Selective repression of RNA polymerase II by microinjected phosvitin

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Nuclear protein phosphorylation regulates gene transcription.
• Casein kinase NII plays a role in phosphorylating nuclear proteins.
• RNA polymerase II activity is crucial for gene expression.

Purpose of the Study:

• To investigate if phosvitin competes with endogenous substrates for casein kinase NII.
• To determine the effect of phosvitin on nuclear protein phosphorylation and RNA polymerase II activity.
• To explore the relationship between GTP-dependent phosphorylation and transcription.

Main Methods:

• Microinjection of phosvitin into salivary gland cell nuclei.
• Analysis of 32P-incorporation from ATP and GTP into nuclear proteins.
• Measurement of hnRNA, Balbiani ring RNA, and 38 S RNA synthesis.
• Heparin sensitivity assays to identify casein kinase NII activity.

Main Results:

• Phosvitin is phosphorylated by endogenous nuclear kinases using both ATP and GTP.
• GTP-dependent phosphorylation of specific nuclear proteins (42 kDa and 115 kDa) was significantly reduced.
• RNA polymerase II-mediated transcription of hnRNA and Balbiani ring RNA decreased by 80-90%.
• RNA polymerase I-mediated transcription of 38 S RNA was unaffected.

Conclusions:

• Phosvitin competes with endogenous substrates for GTP-dependent phosphorylation by casein kinase NII.
• Inhibition of specific protein phosphorylation by phosvitin causally affects RNA polymerase II activity.
• GTP-dependent phosphorylation of nuclear proteins is essential for regulating RNA polymerase II-dependent transcription.