Immunoelectron microscopic localization of snRNPs

Garner

Biochemical and Biophysical Research Communications

et al. 2007

Nanosecond electric pulses generate nanopores in the interior membranes of cells and modulate cellular functions. Here, we used confocal microscopy and flow cytometry to observe Smith antigen antibody (Y12) binding to nuclear speckles, known as small nuclear ribonucleoprotein particles (snRNPs) or intrachromatin granule clusters (IGCs), in Jurkat cells following one or five 10 ns, 150 kV/cm pulses. Using confocal microscopy and flow cytometry, we observed changes in nuclear speckle labeling that suggested a disruption of pre-messenger RNA splicing mechanisms. Pulse exposure increased the nuclear speckled substructures by 2.5-fold above basal levels while the propidium iodide (PI) uptake in pulsed cells was unchanged. The resulting nuclear speckle changes were also cell cycle dependent. These findings suggest that 10 ns pulses directly influenced nuclear processes, such as the changes in the nuclear RNA-protein complexes. Published by Elsevier Inc.Keywords: Nucleus; Nuclear speckles; Small nuclear ribonucleoprotein particles/intrachromatin granule clusters; Intracellular electromanipulation; Nanosecond electric pulsesWe have been exploring a new nanosecond (ns) time domain in cell biology. When cells are exposed to intense (50-300 kV/cm) pulsed electric fields (PEFs) with rise times faster than the time it takes for charges on the plasma membrane to redistribute, the electric field can penetrate into the interior of the cell and organelles. If these fields exceed the breakdown threshold of these membranes, nanopores will form in the organelle membranes [1,2]. To better understand the mechanism involved, mathematical models for the cellular electropermeabilization due to nsPEFs have been developed [3,4]. The resulting changes in cellular structures and functions differ significantly from traditional electroporation [microseconod to millisecond pulsed electric fields (PEFs)] [1,5,6]. Increased permeabilization of the plasma membrane, typically demonstrated by measuring propidium iodide (PI) uptake, due to nsPEFs was delayed compared to traditional electroporation [5,6], indicating that it was an indirect effect. These nsPEFs 0006-291X/$ -see front matter Published by Elsevier Inc.

Section: Discussionmentioning

confidence: 99%

Nanosecond electric pulses penetrate the nucleus and enhance speckle formation

Garner

Biochemical and Biophysical Research Communications

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

“…The bn2-containing aggregates were reminiscent of the speckles in which splicing factors are concentrated (15)(16)(17). One splicing factor widely used for the detection of speckles is SC35 (16)(17)(18).…”

Section: Resultsmentioning

confidence: 99%

Basonuclins 1 and 2, whose genes share a common origin, are proteins with widely different properties and functions

Vanhoutteghem

Djian

2006

Basonuclin (bn) 1 possesses three separated pairs of zinc fingers and a nuclear localization signal. It is largely confined to the basal cells of stratified squamous epithelia and to reproductive germ cells. bn1 can shuttle between the nucleus and the cytoplasm, and its location is correlated with the proliferative potential of the cell. The recently discovered bn2 also possesses three separated pairs of zinc fingers and a nuclear localization signal. Conservation of the zinc fingers and the nuclear localization signal by bn1 and bn2 indicates a common origin. However, in contrast to bn1, bn2 is found in virtually every cell type and is confined to the nucleus. Bn2 but not bn1 colocalizes with SC35 in nuclear speckles and, therefore, is likely to have a function in nuclear processing of mRNA.RNA processing ͉ speckles ͉ zinc fingers B asonuclin (bn1) is a zinc finger protein with highly restricted tissue distribution: It is found mainly in basal keratinocytes of stratified squamous epithelium and in reproductive germ cells (1-5). bn1 possesses three separated pairs of zinc fingers, a nuclear localization signal (NLS) and a serine-rich region that has been called the serine stripe (1). Although the evidence is not conclusive, it seems that the function of bn1 is related to the potential for cell proliferation (6). The only known function of bn1 is that of a transcription factor in the synthesis of ribosomal RNA (7, 8), but it is possible that bn1 possesses a nucleoplasmic function in the regulation of expression of genes transcribed by RNA polymerase II (9).A gene encoding a second basonuclin (bn 2) recently has been discovered (10, 11). Although the deduced amino acid sequences of bn1 and bn2 are only slightly Ͼ40% identical, bn2 possesses all of the characteristic features of bn1 described above. The bn2 mRNA is abundant in cell types that possess bn1 but is also found in tissues that lack bn1, such as kidney, intestine, and uterus. The genes for bn1 and bn2 differ greatly in size and are located on different chromosomes, but it is clear that they have a common evolutionary origin. The evolutionary conservation of bn2 is much greater than that of bn1. It has been postulated that the gene for bn2 is the older of the two. After its duplication, the gene for bn1 was free to evolve in other directions, whereas the gene for bn2 remained virtually invariant (9, 11).Previous work on immunocytological detection of bn1 was carried out with polyclonal antisera raised against most or all of the bn1 protein (6, 12). In view of the similarities between bn1 and bn2, it was possible that these antisera did not distinguish between the two basonuclins. To detect each basonuclin independently, we generated antibodies to specific determinants not shared by bn1 and bn2. Using these antibodies, we show that, although the two basonuclins are of common origin, they possess widely different functions, because bn2 is likely to have a function in pre-mRNA processing. ResultsEvolutionary Conservation of bn2. We had reported that bn2 was e...

“…For most of these analyses anti-Sm antibodies were used, which recognize proteins which are common to all splicing snRNPs. The snRNP accumulations are found to colocalize with the interchromatin granules, the perichromatin fibrils or the coiled bodies (Spector et al 1983, Fakan etal. 1984, BachmannetaT.…”

Section: Introductionmentioning

confidence: 96%

“…Visualization of major components of the splicing machinery-the U1, U2, U4/U6 and U5 snRNP (small nuclear ribonucleoprotein particles) proteins-by means of indirect immunofluorescence-revealed a wide nuclear distribution and, in addition, a speckled nuclear staining with 20 to 50 speckles consisting of higher snRNP concentrations (Mattioli & Reichlin 1971, Northway & Tan 1972, Lerner et al 1981, Spector et al 1983, Spector 1984, Reuter et al 1984, Bachmann et al 1986, Nyman et al 1986, Verheijen et al 1986. For most of these analyses anti-Sm antibodies were used, which recognize proteins which are common to all splicing snRNPs.…”

Section: Introductionmentioning

confidence: 99%

Evidence for a nuclear compartment of transcription and splicing located at chromosome domain boundaries

et al. 1993

The nuclear topography of splicing snRNPs, mRNA transcripts and chromosome domains in various mammalian cell types are described. The visualization of splicing snRNPs, defined by the Sm antigen, and coiled bodies, revealed distinctly different distribution patterns in these cell types. Heat shock experiments confirmed that the distribution patterns also depend on physiological parameters. Using a combination of fluorescence in situ hybridization and immunodetection protocols, individual chromosome domains were visualized simultaneously with the Sm antigen or the transcript of an integrated human papilloma virus genome. Three-dimensional analysis of fluorescence-stained target regions was performed by confocallaser scanning microscopy. RNA transcripts and components of the splicing machinery were found to be generally excluded from the interior of the territories occupied by the individual chromosomes. Based on these findings we present a model for the functional compartmentalization of the cell nucleus. According to this model the space between chromosome domains, including the surface areas of these domains, defines a three-dimensional networklike compartment, termed the interchromosome domain (ICO) compartment, in wh ich transcription and splicing of mRNA occurs.