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Structural/functional properties of a mammalian multi-component structure containing all major spliceosomal small nuclear ribonucleoprotein particles.

ABSTRACT: An approx. 40 S multi-component structure, consisting of all major spliceosomal small nuclear ribonucleoprotein particles (snRNP) (U1, U2, U4/U6 and U5) in stable association with a large number of polypeptides, mainly in the range 50-210 kDa, has been reported to exist within rat liver nuclear extracts [Guialis, Moraitou, Patrinou-Georgoula and Dangli (1991) Nucleic Acids Res. 19, 287-296]. Using a new polyclonal antibody recognizing a 63 kDa protein component of the complex, this multi-snRNP assembly was detected within rat liver nuclear extracts as efficiently as with the antibody for the U2 snRNP-specific B' polypeptide. The 63 kDa protein was found to correspond to the 66 kDa subunit of the splicing factor SF3a, a known integral component of the HeLa 17 S U2 snRNP. Anti-2,2,7-trimethylguanosine affinity chromatography was an easy and efficient way of purifying the multi-snRNP complex from rat liver 40 S heterogeneous nuclear ribonucleoprotein particle (hnRNP)-containing sucrose gradient fractions. By subsequent glycerol-gradient sedimentation, all known snRNP forms active in RNA splicing were identified among its constituents. A complex structurally similar to the rat multi-snRNP was also identified in HeLa nuclear extracts. Preservation of hnRNP-snRNP interactions was observed within HeLa 40 S fractions. Moreover, these fractions were capable of restoring splicing activity when applied in reconstitution studies to supplement a micrococcal nuclease-treated splicing extract.

SUBMITTER: Moraitou M

PROVIDER: S-EPMC1219461 | biostudies-other | 1998 May

REPOSITORIES: biostudies-other

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Project description:A hallmark of systemic lupus erythematosus (SLE) is the appearance of autoantibodies to nuclear antigens, including autoantibodies directed to the heterogeneous nuclear ribonucleoprotein A2 (hnRNP-A2), which occur in 20% to 30% of SLE patients as well as in animal models of this disease. To investigate the underlying cellular reactivity and to gain further insight into the nature and potential pathogenic role of this autoimmune response we characterized the T cell reactivity against hnRNP-A2 in patients with SLE in comparison to healthy controls. Cellular proliferation of peripheral blood T cells to hnRNP-A2 was determined by [3H]thymidine incorporation and T cell clones (TCCs) specific for hnRNP-A2 were grown by limiting dilution cloning; IFNgamma, IL-4 and IL-10 in culture supernatants were measured by ELISA. Bioactivity of culture supernatants was determined by incubation of anti-CD3/anti-CD28 stimulated peripheral blood CD4+ T cells with supernatants of TCCs. Stimulation assays performed with peripheral blood mononuclear cells of 35 SLE patients and 21 healthy controls revealed pronounced proliferative responses in 66% of SLE patients and in 24% of the controls, which were significantly higher in SLE patients (p < 0.00002). Furthermore, hnRNP-A2 specific TCCs generated from SLE patients (n = 22) contained a relatively high proportion of CD8+ clones and mostly lacked CD28 expression, in contrast to TCCs derived from healthy controls (n = 12). All CD4+ TCCs of patients and all control TCCs secreted IFNgamma and no IL-4. In contrast, CD8+ TCCs of patients secreted very little IFNgamma, while production of IL-10 did not significantly differ from other T cell subsets. Interestingly, all CD8+ clones producing IL-10 in large excess over IFNgamma lacked expression of CD28. Functional assays showed a stimulatory effect of the supernatants derived from these CD8+ CD28- hnRNP-A2 specific TCCs that was similar to that of CD4+ CD28+ clones. Taken together, the pronounced peripheral T cell reactivity to hnRNP-A2 observed in the majority of SLE patients and the distinct phenotype of patient-derived CD8+ TCCs suggest a role for these T cells in the pathogenesis of SLE.

Project description:Active pre-mRNA splicing occurs co-transcriptionally, and takes place throughout the nucleoplasm of eukaryotic cells. Splicing decisions are controlled by networks of nuclear RNA-binding proteins and their target sequences, sometimes in response to signalling pathways. Sam68 (Src-associated in mitosis 68 kDa) is the prototypic member of the STAR (Signal Transduction and Activation of RNA) family of RNA-binding proteins, which regulate splicing in response to signalling cascades. Nuclear Sam68 protein is concentrated within subnuclear organelles called SLM/Sam68 Nuclear Bodies (SNBs), which also contain some other splicing regulators, signalling components and nucleic acids.We used proteomics to search for the major interacting protein partners of nuclear Sam68. In addition to Sam68 itself and known Sam68-associated proteins (heterogeneous nuclear ribonucleoproteins hnRNP A1, A2/B1 and G), we identified hnRNP L as a novel Sam68-interacting protein partner. hnRNP L protein was predominantly present within small nuclear protein complexes approximating to the expected size of monomers and dimers, and was quantitatively associated with nucleic acids. hnRNP L spatially co-localised with Sam68 as a novel component of SNBs and was also observed within the general nucleoplasm. Localisation within SNBs was highly specific to hnRNP L and was not shared by the closely-related hnRNP LL protein, nor any of the other Sam68-interacting proteins we identified by proteomics. The interaction between Sam68 and hnRNP L proteins was observed in a cell line which exhibits low frequency of SNBs suggesting that this association also takes place outside SNBs. Although ectopic expression of hnRNP L and Sam68 proteins independently affected splicing of CD44 variable exon v5 and TJP1 exon 20 minigenes, these proteins did not, however, co-operate with each other in splicing regulation of these target exons.Here we identify hnRNP L as a novel SNB component. We show that, compared with other identified Sam68-associated hnRNP proteins and hnRNP LL, this co-localisation within SNBs is specific to hnRNP L. Our data suggest that the novel Sam68-hnRNP L protein interaction may have a distinct role within SNBs.

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Structural/functional properties of a mammalian multi-component structure containing all major spliceosomal small nuclear ribonucleoprotein particles.

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