Cloning of the human cDNA for the U1 RNA-associated 70K protein.

Abstract

Anti‐RNP sera were used to isolate a cDNA clone for the largest polypeptide of the U1 snRNP, a protein of mol. wt 70 kd designated 70K, from a human liver cDNA library constructed in the expression vector pEX1. The cro‐beta‐galactosidase‐70K fusion protein reacted with various anti‐RNP patient sera, a rabbit anti‐70K antiserum, as well as with a monoclonal antibody specific for this protein. The sequences of four 70K peptides were determined and they match parts of the deduced amino acid sequence of the 1.3 kb insert of p70.1 indicating that it is a genuine 70K cDNA. Screening of a new cDNA library constructed from polysomal mRNA of HeLa cells with the p70.1 clone yielded an overlapping clone, FL70K, which was 2.7 kb long and covered the complete coding and 3′‐untranslated sequence of the 70K protein in addition to 680 nucleotides upstream of the putative initiation codon, The predicted mol. wt of the encoded protein is approximately 70 kd. Amino acid analysis of the purified HeLa 70K protein yielded values close or identical to those deduced from the nucleotide sequence of the full‐length cDNA. The 70K protein is rich in arginine (20%) and acidic amino acids (18%). Extremely hydrophilic regions containing mixed‐charge amino acid clusters have been identified at the carboxyl‐terminal half of the protein, which may function in RNA binding. A sequence comparison with two recently cloned RNA binding proteins revealed homology with one region in the U1 RNP 70K protein. This domain may also be responsible for RNA binding.

Human autoantibodies against the 70‐kd polypeptide of u1 small nuclear rnp are associated with hla‐dr4 among connective tissue disease patients

“…Our findings of the association of HLA-DR4 with anti-U 1-70-kd autoantibodies (Table 2) and with the presence of select clinical features (Table 3) present in the anti-U 1-70-kd autoantibody positive CTD patients, compared with anti-U I-70-kd autoantibody negative SLE patients, are consistent with such a model. Recent studies examining the fine specificities of autoantibodies reactive with the U1-70-kd protein antigen also support the proposed model (13,20,(33)(34)(35)(36)(37)(38)(39). We and others have used a recombinant U 1-70-kd fusion protein and deletion mutants derived from the fusion protein for epitope mapping of the fine specificity of human anti-U 1-70-kd autoantibodies (13,20,(33)(34)(35)(36)(37)(38)(39).…”

“…Recent studies examining the fine specificities of autoantibodies reactive with the U1-70-kd protein antigen also support the proposed model (13,20,(33)(34)(35)(36)(37)(38)(39). We and others have used a recombinant U 1-70-kd fusion protein and deletion mutants derived from the fusion protein for epitope mapping of the fine specificity of human anti-U 1-70-kd autoantibodies (13,20,(33)(34)(35)(36)(37)(38)(39). Using an ELISA (13.20,33) and immunoblotting techniques (34)(35)(36)(37)(38)(39).…”

Cloning of B cell lymphoma-associated antigens using modified phage-displayed expression cDNA library and immunized patient sera

“…The sequence of the second peptide, FL-aa-18, matched to alternative frame of U1-snRNP 70K (NM_003089) (Theissen et al, 1986), but its deduced peptide sequence was in a different reading frame (Fig. 6A).…”

Human autoantibody secreted by immortalized lymphocyte cell line against the 68k polypeptide of the u1 small nuclear ribonucleoprotein

“…MAbs to polypeptides in these complexes are very useful in the investigation of the biochemical and antigenic structures of the complexes. Cloning of complementary DNA encoding the 68K polypeptide has led to new information regarding the nucleic acid sequence and inferred amino acid sequence of this polypeptide (37,38). In addition, a region of homology has been found in the 68K polypeptide that is cross-reactive with retroviral p30gag protein antigen, which could indicate a role for retroviruses in the initiation of autoimmunity (37).…”