Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome

Zheng, Fu; Yu, Chenxin; Zhou, Xinyue; Zou, Peng

doi:10.1101/2022.08.01.502286

Cited by 5 publications

(6 citation statements)

References 27 publications

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“…This inclusive labeling feature of TFPy aligns well with its main accumulation at IMM and the relatively larger diffusion radius of 1 O 2 compared to the distance between OMM and IMM. The 1 O 2 -driven mechanism is consistent with those of previously reported synthetic and genetic encoded PSs, 9,10,57 thus allowing for both RNA and protein proximity labeling (Figure S30). Consequently, we envision that such a small molecule-based approach for mitochondria-associated proximity labeling can offer a compressive evaluation of mitochondrial status in various cell lines of interest.…”

Section: ■ Conclusionsupporting

confidence: 87%

Mitochondria-Associated Transcriptome Profiling via Localizable Aggregation-Induced Emission Photosensitizers in Live Cells

Liang,

Han,

Zhuang

et al. 2024

ACS Chem. Biol.

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In recent decades, there has been increasing interest in studying mitochondria through transcriptomic research. Various exogenous fusion proteinbased proximity labeling methods have been reported that focus on the site of one particular protein/peptide and might also influence the corresponding localization or interactome. To enable unbiased and high spatial-resolution profiling of mitochondria-associated transcriptomes in live cells, a flexible RNA proximity labeling approach was developed using aggregation-induced emission (AIE) type photosensitizers (PSs) that possess great mitochondria-targeting capabilities. Their accumulation in an enclosed mitochondrial environment tends to enhance the fluorescence emission and reactive oxygen species generation. By comparing the in vitro optical properties, photosensitization processes, as well as the in cellulo mitochondrial specificity and RNA labeling performance of four AIE PSs, high-throughput sequencing analysis was conducted using TFPymediated RNA proximity labeling in live HeLa cells. This approach successfully captured a comprehensive list of transcripts, including mitochondria-encoded RNAs, as well as some nuclear-derived RNAs located at the outer mitochondrial membrane and interacting organelles. This small molecule-based proximity labeling method bypasses complex genetic manipulation and transfection steps, making it readily applicable for diverse research purposes.

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Section: ■ Conclusionsupporting

confidence: 87%

Mitochondria-Associated Transcriptome Profiling via Localizable Aggregation-Induced Emission Photosensitizers in Live Cells

Liang,

Han,

Zhuang

et al. 2024

ACS Chem. Biol.

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“…Shortly after this work was published as a preprint, two related studies appeared, describing a light-dependent PL method using the FMN-binding variant, miniSOG ( 51 , 52 ). Along with the similarities between the approaches, LITag stands out for its superior temporal resolution, as it accomplishes labeling in a few seconds compared to several minutes.…”

Section: Discussionmentioning

confidence: 99%

A genetically encoded photoproximity labeling approach for mapping protein territories

Hananya

Koren

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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Studying dynamic biological processes requires approaches compatible with the lifetimes of the biochemical transactions under investigation, which can be very short. We describe a genetically encoded system that allows protein neighborhoods to be mapped using visible light. Our approach involves fusing an engineered flavoprotein to a protein of interest. Brief excitation of the fusion protein leads to the labeling of nearby proteins with cell-permeable probes. Mechanistic studies reveal different labeling pathways are operational depending on the nature of the exogenous probe that is employed. When combined with quantitative proteomics, this photoproximity labeling system generates “snapshots” of protein territories with high temporal and spatial resolution. The intrinsic fluorescence of the fusion domain permits correlated imaging and proteomics analyses, a capability that is exploited in several contexts, including defining the protein clients of the major vault protein. The technology should be broadly useful in the biomedical area.

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“…Future study that combines proteomic and transcriptomic data in the same cell line with the same bait protein could help elaborate the relationship between m 6 A-mRNA enrichment and m 6 A-binding protein localization during various stages of SG assembly/disassembly. Such multipronged approach would require photoactivatable protein labeling with miniSOG, which has been recently demonstrated by us and other groups [41][42][43] .…”

Section: Discussionmentioning

confidence: 99%

Profiling stress-triggered RNA condensation with photocatalytic proximity labeling

Zou

Ren

Wei

et al. 2023

Preprint

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Stress granules (SGs) are highly dynamic cytoplasmic membrane-less organelles that assemble when cells are challenged by stress. At different stages of assembly, various RNA molecules are sorted into SGs where they play important roles in maintaining the structural stability of SGs and regulating gene expression. Despite recent efforts of fluorescence microscopy and biochemical fractionation analysis, there still lacks a complete description of the dynamic changes in the molecular inventory of SG components during different stages of assembly and disassembly. In this study, we applied a proximity-dependent RNA labeling method, CAP-seq, to comprehensively investigate the content of local transcriptome in SGs, in the context of live mammalian cells. CAP-seq captures 457 and 822 RNAs in arsenite- and sorbitol-induced SGs in HEK293T cells, respectively, revealing that SG enrichment is positively correlated with RNA length and AU content, but negatively correlated with translation efficiency. The high spatial specificity of CAP-seq dataset is validated by single-molecule FISH imaging. We further applied CAP-seq profile RNA components in microscopically invisible SG cores, both before stress and after recovery from stress, thus mapping the dynamic changes in SG-proximal transcriptome along the time course of granule assembly/disassembly processes. Our data portray a model of AU-rich and translationally repressed SG nanostructure that are memorized long after the removal of stress.

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Genetically encoded photocatalytic protein labeling enables spatially-resolved profiling of intracellular proteome

Cited by 5 publications

References 27 publications

Mitochondria-Associated Transcriptome Profiling via Localizable Aggregation-Induced Emission Photosensitizers in Live Cells

Mitochondria-Associated Transcriptome Profiling via Localizable Aggregation-Induced Emission Photosensitizers in Live Cells

A genetically encoded photoproximity labeling approach for mapping protein territories

Profiling stress-triggered RNA condensation with photocatalytic proximity labeling

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