Selective repression of RNA polymerase II by Microinjected phosvitin

Abstract

We have used a microinjection technique to examine whether injected phosvitin, in its capacity as substrate for casein kinase NII, could compete out the endogenous phosphorylation of some nuclear phosphoproteins with regulatory potential and thereby interfere with the activity of RNA polymerase II. Phosphorylation, which utilizes ATP as phosphate donor, was separated from phosphorylation which uses GTP. Phosvitin introduced into nuclei of salivary gland cells becomes phosphorylated by the endogenous nuclear protein kinase(s) and incorporates phosphates from ATP as well as from GTP. The phosphorylation of nuclear proteins and phosvitin is heparin-sensitive, indicating that they are phosphorylated by casein kinase NII. Microinjected phosvitin does not seem to affect the incorporation of phosphate groups from ATP into nuclear proteins, but protein phosphorylation by GTP is influenced. Apart from a minor overall reduction of 32P-incorporation, the phosphorylation of a 42 kDa nuclear protein, a putative transcription stimulatory factor, and of a 115 kDa nuclear protein was competed out by 70%-80% compared with the control value obtained in the absence of phosvitin. Parallel analyses of DNA transcription in phosvitin-injected nuclei showed that the RNA polymerase II-mediated synthesis of hnRNA and Balbiani ring RNA was diminished by 80% and 90%, respectively. In contrast, the transcription of nucleolar pre-ribosomal 38 S RNA by RNA polymerase I remained unaffected. The inhibitory effect of injected phosvitin could be reversed by in vitro phosphorylation of phosvitin prior to injection, using isolated nuclei as source of protein kinase(s). Taken together, the results suggest a causal relationship between the modification of the GTP-dependent phosphorylation of specific non-histone proteins and the activity of RNA polymerase II.

Microinjection of anti-topoisomerase I immunoglobulin G into nuclei of Chironomus tentans salivary gland cells leads to blockage of transcription elongation.

“…It is presently unclear why low-activity genes are less vulnerable to treatment with anti-topoisomerase I IgG. One possibility is that the antibodies are less accessible to topoisomerase I molecules associated with only partially decondensed hnRNA (10), did not reveal any significant alteration in the morphology of polytene chromosomes and of the Balbiani rings. Likewise, no effect of the salt medium on DNA transcription was detected with or without control proteins.…”

“…New pipettes were used for each solution. The fixed positions of the cells within the gland enabled us to keep track of immune-and preimmune-IgG-injected cells (10). Anti-topoisomerase I IgG and preimmune IgG were solubilized in 20 mM KCI, including 28 mM NaCl and 20 mM Tris hydrochloride (pH 8.0), and injected into the nuclei.…”

Kinetics of inhibition by 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole on calf thymus casein kinase II

“…Unphosphorylated phosvitin was able to inhibit transcription after microinjection of Chironomus cells, whereas phosphorylated phosvitin was not. Presumably, all casein kinase II activity was shunted to this exogenous substrate and was thus unable to participate in the transcription process (Egyhazi & Pigon, 1986). Thus, although the phosphorylation targets for casein kinase within the transcriptional machinery have not yet been identified, the role of this enzyme in the DRB inhibition of specific transcription in vitro appears to be well established (Egyhazi & Pigon, 1986;Zandomeni et al, 1986).…”

Microinjection of Anti-Topoisomerase I Immunoglobulin G Into Nuclei of Chironomus Tentans Salivary Gland Cells Leads tO Blockage of Transcription Elongation

“…The appearance and transcription of the highly expanded giant Balbiani ring puffs are sensitive indicators of disturbances in chromosome function. Visual inspection of salivary gland nuclei after injection of salt medium with or without antibody, or with other control proteins like casein and actin (10), did not reveal any significant alteration in the morphology of polytene chromosomes and of the Balbiani rings. Likewise, no effect of the salt medium on DNA transcription was detected with or without control proteins.…”

“…New pipettes were used for each solution. The fixed positions of the cells within the gland enabled us to keep track of immuneand preimmune-IgG-injected cells (10). Anti-topoisomerase I IgG and preimmune IgG were solubilized in 20 mM KCI, including 28 mM NaCl and 20 mM Tris hydrochloride (pH 8.0), and injected into the nuclei.…”