At least three linear regions but not the zinc-finger domain of U1C protein are exposed at the surface of the protein in solution and on the human spliceosomal U1 snRNP particle

Institut de Biologie Moléculaire et Cellulaire, UPR 9021 CNRS, 15 rue Descartes, 67000 Strasbourg, France.
Nucleic Acids Research (Impact Factor: 9.11). 01/1999; 26(23):5486-91. DOI: 10.1093/nar/26.23.5486
Source: PubMed


No structural information on U1C protein either in its free state or bound to the spliceosomal U1 small nuclear ribonucleoprotein (snRNP) particle is currently available. Using rabbit antibodies raised against a complete set of 15 U1C overlapping synthetic peptides (16-30 residues long) in different immunochemical tests, linear regions exposed at the surface of free and U1 snRNP-bound U1C were identified. Epitopes within at least three regions spanning residues 31-62, 85-103 and 116-159 were recognized on free and plastic-immobilized recombinant human U1C expressed in Escherichia coli, on in vitro translated U1C protein and on U1C bound to the U1 snRNP particle present in HeLa S100 extract. Using a zinc affinity labeling method, we further showed that the N-terminal U1C peptide containing a zinc-finger motif (peptide 5-34) effectively binds65Zn2+. The N-terminal region of U1C, which is functional in U1 snRNP assembly, is apparently not located at the surface of the U1 snRNP particle.

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    • "Anti-U1A antibodies (856; Kambach and Mattaj, 1992), anti-TIA-1 (anti-2G9; Anderson et al., 1990), anti-U170k (16H3; Neugebauer et al., 1995) and anti-U1C (Dumortier et al., 1998) were bound to protein A±Sepharose beads and incubated in 15 ml of nuclear extracts complemented with buffer D to a total volume of 25 ml for 30 min on ice. After addition of 60 ml of IPP 150 buffer (10 mM Tris pH 8.0, 150 mM NaCl, 0.1% NP-40), the reaction was incubated for 2 h on a rotating wheel at 4°C. "
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