RESUMEN
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.
Asunto(s)
Técnicas de Silenciamiento del Gen , Proteínas de Unión al ARN , Línea Celular , Proteínas de Unión al ARN/genética , Plásmidos/genética , Ubiquitina-Proteína LigasasRESUMEN
Members of the arginine-serine-rich protein family (SR proteins) are multifunctional RNA-binding proteins that have emerged as key determinants for mRNP formation, identity and fate. They bind to pre-mRNAs early during transcription in the nucleus and accompany bound transcripts until they are translated or degraded in the cytoplasm. SR proteins are mostly known for their essential roles in constitutive splicing and as regulators of alternative splicing. However, many additional activities of individual SR proteins, beyond splicing, have been reported in recent years. We will summarize the different functions of SR proteins and discuss how multifunctionality can be achieved. We will also highlight the difficulties of studying highly versatile SR proteins and propose approaches to disentangle their activities, which is transferrable to other multifunctional RBPs.
Asunto(s)
Precursores del ARN , Empalme del ARN , Empalme Alternativo , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Serina/genéticaRESUMEN
Here we present a research of bacterial adhesion to two most often used materials in modern kitchens, namely food grade ceramics and Teflon. To test the bacterial adhesion on kitchen worktops Escherichia coli, Pseudomonas aeruginosa and Campylobacter jejuni were used as the most common foodborne contaminants. Contact angle, roughness and streaming potential measurements were used for surface characterization. Crystal violet staining and scanning electron microscopy were applied for bacterial adhesion analysis. We showed that the adhesion of tested bacteria strains was lower on the Teflon surface compared to the ceramics. The hydrophobicity of the surface substantially contributed to the bacterial adhesion rate. On the other hand, the surface roughness and charge did not play a crucial role in the adhesion process.