hGRAD – a versatile ʹone-fits-allʹ system for the acute depletion of RNA binding proteins in nuclear condensates
bioRxiv (Cold Spring Harbor Laboratory)(2023)
摘要
Nuclear RNA binding proteins (RBPs) are difficult to study because they often belong to large protein families and form extensive networks of auto- and cross- regulation. They are highly abundant and often localize to condensates with a slow turnover, requiring long depletion times or knockouts that cannot distinguish between direct and indirect or compensatory effects.
Here, we developed a system that is optimized for the rapid degradation of nuclear RBPs, called hGRAD. It comes as a ʹone-fits-allʹ plasmid, and integration into any cell line that expresses endogenously GFP-tagged proteins allows an inducible, rapid and complete knockdown. We show that the nuclear RBPs SRSF3, SRSF5, SRRM2 and NONO are completely cleared from nuclear speckles and paraspeckles within two hours. hGRAD works in various cell types, is more efficient than other methods and does not require the expression of exogenous ubiquitin ligases. Combining SRSF5 hGRAD degradation with Nascent-seq uncovered highly dynamic transient transcript changes, compensatory mechanisms and that SRSF5 promotes transcript stability.
### Competing Interest Statement
The authors have declared no competing interest.
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关键词
rna binding proteins,nuclear,one-fits-all
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