Physiological importance of RNA and protein mobility in the cell nucleus

Trafficking of proteins and RNAs is essential for cellular function and homeostasis. While it has long been appreciated that proteins and RNAs move within cells, only recently has it become possible to visualize trafficking events in vivo. Analysis of protein and RNA motion within the cell nucleus have been particularly intriguing as they have revealed an unanticipated degree of dynamics within the organelle. These methods have revealed that the intranuclear trafficking occurs largely by energy-independent mechanisms and is driven by diffusion. RNA molecules and non-DNA binding proteins undergo constrained diffusion, largely limited by the spatial constraint imposed by chromatin, and chromatin binding proteins move by a stop-and-go mechanism where their free diffusion is interrupted by random association with the chromatin fiber. The ability and mode of motion of proteins and RNAs has implications for how they find nuclear targets on chromatin and in nuclear subcompartments and how macromolecular complexes are assembled in vivo. Most importantly, the dynamic nature of proteins and RNAs is emerging as a means to control physiological cellular responses and pathways.     

The effect of kainic acid administration on the cytoplasmic RNA content of nucleus arcuatus neurons in mice

A single intramuscular injection of kainic acid (12 micrograms/g body weight, dissolved in 0.1 ml of aqua pro injectione) was administered to adult, sexually mature, male mice. Control mice received equivalent amounts of aqua pro injectione only. Experimental animals were killed 24, 72, 120, and 192 h after the injection, and those of the control group simultaneously with the final experimental one. As determined cytophotometrically, in all the experimental groups a significant increase in the cytoplasmic RNA content of nucleus arcuatus neurons took place, this increase being highest 72 h after kainic acid treatment.     

Direct analysis of RNA in whole cell and cytoplasmic extracts by gel electrophoresis

Procedures are presented by which whole cell, cytoplasmic, or nuclear extracts can be subjected to gel electrophoresis for the separation of the various RNA species, which are then analyzed by conventional blotting and hybridization techniques. Since the methods for preparing the extracts do not involve precipitation or two-phase extraction steps, the minimum number of cells that can be processed is limited only by the sensitivity of detection for specific RNA species. Multiple small or large aliquots of tissue culture cells can be quickly prepared. Cell preparations with high RNase levels, such as resting human lymphocytes or HL60, can be processed reliably with these procedures.     

Precursors of U4 small nuclear RNA

The processing and ribonucleoprotein assembly of U4 small nuclear RNA has been investigated in HeLa cells. After a 45-min pulse label with [3H]uridine, a set of apparently cytoplasmic RNAs was observed migrating just behind the gel electrophoretic position of mature U4 RNA. These molecules were estimated to be one to at least seven nucleotides longer than mature U4 RNA. They reacted with Sm autoimmune patient sera and a monoclonal Sm antibody, indicating their association with proteins characteristic of small nuclear ribonucleoprotein complexes. The same set of RNAs was identified by hybrid selection of pulse-labeled RNA with cloned U4 DNA, confirming that these are U4 RNA sequences. No larger nuclear precursors of these RNAs were detected. Pulse-chase experiments revealed a progressive decrease in the radioactivity of the U4 precursor RNAs coincident with an accumulation of labeled mature U4 RNA, confirming a precursor-product relationship.     

Drug-induced migration of cytoplasmic ornithine decarboxylase into rat liver nucleus in not related to increased RNA polymerase activity

1-Methyl-3-isobutylxantine (MIX) caused rapid increases in cytoplasmic and nuclear ornithine decarboxylase (ODC) activity as well as increases in RNA polymerases I and II. MIX also significantly increased labeling of nuclear proteins with [³H]-leucine while causing only a slight rise in the labeling of the cytoplasm. Cycloheximide prevented the MIX-induced increases in cytoplasmic ODC, RNA polymerases I and II, and radioactive labeling of cytoplasmic and nuclear proteins. Cycloheximide did not prevent the MIX-induced change in nuclear ODC. These data suggest that cytoplasmic ODC migrated in to the nucleus after MIX treatment but this migration was not correlated with increased RNA polymerase activity.     

Precursors of 5 S ribosomal RNA in Bacillus subtilis

Bacillus subtilis 168 accumulates subnormal quantities of mature 5 S ribo-somal RNA in the presence of inhibitors of protein synthesis, such as chloramphenicol, or during pulse-labeling experiments. However, two RNA species, evidently precursors of m5 rRNA and therefore designated as p5_A and p5_B, do accumulate under these conditions. These RNA species are substantially longer than B. subtilis m5 rRNA: p5_A is about 179 nucleotides in length and p5_B is composed of approximately 152 nucleotides. The sum of p5_A, p5_B and m5 rRNA accumulating in the absence of protein synthesis, less excess chain length associated with p5_A and p5_B, equals the expected quantities of m5 rRNA in growing cells. p5_A and p5P_B both contain all t₁ RNase-generated oligonucleotides characteristic of m5 rRNA plus additional sequences. At least the 5′ termini of p5_A and p5_B differ from that of m5. If chloramphenicol is removed from a culture in which p5_A and p5_B have accumulated and further RNA synthesis is inhibited, then a quantitative reciprocal loss of p5_A and p5_B occurs as m5 rRNA accumulates. No evidence suggests any p5_A to p5_B transition under these conditions.     

Changes in the cytoplasmic RNA content in neurons of the nucleus suprachiasmaticus following a single dose of kainic acid

The Cytoplasmic RNA content in neurons of the nucleus suprachiasmaticus was determined cytophotometrically in adult mice following injection of kainic acid. A dose of 12 mg/kg b. w. caused a significant increase in the cytoplasmic RNA content at least between 1 and 15 days postinjection.     

RNA polymerase in normal avian erythrocytes

Significant RNA polymerase activity was demonstrated in isolated nuclei from mature avian erythrocytes. This activity was shown to have characteristics common to mammalian systems, including sensitivity to α-amanatin. A crude fraction of RNA polymerase was solubilized from these nuclei and characterized to provide further support for the existence of the enzyme in these cells.     

Processing of RNA by cytoplasmic extract

Nuclei are isolated from HeLa cells by a low speed centrifugation procedure. These nuclei can carry out RNA synthesis at 30°. The effect of cytoplasmic extract on the transport and processing of RNA is measured. In the presence of cytoplasmic extract the precursor to 4S RNA is processed to 4S RNA and an RNA species of 7–9S is transported outside the nuclei.Abbreviations HEPES N-2-Hydroxymethylpiperazine-N'-2'-ethanesulfonic acid - DTT dithiothreitol - cDNA complementary DNA     

Precursors of chloroplast ribosomal RNA in Euglena gracilis

Incorporation of ³²P into mature chloroplast rRNA species of MW 1.1 × 101 and 0.56 × 10⁶ has been followed in Euglena gracilis by pulse and pulse chase experiments. Mature rRNA species have precursors of MW 1.16 × 10⁶ ± 0.01 × 10⁶ and 0.64 × 106 ± 0.01 × 10⁶ resp. These precursors have base composition and hydridization properties similar to those of the mature, rRNA species. No evidence of a single common precursor to these molecules was found. Rifampicin did not affect the synthesis of chloroplast rRNA.     

c-fos expression in the putative avian suprachiasmatic nucleus

c-fos induction was investigated as a potential component in the avian photic entrainment pathway and as a possible means of locating the central pacemaker in birds. In both quail (Coturnix coturnix japonica) and starlings (Sturnus vulgaris) exposure to 1 h of light induced Fos-lir in the visual suprachiasmatic nucleus but not in the medial suprachiasmatic nucleus. However, the degree of c-fos induction in the visual suprachiasmatic nucleus was similar at different circadian times despite the fact that the light pulses caused differential phase shifts in the locomotor rhythm. For golden hamsters the same experiment resulted in significantly different levels of Fos-lir in the suprachiasmatic nucleus, as well as different phase shifts. Starlings and hamsters were also entrained to T-cycles that caused a large daily phase shift (T = 21.5 h in starlings, T = 22.67 hours in hamsters), or no daily phase shift (T = free running period). No difference in the induced levels of Fos-lir in the visual suprachiasmatic nucleus region was observed between the two groups of starlings, but in hamsters there were significantly different levels of Fos-lir in the suprachiasmatic nucleus between the two groups. Accepted: 15 November 1996     

Fibroblast growth factor-2 stimulates cell proliferation and decreases sexually dimorphic cell death in an avian song control nucleus

The neural system controlling song in birds has proven a useful model for investigating how neuronal growth and survival are regulated by sexual differentiation. The present study focused on one song control area, the robust nucleus of the archistriatum (RA), and explored how sex differences in the proliferation of putative glia cells in this region influence sexually dimorphic cell survival. In zebra finches (Poephila guttata), RA neuron death is much greater in young females than in males, resulting in marked sex differences in RA neuron number. An earlier study indicated that just prior to this sexually dimorphic neuron death the proliferation of putative glia cells within the RA is significantly lower in females than in males and remains so throughout the peak of neuron death. This suggests that sex differences in glia (or glia-derived molecules) might regulate neuron survival during sexual differentiation of the RA. To determine whether increased cell proliferation within the RA favors increased cell survival, we infused the potent glia mitogen fibroblast growth factor-2 (FGF-2) into the RA unilaterally in young females. We find that FGF-2 infusions increase RA cell proliferation and concurrently decrease the incidence of degenerating RA cells, results consistent with the hypothesis that glia exert neurotrophic effects on RA neurons during sexual differentiation. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 573–581, 1998