We report the identification of a transcription elongation factor from HeLa cell nuclear extracts that causes pausing of RNA polymerase II (Pol II) in conjunction with the transcription inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB). This factor, termed DRB sensitivity-inducing factor (DSIF), is also required for transcription inhibition by H8. DSIF has been purified and is composed of 160-kD (p160) and 14-kD (p14) subunits. Isolation of a cDNA encoding DSIF p160 shows it to be a homolog of the Saccharomyces cerevisiae transcription factor Spt5. Recombinant Supt4h protein, the human homolog of yeast Spt4, is functionally equivalent to DSIF p14, indicating that DSIF is composed of the human homologs of Spt4 and Spt5. In addition to its negative role in elongation, DSIF is able to stimulate the rate of elongation by RNA Pol II in a reaction containing limiting concentrations of ribonucleoside triphosphates. A role for DSIF in transcription elongation is further supported by the fact that p160 has a region homologous to the bacterial elongation factor NusG. The combination of biochemical studies on DSIF and genetic analysis of Spt4 and Spt5 in yeast, also in this issue, indicates that DSIF associates with RNA Pol II and regulates its processivity in vitro and in vivo.

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Dsif, a novel transcription elongation factor that regulates rna polymerase ii processivity, is composed of human spt4 and spt5 homologs

An Immediate Response of Ribosomal Transcription to Growth Factor Stimulation in Mammals Is Mediated by ERK Phosphorylation of UBF

A monoclonal IgG2a antinucleolar autoantibody (72B9) was obtained by fusion of spleen cells from a (New Zealand black x New Zealand white)F1 mouse with myeloma cells (P3x63Ag8.653). Antibody 72B9 recognized a highly conserved nucleolar antigen present in both animal and plant cells. The staining pattern produced by antibody 72B9 in different cell substrates was identical with those obtained by scleroderma antibodies reactive with a basic (pI 8.5) nucleolar protein of Mr 34,000, which is associated with the U3 RNP particle. Western blotting further confirmed its reactivity with this scleroderma-related U3 RNP protein.

Monoclonal autoantibody from a (New Zealand black x New Zealand white)F1 mouse and some human scleroderma sera target an Mr 34,000 nucleolar protein of the U3 RNP particle.

The nucleolus is the ribosome factory of the cells. This is the nuclear domain where ribosomal RNAs are synthesized, processed, and assembled with ribosomal proteins. Here we describe the classical tripartite organization of the nucleolus in mammals, reflecting ribosomal gene transcription and pre-ribosomal RNA (pre-rRNA) processing efficiency: fibrillar center, dense fibrillar component, and granular component. We review the nucleolar organization across evolution from the bipartite organization in yeast to the tripartite organization in humans. We discuss the basic principles of nucleolar assembly and nucleolar structure/function relationship in RNA metabolism. The control of nucleolar assembly is presented as well as the role of pre-existing machineries and pre-rRNAs inherited from the previous cell cycle. In addition, nucleoli carry many essential extra ribosomal functions and are closely linked to cellular homeostasis and human health. The last part of this review presents recent advances in nucleolar dysfunctions in human pathology such as cancer and virus infections that modify the nucleolar organization.  2010 John Wiley & Sons, Ltd. WIREs RNA 2010 1 415‐431

The nucleolus: structure/function relationship in RNA metabolism

Autoantibodies to components of the nucleolus are a unique serological feature of patients with scleroderma. There are autoantibodies of several specificities; one type produces a speckled pattern of nucleolar staining in immunofluorescence. In actinomycin D and 5,6-dichloro-beta-D-ribofuranosylbenzimidazole-treated Vero cells, staining was restricted to the fibrillar and not the granular regions. By double immunofluorescence, specific rabbit anti-RNA polymerase I antibodies stained the same fibrillar structures in drug-segregated nucleoli as scleroderma sera. Scleroderma sera immunoprecipitated 13 polypeptides from [35S]methionine-labeled HeLa cell extract with molecular weights ranging from 210,000 to 14,000. Similar polypeptides were precipitated by rabbit anti-RNA polymerase I antibodies, and their common identities were confirmed in immunoabsorption experiments. Microinjection of purified IgG from a patient with speckled nucleolar staining effectively inhibited ribosomal RNA transcription. Autoantibodies to RNA polymerase I were restricted to certain patients with scleroderma and were not found in other autoimmune diseases.

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Autoantibody to RNA polymerase I in scleroderma sera.

A number of chemicals, mostly adenosine analogues, cause the nucleolus of the chick embryo fibroblast to lose material and unravel over a period of several hours into beaded strands termed nucleolar necklaces (NN). The results of analyses of the fibroblasts, treated with the NN-forming chemical dichlororibobenzimidazole (DRB), suggests that the following biochemical alterations occur: DRB almost completely prevents the increase in both messenger RNA (mRNA) and heterogeneous nuclear RNA. It interferes with ribosome synthesis by decreasing the rate of 45S ribosomal RNA (rRNA) accumulation by 50%, slowing the rate of 18S rRNA appearance by 50%, and causing an extensive degradation (80%) of the 32S and 28S rRNA-containing preribisomes. Most of this preribosome degration probably occurs at or before the 32S rRNA preribosome stage. The degradation of these preribosomes appears to be due to the formation of defective 45S rRNA preribosomes rather than to a direct DRB interference with preribosome processing enzyme action. DRB inhibits total cellular RNA synthesis in less than 15 min, suggesting a direct interference with RNA synthesis. DRB also inhibits the uptake of nucleosides into the cell. DRB in the concentrations used does not appear to directly interfere with the translation of mRNA (i.e., protein synthesis). Other NN-forming adenoside analogues and high concentrations of adenosine (2 mM) cause biochemical alterations similar to those produced by DRB. To explain the preribosome degradation, we propose the hypothesis that DRB inhibits the synthesis of mRNA; as a consequence, some of the preribosomal proteins that normally coat the 32S rRNA portion of the 45S precursor RNA become limiting, and this defective portion is then subject to degradation by nucleases.

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Nucleolar necklaces in chick embryo fibroblast cells. I. Formation of necklaces by dichlororibobenzimidazole and other adenosine analogues that decrease RNA synthesis and degrade preribosomes.

D Granick

Papers

Nucleolar necklaces in chick embryo fibroblast cells. I. Formation of necklaces by dichlororibobenzimidazole and other adenosine analogues that decrease RNA synthesis and degrade preribosomes.