018; 3 (10): 1078-1079

  View details for DOI 10.1038/s41564-018-0250-3

  View details for PubMedID 30254331

• Kasugamycin potentiates rifampicin and limits emergence of resistance in Mycobacterium tuberculosis by specifically decreasing mycobacterial mistranslation. eLife Chaudhuri, S., Li, L., Zimmerman, M., Chen, Y., Chen, Y. X., Toosky, M. N., Gardner, M., Pan, M., Li, Y. Y., Kawaji, Q., Zhu, J. H., Su, H. W., Martinot, A. J., Rubin, E. J., Dartois, V. A., Javid, B. 2018; 7

  Abstract

  Most bacteria use an indirect pathway to generate aminoacylated glutamine and/or asparagine tRNAs. Clinical isolates of Mycobacterium tuberculosis with increased rates of error in gene translation (mistranslation) involving the indirect tRNA-aminoacylation pathway have increased tolerance to the first-line antibiotic rifampicin. Here, we identify that the aminoglycoside kasugamycin can specifically decrease mistranslation due to the indirect tRNA pathway. Kasugamycin but not the aminoglycoside streptomycin, can limit emergence of rifampicin resistance in vitro and increases mycobacterial susceptibility to rifampicin both in vitro and in a murine model of infection. Moreover, despite parenteral administration of kasugamycin being unable to achieve the in vitro minimum inhibitory concentration, kasugamycin alone was able to significantly restrict growth of Mycobacterium tuberculosis in mice. These data suggest that pharmacologically reducing mistranslation may be a novel mechanism for targeting bacterial adaptation.

  View details for DOI 10.7554/eLife.36782

  View details for PubMedID 30152756

  View details for PubMedCentralID PMC6160228

• The essential mycobacterial amidotransferase GatCAB is a modulator of specific translational fidelity. Nature microbiology Su, H. W., Zhu, J. H., Li, H., Cai, R. J., Ealand, C., Wang, X., Chen, Y. X., Kayani, M. U., Zhu, T. F., Moradigaravand, D., Huang, H., Kana, B. D., Javid, B. 2016; 1 (11): 16147

  Abstract

  Although regulation of translation fidelity is an essential process1-7, diverse organisms and organelles have differing requirements of translational accuracy8-15, and errors in gene translation serve an adaptive function under certain conditions16-20. Therefore, optimal levels of fidelity may vary according to context. Most bacteria utilize a two-step pathway for the specific synthesis of aminoacylated glutamine and/or asparagine tRNAs, involving the glutamine amidotransferase GatCAB21-25, but it had not been appreciated that GatCAB may play a role in modulating mistranslation rates. Here, by using a forward genetic screen, we show that the mycobacterial GatCAB enzyme complex mediates the translational fidelity of glutamine and asparagine codons. We identify mutations in gatA that cause partial loss of function in the holoenzyme, with a consequent increase in rates of mistranslation. By monitoring single-cell transcription dynamics, we demonstrate that reduced gatCAB expression leads to increased mistranslation rates, which result in enhanced rifampicin-specific phenotypic resistance. Consistent with this, strains with mutations in gatA from clinical isolates of Mycobacterium tuberculosis show increased mistranslation, with associated antibiotic tolerance, suggesting a role for mistranslation as an adaptive strategy in tuberculosis. Together, our findings demonstrate a potential role for the indirect tRNA aminoacylation pathway in regulating translational fidelity and adaptive mistranslation.

  View details for DOI 10.1038/nmicrobiol.2016.147

  View details for PubMedID 27564922