TRIM21 E3 ubiquitin ligase screen for stress granule regulation

PRIMARY OUTCOME

identification of TRIM21 as stress granule regulator

EXPECTED OUTCOMES

Quantitative predictions: (1) Screen: TRIM21 knockdown increases SG count 2-3 fold and SG size 1.5-2 fold vs. scrambled. Other E3s show <30% change. (2) TRIM21 localization: Pearson R=0.6-0.8 with G3BP1 in arsenite-treated cells (p<0.001). (3) TRIM21 OE: SG count reduced 60-70% vs. control vector. (4) Dose-response: SG inhibition by TRIM21 OE is strongest at low arsenite (0.1-0.3 mM, 80% reduction) but partially overcome at high stress (1 mM, 40% reduction). (5) ALS models: TRIM21 OE reduces pathological SG by 50-60% in ALS-FUS and ALS-TDP43 lines. (6) TRIM21-C16A mutant has no effect on SG (equivalent to empty vector control). (7) Time course: SG peak at 1h in control; with TRIM21 OE, SG form later (peak at 2-3h) and dissolve faster (90% gone by 4h vs. 50% in control).

SUCCESS CRITERIA

Primary: TRIM21 knockdown increases SG count >2-fold vs. scrambled siRNA (p<0.001, Welch t-test, n=3 experiments with 100 cells each). Secondary: (1) TRIM21 overexpression reduces SG count >50% (p<0.001). (2) Colocalization with G3BP1: Pearson R>0.5 (p<0.001 vs. randomized controls). (3) Catalytic mutant C16A shows no SG suppression (p>0.1 vs. empty vector). (4) Effect reproduces in ALS disease models (>40% SG reduction, p<0.01). (5) Consistency across independent siRNAs (3/3 show same direction, 2/3 reach significance). (6) Z-factor for screen assay >0.5 (validates robust assay window). Hit validation: Same effect with 2nd stressor (heat shock).

PROTOCOL

Screen design: Test 6 E3 ligases enriched in SG proteomics: TRIM21, TRIM32, STUB1, HUWE1, UBR4, MARCH5. Cells: U2OS stably expressing mCherry-G3BP1 (SG marker). Knockdown: siRNA pools (Dharmacon, 50 nM, 72h), verify >70% knockdown by qPCR and Western blot. Stress: 0.5 mM sodium arsenite (1h), or ALS patient-derived iPSC neurons (endogenous SG). Imaging: Automated confocal microscopy (Operetta CLS), 10 fields per well, 3 wells per condition. Quantify: SG count, size, intensity per cell. Hit threshold: >50% change vs. scrambled siRNA (p<0.01). TRIM21 validation: (1) Knockdown (3 independent siRNAs) and overexpression (lentivirus, FLAG-TRIM21). (2) Dose-response: arsenite 0-1 mM, time course 0-4h. (3) Disease model: ALS-FUS, ALS-TDP43, OPMD (PABPN1) patient lines. (4) Localization: Immunostain endogenous TRIM21 in arsenite-treated cells, measure colocalization with G3BP1 (Pearson coefficient). (5) Catalytic requirement: WT vs C16A mutant. Controls: (1) No stress (baseline SG). (2) Scrambled siRNA. (3) Positive control: G3BP1 knockdown (abolishes SG). (4) Heat shock (42°C, 1h) as alternative stressor.

LINKED HYPOTHESES

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[TRIM21 E3 ubiquitin ligase screen for stress granule regulation](http://scidex.ai/artifact/exp-b6c4a13e-3b7b-41fa-9e4d-eacfb11bb61f)

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http://scidex.ai/artifact/exp-b6c4a13e-3b7b-41fa-9e4d-eacfb11bb61f

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