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La interacts directly with miR-122 through LaM and RRM1. (A) Sequences for miR-122 and miR-190. (B) EMSAs with purified La 1–408 and 5′ fluorescently labeled miR-122 or miR-190. La 1–408 was titrated from 244 pM to 2 µM. The migration of each miRNA was detected by in-gel fluorescence. The fraction bound was quantified as a function of the exhaustion of free miRNA. (C) Schematic illustrating the four functional domains of La. (D) EMSAs with 5′ fluorescently labeled miR-122 and purified La 1–334 , La 1–224 , or La 1–103 . The titration range of each La truncation is indicated above each gel. miR-122 migration was detected by in-gel fluorescence. The fraction bound was quantified as a function of the exhaustion of free miR-122. (E) Quantification of showing the calculated K d values. (F) EMSAs with 5′ fluorescently labeled miR-122 and purified La 104–408 , La 225–408 , or La 335–408 . Each La truncation was titrated from 244 pM to 2 µM. miR-122 migration was detected by in-gel fluorescence. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: p62 sorts Lupus La and selected microRNAs into breast cancer-derived exosomes

doi: 10.1083/jcb.202503087

Figure Lengend Snippet: La interacts directly with miR-122 through LaM and RRM1. (A) Sequences for miR-122 and miR-190. (B) EMSAs with purified La 1–408 and 5′ fluorescently labeled miR-122 or miR-190. La 1–408 was titrated from 244 pM to 2 µM. The migration of each miRNA was detected by in-gel fluorescence. The fraction bound was quantified as a function of the exhaustion of free miRNA. (C) Schematic illustrating the four functional domains of La. (D) EMSAs with 5′ fluorescently labeled miR-122 and purified La 1–334 , La 1–224 , or La 1–103 . The titration range of each La truncation is indicated above each gel. miR-122 migration was detected by in-gel fluorescence. The fraction bound was quantified as a function of the exhaustion of free miR-122. (E) Quantification of showing the calculated K d values. (F) EMSAs with 5′ fluorescently labeled miR-122 and purified La 104–408 , La 225–408 , or La 335–408 . Each La truncation was titrated from 244 pM to 2 µM. miR-122 migration was detected by in-gel fluorescence. Source data are available for this figure: .

Article Snippet: The TaqMan miRNA assay ID for hsa-miR-122-5p was 477855_mir (Life Technologies).

Techniques: Purification, Labeling, Migration, Fluorescence, Functional Assay, Titration

Competition EMSAs between truncated La proteins. (A) EMSA with 5′ fluorescently labeled miR-122, 2 nM La 1–334 , and increasing concentrations of La 335–408 . La 335–408 was titrated from 448 pM to 2 µM. miR-122 migration was detected using in-gel fluorescence. (B) EMSA with 5′ fluorescently labeled miR-122, 2 nM La 1–224 , and increasing concentrations of La 225–408 . La 225–408 was titrated from 448 pM to 2 µM. miR-122 migration was detected using in-gel fluorescence. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: p62 sorts Lupus La and selected microRNAs into breast cancer-derived exosomes

doi: 10.1083/jcb.202503087

Figure Lengend Snippet: Competition EMSAs between truncated La proteins. (A) EMSA with 5′ fluorescently labeled miR-122, 2 nM La 1–334 , and increasing concentrations of La 335–408 . La 335–408 was titrated from 448 pM to 2 µM. miR-122 migration was detected using in-gel fluorescence. (B) EMSA with 5′ fluorescently labeled miR-122, 2 nM La 1–224 , and increasing concentrations of La 225–408 . La 225–408 was titrated from 448 pM to 2 µM. miR-122 migration was detected using in-gel fluorescence. Source data are available for this figure: .

Article Snippet: The TaqMan miRNA assay ID for hsa-miR-122-5p was 477855_mir (Life Technologies).

Techniques: Labeling, Migration, Fluorescence

p62 is required for the secretion of miR-122. (A) RT-qPCR analysis of miR-122 from HSP fractions isolated from the conditioned medium of MDA-MB-231 cells expressing HA-CD63-mEGFP ECL1 , ZIM3-KRAB-dCas9, and either sgNT or sgSQSTM1. Statistical significance was performed using an unpaired two-tailed t test ( n = 3; ***P < 0.001).

Journal: The Journal of Cell Biology

Article Title: p62 sorts Lupus La and selected microRNAs into breast cancer-derived exosomes

doi: 10.1083/jcb.202503087

Figure Lengend Snippet: p62 is required for the secretion of miR-122. (A) RT-qPCR analysis of miR-122 from HSP fractions isolated from the conditioned medium of MDA-MB-231 cells expressing HA-CD63-mEGFP ECL1 , ZIM3-KRAB-dCas9, and either sgNT or sgSQSTM1. Statistical significance was performed using an unpaired two-tailed t test ( n = 3; ***P < 0.001).

Article Snippet: The TaqMan miRNA assay ID for hsa-miR-122-5p was 477855_mir (Life Technologies).

Techniques: Quantitative RT-PCR, Isolation, Expressing, Two Tailed Test

p62 mediates the secretion of miR-122 and other exosomal miRNAs. (A) Volcano plot showing miRNAs present in immunoisolated sgNT exosomes compared with sgSQSTM1 exosomes ( n = 3). (B) Volcano plot showing miRNAs present in HA-CD63-mEGFP ECL1 sgNT cells compared with sgSQSTM1 cells ( n = 3). (C) Changes in the intracellular and exosomal levels of miRNAs of interest from HA-CD63-mEGFP ECL1 sgNT and sgSQSTM1 cells and immunopurified exosomes ( n = 3). Mapped miRNA reads were normalized to the total number of miRNA reads in each sample.

Journal: The Journal of Cell Biology

Article Title: p62 sorts Lupus La and selected microRNAs into breast cancer-derived exosomes

doi: 10.1083/jcb.202503087

Figure Lengend Snippet: p62 mediates the secretion of miR-122 and other exosomal miRNAs. (A) Volcano plot showing miRNAs present in immunoisolated sgNT exosomes compared with sgSQSTM1 exosomes ( n = 3). (B) Volcano plot showing miRNAs present in HA-CD63-mEGFP ECL1 sgNT cells compared with sgSQSTM1 cells ( n = 3). (C) Changes in the intracellular and exosomal levels of miRNAs of interest from HA-CD63-mEGFP ECL1 sgNT and sgSQSTM1 cells and immunopurified exosomes ( n = 3). Mapped miRNA reads were normalized to the total number of miRNA reads in each sample.

Article Snippet: The TaqMan miRNA assay ID for hsa-miR-122-5p was 477855_mir (Life Technologies).

Techniques:

Schematic depicting the current model of p62-dependent exosome secretion. Upon ATG7-dependent conjugation of LC3-II to late endosomes, p62 localizes to late endosomes and recruits cytoplasmic RBPs that are bound to selected miRNAs (such as La bound to miR-122 via LaM and RRM1). The RNP complexes are then sequestered into ILVs and secreted within exosomes upon fusion of MVBs with the plasma membrane.

Journal: The Journal of Cell Biology

Article Title: p62 sorts Lupus La and selected microRNAs into breast cancer-derived exosomes

doi: 10.1083/jcb.202503087

Figure Lengend Snippet: Schematic depicting the current model of p62-dependent exosome secretion. Upon ATG7-dependent conjugation of LC3-II to late endosomes, p62 localizes to late endosomes and recruits cytoplasmic RBPs that are bound to selected miRNAs (such as La bound to miR-122 via LaM and RRM1). The RNP complexes are then sequestered into ILVs and secreted within exosomes upon fusion of MVBs with the plasma membrane.

Article Snippet: The TaqMan miRNA assay ID for hsa-miR-122-5p was 477855_mir (Life Technologies).

Techniques: Conjugation Assay, Clinical Proteomics, Membrane