hGRAD: A versatile "one-fits-all" system to acutely deplete RNA binding proteins from condensates.
J Cell Biol
; 223(2)2024 02 05.
Article
em En
| MEDLINE
| ID: mdl-38108808
ABSTRACT
Nuclear RNA binding proteins (RBPs) are difficult to study because they often belong to large protein families and form extensive networks of auto- and crossregulation. They are highly abundant and many 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 "one-fits-all" plasmid, and integration into any cell line with endogenously GFP-tagged proteins allows for 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 2 h. hGRAD works in various cell types, is more efficient than previous methods, and does not require the expression of exogenous ubiquitin ligases. Combining SRSF5 hGRAD degradation with Nascent-seq uncovered transient transcript changes, compensatory mechanisms, and an effect of SRSF5 on transcript stability.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Ligação a RNA
/
Técnicas de Silenciamento de Genes
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article