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(A) A combined proteomic and transcriptomic strategy was employed to search for tripartite MetaAggregates composed of RNA, RBP, and amyloids in ALS brains. Single-cell RNA-seq data from iPSC-derived motor neurons were analyzed to identify disease-associated ELAVL4 and biotinylated rG4-containing RNA sequences <t>(IGFBP2-rG4).</t> A synthetic photo-crosslinkable probe mimicking the tripartite MetaAggregate was generated using psoralen and diazirine chemistry, and used to pull-down binding proteins via NeutrAvidin beads. (B) In the workflow, the probe was incubated with brain lysates from three ALS patients or three healthy controls to capture the associated aggregative proteins. Protein identities were subsequently determined via LC-MS/MS. PCA incorporating amyloidogenicity-related scores was conducted to prioritize candidate proteins, followed by docking simulations with ELAVL4 to assess their structural stability and interaction interfaces.
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(A) A combined proteomic and transcriptomic strategy was employed to search for tripartite MetaAggregates composed of RNA, RBP, and amyloids in ALS brains. Single-cell RNA-seq data from iPSC-derived motor neurons were analyzed to identify disease-associated ELAVL4 and biotinylated rG4-containing RNA sequences <t>(IGFBP2-rG4).</t> A synthetic photo-crosslinkable probe mimicking the tripartite MetaAggregate was generated using psoralen and diazirine chemistry, and used to pull-down binding proteins via NeutrAvidin beads. (B) In the workflow, the probe was incubated with brain lysates from three ALS patients or three healthy controls to capture the associated aggregative proteins. Protein identities were subsequently determined via LC-MS/MS. PCA incorporating amyloidogenicity-related scores was conducted to prioritize candidate proteins, followed by docking simulations with ELAVL4 to assess their structural stability and interaction interfaces.
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(A) A combined proteomic and transcriptomic strategy was employed to search for tripartite MetaAggregates composed of RNA, RBP, and amyloids in ALS brains. Single-cell RNA-seq data from iPSC-derived motor neurons were analyzed to identify disease-associated ELAVL4 and biotinylated rG4-containing RNA sequences <t>(IGFBP2-rG4).</t> A synthetic photo-crosslinkable probe mimicking the tripartite MetaAggregate was generated using psoralen and diazirine chemistry, and used to pull-down binding proteins via NeutrAvidin beads. (B) In the workflow, the probe was incubated with brain lysates from three ALS patients or three healthy controls to capture the associated aggregative proteins. Protein identities were subsequently determined via LC-MS/MS. PCA incorporating amyloidogenicity-related scores was conducted to prioritize candidate proteins, followed by docking simulations with ELAVL4 to assess their structural stability and interaction interfaces.
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(A) A combined proteomic and transcriptomic strategy was employed to search for tripartite MetaAggregates composed of RNA, RBP, and amyloids in ALS brains. Single-cell RNA-seq data from iPSC-derived motor neurons were analyzed to identify disease-associated ELAVL4 and biotinylated rG4-containing RNA sequences (IGFBP2-rG4). A synthetic photo-crosslinkable probe mimicking the tripartite MetaAggregate was generated using psoralen and diazirine chemistry, and used to pull-down binding proteins via NeutrAvidin beads. (B) In the workflow, the probe was incubated with brain lysates from three ALS patients or three healthy controls to capture the associated aggregative proteins. Protein identities were subsequently determined via LC-MS/MS. PCA incorporating amyloidogenicity-related scores was conducted to prioritize candidate proteins, followed by docking simulations with ELAVL4 to assess their structural stability and interaction interfaces.

Journal: bioRxiv

Article Title: Proteomic and in silico dissection of MetaAggregates in amyotrophic lateral sclerosis brains

doi: 10.1101/2025.07.09.663594

Figure Lengend Snippet: (A) A combined proteomic and transcriptomic strategy was employed to search for tripartite MetaAggregates composed of RNA, RBP, and amyloids in ALS brains. Single-cell RNA-seq data from iPSC-derived motor neurons were analyzed to identify disease-associated ELAVL4 and biotinylated rG4-containing RNA sequences (IGFBP2-rG4). A synthetic photo-crosslinkable probe mimicking the tripartite MetaAggregate was generated using psoralen and diazirine chemistry, and used to pull-down binding proteins via NeutrAvidin beads. (B) In the workflow, the probe was incubated with brain lysates from three ALS patients or three healthy controls to capture the associated aggregative proteins. Protein identities were subsequently determined via LC-MS/MS. PCA incorporating amyloidogenicity-related scores was conducted to prioritize candidate proteins, followed by docking simulations with ELAVL4 to assess their structural stability and interaction interfaces.

Article Snippet: A biotinylated 35-nt RNA sequence from IGFBP2 in D-PBS(−) (GGGGGAGGAAGGGGGUU-GUGGUCGGGGAGCUGGGG; IGFBP2-rG4, FASMAC, Kanagawa, Japan) was added to the fraction containing His-ELAVL4.

Techniques: RNA Sequencing, Derivative Assay, Generated, Binding Assay, Incubation, Liquid Chromatography with Mass Spectroscopy

(A) The brain sections from the temporal tip were stained with an anti-ELAVL4 antibody to assess its neuronal localization. Cytoplasmic accumulation (arrows) of ELAVL4 was detected in all ALS brain sections. (B) CD spectra of IGFBP2-rG4 (5 μM) in the presence of 100 mM KCl (solid line) or LiCl (dotted line). The guanine-rich sequence of IGFBP2-rG4 is shown. A characteristic G4 spectrum was prominently observed in the presence of the G4-promoting agent KCl, but not with the control LiCl. (C) Western blot of the covalent complex between ELAVL4 and IGFBP2-rG4 via psoralen/diazirine-mediated photo-crosslinking. Complexes (solid arrow) as well as unreacted ELAVL4 (dashed arrow) were immunoblotted in the photo reaction mixture (UV: +) using an anti-ELAVL4 antibody.

Journal: bioRxiv

Article Title: Proteomic and in silico dissection of MetaAggregates in amyotrophic lateral sclerosis brains

doi: 10.1101/2025.07.09.663594

Figure Lengend Snippet: (A) The brain sections from the temporal tip were stained with an anti-ELAVL4 antibody to assess its neuronal localization. Cytoplasmic accumulation (arrows) of ELAVL4 was detected in all ALS brain sections. (B) CD spectra of IGFBP2-rG4 (5 μM) in the presence of 100 mM KCl (solid line) or LiCl (dotted line). The guanine-rich sequence of IGFBP2-rG4 is shown. A characteristic G4 spectrum was prominently observed in the presence of the G4-promoting agent KCl, but not with the control LiCl. (C) Western blot of the covalent complex between ELAVL4 and IGFBP2-rG4 via psoralen/diazirine-mediated photo-crosslinking. Complexes (solid arrow) as well as unreacted ELAVL4 (dashed arrow) were immunoblotted in the photo reaction mixture (UV: +) using an anti-ELAVL4 antibody.

Article Snippet: A biotinylated 35-nt RNA sequence from IGFBP2 in D-PBS(−) (GGGGGAGGAAGGGGGUU-GUGGUCGGGGAGCUGGGG; IGFBP2-rG4, FASMAC, Kanagawa, Japan) was added to the fraction containing His-ELAVL4.

Techniques: Staining, Circular Dichroism, Sequencing, Control, Western Blot