Molecular cloning of the cDNA for the human U2 snRNA-specific A' protein

• Published in: Nucleic acids research Vol. 17; no. 5; pp. 1893 - 1906
• Main Authors: SILLEKENS, P. T. G, BEIJER, R. P, HABETS, W. J, VAN VENROOIJ, W. J
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 11.03.1989
• Subjects: Amino Acid Sequence; Base Sequence; Biological and medical sciences; Cloning, Molecular - methods; DNA - isolation & purification; Fundamental and applied biological sciences. Psychology; genes; Genes. Genome; HeLa Cells; Humans; Immune Sera; Immunoblotting; man; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; ribonucleoproteins; RNA, Small Nuclear - genetics; RNA, Small Nuclear - immunology; RNA, Small Nuclear - isolation & purification; snRNA; Human; Ribonucleoprotein; sn-RNA; Messenger RNA; In vitro translation; Nucleotide sequence; Homology; Molecular cloning; Hela cell line
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/17.5.1893

The A' polypeptide is one of the protein constituents of the U2 snRNP particle. A potentially full-length cDNA clone containing the complete coding sequence for this U2 snRNP-specific protein was isolated by screening of a human lambda gt11 expression vector library with an autoimmune anti-(U1,U2)RNP serum. Monospecific antibodies, eluted from the 140-150 kD fusion protein of this cDNA recombinant, specifically recognized the A' protein on immunoblots and immunoprecipitated U2 snRNP particles from nuclear extracts. The identity of the clone was confirmed by in vitro translation of hybrid-selected mRNA or an RNA transcript synthesized from the cDNA insert. RNA blot analysis showed strong hybridization to a single polyadenylated transcript of 1.3 kb in human cells. The nucleotide sequence of the 1054 bp cDNA contains an open reading frame of 756 bp encoding a polypeptide of 255 amino acids with a predicted molecular weight of 28,444 D. The coding sequence is preceded by a 49 bp 5'-untranslated region and followed by a 226 bp 3'-untranslated region containing a single polyadenylation signal. Most striking feature of the deduced primary structure for the A' protein is a leucine-rich region in the amino-terminal half of the polypeptide. In contrast to the other U2 snRNP-specific protein B", the A' protein does not contain segments homologous to the RNP consensus sequences RNP1 and RNP2, common amino acid motifs found in several RNA-binding proteins. In the A' protein, however, the extremely hydrophilic carboxy terminus may constitute an RNA-binding moiety.