Polyamine-mediated phosphorylation of a nucleolar protein from Physarum polycephalum that stimulates rRNA synthesis

Abstract

An acidic nucleolar phosphoprotein with a subunit M, of 70,000 was purified as an a parent dimer of 139,000 from isolated nuclei of the slime mo sarum polycephalum. The protein was purified without the aid of strong dissociating agents after its selective phosphorylation in isolated nuclei by a polyamine-mediated reaction. Its amino acid composition resembled that of a nucleolar phosphoprotein from Novikoff hepatoma ascites cells. The phosphoprotein stimulated rRNA synthesis 5-fold by RNA polymerase I within a nucleolar, ribosomal deoxyribonucleoprotein complex isolated from nucleoli of P. polycephalum. It was also identified as a component of the complex. It bound with high affinity and specificity to the palindromic ribosomal DNA of 38 X 106 Mr from P. polycephalum, which contained two coding sequences for 5.8S, 19S, and 26S rRNA. It also bound to three fragments of ribosomal DNA of Mr 21.2 X 106, 17.1 X 10", and 8.1 X 10", prepared by cleavage with restriction endonucleases HindIII, Pst I, and BamHI, respectively. All of these fragments included the symmetry axis of the palindromic ribosomal DNA. The phosphoprotein that had been treated with alkaline phosphataseagarose to hydrolyze the phosphate groups did not stimulate transcription and did not bind to ribosomal DNA or to the restriction fragments indicated. We have thus isolated a specific phosphoprotein with the capacity to stimulate transcription of a specific set of genes in a eukaryote. These findings suggest that this phosphoprotein may specifically regulate functions of ribosomal DNA in a manner dependent on its degree of phosphorylation.The polyamines, putrescine, spermidine, and spermine, have been implicated in the control and promotion of cell growth processes (1, 2). One favored proposal has linked the polyamines to the metabolism (1), structure (3), and function (3, 4) of the nucleic acids. Specifically, much correlative evidence implicates their involvement in stimulating rRNA synthesis although evidence for a causal relationship has not been conclusive (for reviews see refs. 1, 2, and 5).Recently the polyamines were found to selectively stimulate the phosphorylation of two nonhistone acidic proteins in isolated nucleoli from the slime mold Physarum polycephalum (6). Numerous investigations support the general notion that genetic regulatory molecules of eukaryotic cells are to be found among the nonhistone chromosomal proteins (7). The two nucleolar phosphoproteins were of major interest for further investigation of putative regulation of rRNA synthesis because the genes that code for rRNA in P. polycephalum reside on a satellite DNA that has been extensively characterized. This MATERIALS AND METHODS Preparation of Nuclei and Nucleoli. Nuclei were isolated from 48-hr shake cultures of microplasmodia (6,11). Slime-free nucleoli and nuclei were isolated by the Percoll (Pharmacia) gradient method (10) from synchronous surface cultures 18-21 hr after provision of nutrient medium (8).Purification of the 70,000 Mr Phosphoprotein. The total ...

Interrelations between the maturation of a 100 kDa nucleolar protein and pre rRNA synthesis in CHO cells

“…In "in vivo" experiments, correLations have been estabLished between pre rRNA synthesis and the evolution of given parameters such as: aminoacid pool size (3,4), nucleotide pool size (5), turn over of labile stimulatory protein (6) in particular ornithine decarboxylase (7), and phosphorylation of specific proteins (6). The recent development of "in vitro" systems in which a defined rDNA fragment containing the "in vivo" start site can be transcribed by RNA polymerase I, should allow the characterization of factors involved in the regulation of pre rRNA synthesis (8,9).…”

Purification and properties of a cAMP‐independent nuclear protein kinase from Dictyostelium discoideum

“…A cyclic-nucleotideindependent, polyamine-sensitive protein kinase has been involved in the control of transcription of ribosomal RNA genes by means of phosphorylation of RNA polymerase I [6] and some transcriptional factors [7].…”

Opposing kinetic effects of an acidic nucleolar phosphoprotein from Physarum polycephalum on homologous and heterologous transcription systems

“…Moreover, 77% of the [ 3H] RNA synthesized by the rDNP complex that was stimulated by the phosphoprotein could be competed for by unlabeled 19 S and 26 S rRNA from nitro~e~ulose filters containing bound rDNA [3]. Fig.1.…”

“…The 70 000 Mr phosphoprotein was purified from P. polycephalum plasmodia and stored as in [3]. The [32P]phosphoprotein was prepared by initial phosphorylation in intact isolated nuclei in a polyaminedependent reaction [9] prior to isolation.…”