Intranuclear localization of snRNP antigens.

Abstract

Abstract.Anti-Sm antibodies recognize a group of small, nuclear RNA-protein complexes (snRNPs) containing U 1, U2, U4, U5, and U6 snRNAs. Anti-RNP antibodies only react with U I snRNA-containing complexes.The intranuclear distribution of snRNP particles was studied by double immunofluorescence staining of human fibroblasts. Mouse monoclonal anti-Sm antibodies and polyclonal patient sera reacting with different peptides in the snRNP complexes were used. The immunofluorescence patterns obtained with fluorescein isothiocyanate-conjugated anti-mouse Ig and tetramethylrhodamine isothiocyanate-conjugated anti-human Ig second antibodies were examined using computer analysis of digitized images. With this approach the similarity of different patterns could be visualized and estimated with mathematical methods. It was found that human anti-Sm serum as well as three different anti-RNP sera produced speckled patterns overlapping with the anti-Sm monoclonal pattern. Thus, Sm antigenic intranuclear domains also reacted with anti-RNP antibodies, suggesting a high degree of co-localization of the antigenic structures. A partial overlap was found between speckles detected by mouse anti-Sm antibodies and a human La-antiserum. No significant co-localization occurred between speckles detected by mouse anti-Sm antibodies and speckles detected by human antisera reacting with Scl-70 and centromeric antigens.As the U 1 snRNP complex is believed to play a role in the splicing of RNA polymerase II transcripts, it appears that the speckles detected by Sm and RNP antibodies may be regions of hnRNA synthesis and m.RNA processing. Although no function has been demonstrated for the U2, U4, U5, and U6 snRNPs, the co-localization with the U 1 RNA complexes shown in this report indicate that they too participate in some aspect of mRNA processing. The results suggest that computer-assisted analysis of nuclear immunofluorescence patterns will be a useful tool in studies of the spatial and functional organization of the interphase nucleus. IMMUNE staining of mammalian cells with autoimmune sera frequently results in a speckled nuclear pattern. The two major specificities that produce speckled patterns are known as Sm and RNP. These autoantibodies occur primarily in systemic lupus erythematosus, mixed connective tissue disease, and less frequently in other autoimmune diseases (3,31,35).The Sm and RNP antigenic structures are thus concentrated in certain nuclear domains. Immunochemical and biochemical studies have elucidated the nature of the antigens (1, 11-13, 16, 18, 20, 25, 33, 34, 37, 38). Sm-antibodies were shown to immunoprecipitate RNA-protein complexes containing a class of small nuclear RNAs designated U l, U2, U4, U5, and U6 snRNAs) RNP-antibodies, on the other hand, immunoprecipitated only U 1 RNA-containing complexes (18). Proteins were required for antigenicity (18). The fact that small, U3 is nucleolar and not precipitated by Sm/RNP antibodies. nuclear RNA-protein complex (snRNP) 2 particles containing only U l RNA can be immunoprecip...

The chimeric oncoproteins E2A-PBX1 and E2A-HLF are concentrated within spherical nuclear domains

“…In particular, subcellular detection of polyadenylated mRNA by in situ hybridization produces a signal distribution characterized by numerous intranuclear speckles, termed`transcript domains'. These have been shown to colocalize with speckles produced on immunodetection of small nuclear ribonuclear protein (snRNP) particles involved in pre-mRNA splicing (Nyman et al, 1986;Spector, 1984;Verheijen et al, 1986;Reuter et al, 1984;Carter et al, 1991;Carme-Fonseca et al, 1992). Splicing and other RNA processing components may also be distributed at lower concentration diusely throughout the nucleoplasm exclusive of nucleoli.…”

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

“…By analogy to the fact that the r-DNA is localized and transcribed in the nucleolus, where the formation of the ribosome subunits takes place, it has been postulated that other functional compartments exist in cell nuclei (Blobel 1985, Nyman et al 1986, Hochstrasser & Sedat 1987, Spector 1990, Manuelidis 1990, Carter et al 1991. Hutchison and Weintraub (1985) found DNAse hypersensitive sites in the nuclear periphery, and cbncluded that active RNApolymerase 11 genes are preferentially localized in these regions.…”

“…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.…”

Repositioning of Muscle-specific Genes Relative to the Periphery of SC-35 Domains during Skeletal Myogenesis

“…SC-35 domains are subnuclear compartments rich in poly A RNA (Carter et al, 1991;Visa et al, 1993) and certain mRNAs , as well as a variety of RNA metabolic factors, including spliceosome assembly factor SC-35 (Fu and Maniatis, 1990;Blencowe et al, 1994), numerous other splicing factors (Nyman et al, 1986;Spector et al, 1991), a subset of poly (A) polymerase (Schul et al, 1998), a putative RNA helicase (Gee et al, 1997), and a hyperphosphorylated form of RNA polymerase II (Bregman et al, 1995). These 20 -40 globular domains consist of the more prominent (0.5-3 m in diameter) components of the small nuclear ribonucleoprotein "speckled" staining pattern and occupy Ͻ5% of the nuclear volume, lying in a narrow focal plane in cultured human fibroblasts and myoblasts .…”