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A new double-stranded RNA-binding protein that interacts with PKR
1Department of Molecular Biology, Box 1820, Station B, Vanderbilt University, Nashville, TN 37235, USA.
Abstract:
We have identified a 74 kDa double-stranded (ds)RNA-binding protein that shares extensive homology with the mouse spermatid perinuclear RNA-binding (Spnr) protein. p74 contains two dsRNA-binding motifs (dsRBMs) that are essential for preferential binding to dsRNA. Previously, dsRNA-binding proteins were shown to undergo homo- and heterodimerization, raising the possibility that regulation of activity could be controlled by interactions between different family members. Homodimerization is required to activate the dsRNA-dependent protein kinase PKR, whereas hetero-dimerization between PKR and other dsRNA-binding proteins can inhibit kinase activity. We have found that p74 also interacts with PKR, both the wild-type enzyme and a catalytically defective mutant (K296R). While co-expression of p74 and wild-type PKR in the yeast Saccharomyces cerevisiae did not alter PKR activity, co-expression of p74 and the catalytically defective K296R mutant surprisingly resulted in abnormal morphology and cell death in transformants that maintained a high level of p74 expression. These transformants could be rescued by overexpression of the alpha-subunit of wild-type eukaryotic translation initiation factor 2 (eIF2alpha), one of the known substrates for PKR. We hypothesize that competing heterodimers between p74-K296R PKR and eIF2alpha-K296R PKR may control cell growth such that stabilization of the p74-K296R PKR heterodimer induces abnormal morphology and cell death.
Insights
A novel double-stranded RNA-binding protein, p74, interacts with protein kinase R (PKR). Its interaction with a defective PKR mutant (K296R) leads to cell death, suggesting a role in regulating PKR activity and cell growth.
Area of Science:
- Molecular Biology
- Cell Biology
- Virology
Background:
- Double-stranded RNA-binding proteins (dsRBPs) play crucial roles in cellular processes.
- Protein kinase R (PKR) is a key regulator of protein synthesis and cellular stress responses.
- Interactions between dsRBPs and PKR can modulate PKR activity.
Purpose of the Study:
- To identify and characterize a novel dsRBP, p74, and investigate its interaction with PKR.
- To elucidate the functional consequences of p74-PKR interactions on cellular processes.
- To explore the potential role of p74 in PKR-mediated signaling pathways.
Main Methods:
- Identification and characterization of a 74 kDa dsRNA-binding protein (p74) with homology to mouse Spnr.
- Co-expression of p74 with wild-type and mutant PKR (K296R) in Saccharomyces cerevisiae.
- Analysis of cell morphology, viability, and PKR activity in yeast transformants.
- Investigating the effect of eukaryotic translation initiation factor 2 alpha (eIF2alpha) overexpression on cell viability.
Main Results:
- p74 possesses two dsRNA-binding motifs (dsRBMs) essential for dsRNA binding.
- Co-expression of p74 with wild-type PKR did not affect PKR activity.
- Co-expression of p74 with PKR K296R mutant induced abnormal morphology and cell death, which was rescued by eIF2alpha overexpression.
Conclusions:
- p74 interacts with both wild-type and catalytically inactive PKR.
- The interaction between p74 and PKR K296R mutant can lead to cell death, suggesting a dominant-negative effect.
- A hypothesis is proposed where competing heterodimers involving p74-PKR K296R and eIF2alpha-PKR K296R regulate cell growth and survival.