Nucleolar targeting of proteins by the tandem array of basic amino acid stretches identified in the RNA polymerase I-associated factor PAF49

There is accumulating evidence to indicate that the regulation of subnuclear compartmentalization plays important roles in cellular processes. The RNA polymerase I-associated factor PAF49 has been shown to accumulate in the nucleolus in growing cells, but disperse into the nucleoplasm in growth-arrested cells. Serial deletion analysis revealed that amino acids 199-338 were necessary for the nucleolar localization of PAF49. Combinatorial point mutation analysis indicated that the individual basic amino acid stretches (BS) within the central (BS1-4) and the C-terminal (BS5 and 6) regions may cooperatively confer the nucleolar localization of PAF49. Addition of the basic stretches in tandem to a heterologous protein, such as the interferon regulatory factor-3, translocated the tagged protein into the nucleolus, even in the presence of an intrinsic nuclear export sequence. Thus, tandem array of the basic amino acid stretches identified here functions as a dominant nucleolar targeting sequence.     

Phosphorylation of RNA polymerase I-associated polypeptides of Tetrahymena pyriformis

In the ciliate Tetrahymena pyriformis phosphorylation of RNA polymerase I [EC 2.7.7.6] and of polymerase-associated polypeptides was investigated in growing and growth-arrested cultures which differ widely in their rates of rRNA synthesis. Several putative subunits of RNA polymerase I (of 180, 21.5, and 19.5 kDa) and a polymerase-associated polypeptide of 27 kDa were found to be phosphorylated, independent of the growth conditions. However, an additional enzyme-associated polypeptide of 26 kDa was intensively labeled with 32P only after arrestment of growth by starvation. The molar quantities of both phosphorylated, enzyme-associated polypeptides thereby did not differ in growing and growth-arrested cultures, and the specific 32P-labeling of cellular ATP remained nearly unchanged under the different culture conditions. These findings indicate a selective, reversible phosphorylation of the RNA polymerase I-associated 26 kDa polypeptide correlated with conditions of repressed rRNA synthesis induced by the starvation procedure. In vitro phosphorylation in macronuclei isolated from growing and growth-arrested cultures using [gamma-32P]ATP revealed essentially the same pattern of labeling of the enzyme-associated polypeptides of 27 and 26 kDa as it was found in vivo.     

RNA polymerase I-specific subunits promote polymerase clustering to enhance the rRNA gene transcription cycle

RNA polymerase I (Pol I) produces large ribosomal RNAs (rRNAs). In this study, we show that the Rpa49 and Rpa34 Pol I subunits, which do not have counterparts in Pol II and Pol III complexes, are functionally conserved using heterospecific complementation of the human and Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae. Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar assembly can be restored by decreasing ribosomal gene copy number from 190 to 25. Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a fourfold decrease in Pol I loading rate per gene and decreased contact between adjacent Pol I complexes. Therefore, the Rpa34 and Rpa49 Pol I-specific subunits are essential for nucleolar assembly and for the high polymerase loading rate associated with frequent contact between adjacent enzymes. Together our data suggest that localized rRNA production results in spatially constrained rRNA production, which is instrumental for nucleolar assembly.