Purification and characterization of human autoantibodies directed to specific regions on U1RNA; recognition of native U1RNP complexes

Abstract

Antibodies against naked U1RNA can be found in sera from patients with overlap syndromes of systemic lupus erythematosus (SLE) in addition to antibodies directed to the proteins of U1 ribonucleoproteins (U1RNP). We investigated the reactivity of these U1RNA specific autoantibodies with the native U1RNP particle both in vitro and inside the cell. For this purpose a method was developed to purify human autoantibodies directed to specific regions of U1RNA. The antibodies are specifically directed to either stemloop II or stemloop IV of U1RNA and do not crossreact with protein components of U1RNP. Both types of antibody are able to precipitate from cell extracts native U1snRNPs containing most, if not all, protein components. Immunofluorescence patterns indicate that the antigenic sites on the RNA, i.e. the stem of stemloop II and the loop of stemloop IV, are also available after fixation of the cells. Immunoelectron microscopy employing anti-stemloop IV antibodies and purified, complete U1snRNP particles showed that stemloop IV is located within the body of the U1RNP complex, which also comprises the Sm site and the common Sm proteins. The anti-U1RNA autoantibodies described in this paper recognize native U1RNP particles within the cell and can therefore be used as tools to study mechanisms involved in splicing of pre-mRNA.

The U1‐snRNP complex: structural properties relating to autoimmune pathogenesis in rheumatic diseases

“…Four of the U1-C specific antibodies recognized the N-terminal region between residues 30-63. Two crossreactive and one specific antibody bound to the C-terminal portion of U1-C from 98 to 126, where the crossreactive antibodies also recognized the proline-rich region of U1-A from residues 187-204 (72).…”

Differential immunoglobulin class-mediated responses to components of the U1 small nuclear ribonucleoprotein particle in systemic lupus erythematosus and mixed connective tissue disease

“…Previous investigations have demonstrated that SLE and MCTD patients often exhibit 1000-fold greater auto-reactivity to subunits of the U1 small nuclear ribonucleoprotein particle (snRNP) than to any other cellular component 23, 24 . The U1 snRNP is an RNA-protein complex that is responsible for pre-mRNA processing and is composed of 10 proteins (U1–70K, U1A, U1C and seven Smith antigen (Sm) proteins) 25, 26, 27 .…”

Impaired phagocytosis of apoptotic cell material by monocyte-derived macrophages from patients with systemic lupus erythematosus

“…Serologically, the disease is characterized by high serum levels of autoantibodies to various cytoplasmic (6,7) and nuclear (8)(9)(10)(11) antigens. IgG antibodies that recognize double-stranded B-DNA (dsDNA) with high affinity (12,13) are specific for SLE and serve as a diagnostic hallmark of the disease (14).…”