https://www.cell.com/cell/pdf/S0092-8674(00)00188-4.pdf

Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription.

Both long-term and short-term sensitization of the gill and siphon withdrawal reflex in Aplysia involve facilitation of the monosynaptic connections between the sensory and motor neurons. To analyze the relationship between these two forms of synaptic facilitation at the cellular and molecular level, this monosynaptic sensorimotor component of the gill-withdrawal reflex of Aplysia can be reconstituted in dissociated cell culture. Whereas one brief application of 1 microM serotonin produced short-term facilitation in the sensorimotor connection that lasted minutes, five applications over 1.5 hours resulted in long-term facilitation that lasted more than 24 hours. Inhibitors of protein synthesis or RNA synthesis selectively blocked long-term facilitation, but not short-term facilitation, indicating that long-term facilitation requires the expression of gene products not essential for short-term facilitation. Moreover, the inhibitors only blocked long-term facilitation when given during the serotonin applications; the inhibitors did not block the facilitation when given either before or after serotonin application. These results parallel those for behavioral performance in vertebrates and indicate that the critical time window characteristic of the requirement for macromolecular synthesis in long-term heterosynaptic facilitation is not a property of complex circuitry, but an intrinsic characteristic of specific nerve cells and synaptic connections involved in the long-term storage of information.

https://science.sciencemag.org/content/sci/234/4781/1249.full.pdf

A critical period for macromolecular synthesis in long-term heterosynaptic facilitation in Aplysia

The c-myc gene product is a nuclear protein1,2 expressed in a wide variety of cell types3. It has been implicated in the control of normal cell growth as well as transformation4–7, but its exact function is unknown. When the human promyelocytic leukaemia cell line HL60 is treated with retinoic acid, the cells differentiate into granulocytes, and there is a reduction in steady state c-myc RNA of more than 10-fold8. Nuclear runoff assays show that this reduction is caused by a corresponding decrease in the transcription of exon 2. However, only a minor decrease in exon 1 transcription is observed upon differentiation. In undifferentiated HL60 cells there is an approximately 3-fold molar excess of exon 1 transcription over exon 2, and this excess increases to about 15-fold in differentiated cells. This observation suggests that a major component of c-myc transcriptional down-regulation in HL60 cells is at the level of elongation rather than at the level of initiation. The position of the elongation block was mapped to the region of the boundary between exon 1 and intron 1. During HL60 differentiation, a DNase I hypersensitive site in the chromatin about 300 bases downstream of the 5′ end of of intron 1 increases in intensity relative to other sites, possibly reflecting events associated with the termination of transcription. Our runoff analysis also revealed transcription of both strands immediately upstream of exon 1 in HL60 cells. The sense strand transcription of this region produces a novel c-myc RNA which initiates several hundred bases upstream of the previously defined promoters and is found in a variety of cell types.

A block to elongation is largely responsible for decreased transcription of c-myc in differentiated HL60 cells.

Although microRNAs (miRNAs) are key regulators of gene expression in normal human physiology and disease, transcriptional regulation of miRNAs is poorly understood, because most miRNA promoters have not yet been characterized. We identified the proximal promoters of 175 human miRNAs by combining nucleosome mapping with chromatin signatures for promoters. We observe that one-third of intronic miRNAs have transcription initiation regions independent from their host promoters and present a list of RNA polymerase II- and III-occupied miRNAs. Nucleosome mapping and linker sequence analyses in miRNA promoters permitted accurate prediction of transcription factors regulating miRNA expression, thus identifying nine miRNAs regulated by the MITF transcription factor/oncoprotein in melanoma cells. Furthermore, DNA sequences encoding mature miRNAs were found to be preferentially occupied by positioned-nucleosomes, and the 3' end sites of known genes exhibited nucleosome depletion. The high-throughput identification of miRNA promoter and enhancer regulatory elements sheds light on evolution of miRNA transcription and permits rapid identification of transcriptional networks of miRNAs.

http://genesdev.cshlp.org/content/22/22/3172.full.pdf

Chromatin structure analyses identify miRNA promoters

Pre-mRNA splicing is a predominantly co-transcriptional event which involves a large number of essential splicing factors. Within the mammalian cell nucleus, most splicing factors are concentrated in 20-40 distinct domains called speckles. The function of speckles and the organization of cellular transcription and pre-mRNA splicing in vivo are not well understood. We have investigated the dynamic properties of splicing factors in nuclei of living cells. Here we show that speckles are highly dynamic structures that respond specifically to activation of nearby genes. These dynamic events are dependent on RNA polymerase II transcription, and are sensitive to inhibitors of protein kinases and Ser/Thr phosphatases. When single genes are transcriptionally activated in living cells, splicing factors leave speckles in peripheral extensions and accumulate at the new sites of transcription. We conclude that one function of speckles is to supply splicing factors to active genes. Our observations demonstrate that the interphase nucleus is far more dynamic in nature than previously assumed.

The dynamics of a pre-mRNA splicing factor in living cells.

alpha-Amanitin, a toxic substance from the mushroom Amanita phalloides, is a potent inhibitor of DNA-dependent RNA polymerase II (the nucleoplasmic form) from sea urchin, rat liver, and calf thymus. This compound exerts no effect on the activity of polymerase I (nucleolar form) or polymerase III (also nucleoplasmic). The inhibition is due to a specific interaction with polymerase II or with a complex of DNA and polymerase II.

https://science.sciencemag.org/content/sci/170/3956/447.full.pdf

Specific Inhibition of Nuclear RNA Polymerase II by α-Amanitin

DNA-dependent RNA polymerase, DNA-Dependent DNA polymerase, and terminal riboadenylate transferase (TRT) activities have been measured after DEAE-Sephadex chromatography of whole cell extracts prepared from eggs and staged embryos of the urchin, Stronglyocentrotus franciscanus. Activity of each of these three polymerase classes is present in the egg, and the total activity per embryo is constant throughout embryogenesis to the pluteus stage (approximately 1000 cells). Thus the egg appears to contain sufficient DNA polymerase, RNA polymerase, and TRT TRT for embryogenesis. The increases in the synthesis of DNA, RNA and polyadenylated RNA tracts observed after fertilization must be due to the activation of the preexisting egg enzymes. Separation of the egg into nucleate and anucleate halves demonstrates that the RNA polymerases are not restricted to the egg nucleus. During development, the enzymes become progressively more associated with the cell nucleus. The egg extracts contain low activities (approximately 6% total) of RNA polymerase II as measured by sensitivity to alpha-amanitin. This is confirmed by resolution of the RNA polymerase forms I, II, and III by gradient sievorptive elution on DEAE-Sephadex. Later stage embryos contain more nearly equal activities of RNA polymerase, I, II, and III, although the total RNA polymerase activity per embryo is not changed. Additionally, two chromatographicallly distinct species of RNA polymerase III are detected, one of which is observed only in later stages. Thus interconversion of enzymes via addition of new subunits or coordinate synthesis and loss of enzyme species must occur.

Nucleic acid polymerizing enzymes in developing Strongylocentrotus franciscanus embryos.

Iodination of alpha-amanitin at the 7-position in the 6-hydroxy-2-sulfoxytryptophan moiety is effected with 1 equiv of iodine monochloride in methanol. The isolated product shows a lambdamax in methanol at 301 nm, compared with 305 nm for the parent alpha-amanitin; in methanolic 0.01 M NaOH the lambdamax are 330 and 332 nm for the product and parent, respectively. Spectrophotometric titration of the phenolic hydroxyl shows a decrease in pKa from 9.72 (alpha-amanitin) to 7.94 (7 iodo-alpha-amanitin). Appropriate spectrophotometric examination therefore distinguishes between parent and product. Proton magnetic resonance shows two aromatic protons (v4H = 7.57; V5H = 6.90 ppm; j4,5 = 9) in the 7-iodo-alpha-amanitin and three aromatic protons (v4H = 7.64; V5H = 6.78; V7H = 6.94 ppm; j4,5 = 9; J5,7 = 2) in alpha amanitin thus establishing the extent and position of iodine substitution. The 7-iodo-alpha-amanitin effectively inhibits RNA polymerase activity with half-maximal inhibition at 2 X 10(-9) M and 10(-4) M for the sea urchin RNA polymerases II and III, respectively. Addition of [125I]-7-iodo-alpha-amanitin (200 Ci/mmol) to crude extracts from sea urchin blastula, MOPC 315 plasmacytoma, and adult Oregon R Drosophila melanogaster followed by resolution on DEAE-Sephadex demonstrates that the radioactive ligand binds stably and specifically with RNA polymerase II in each of these extracts.

Paul W. Morris

Papers

Specific Inhibition of Nuclear RNA Polymerase II by α-Amanitin

Nucleic acid polymerizing enzymes in developing Strongylocentrotus franciscanus embryos.

Biochemistry of the amatoxins: preparation and characterization of a stably iodinated alpha-amanitin.