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

Department of Biochemistry, Radboud University Nijmegen, Nymegen, Gelderland, Netherlands
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 binds
65Zn2+. 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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Available from: Sylviane Muller
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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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    ABSTRACT: The U1 small nuclear ribonucleoprotein (U1 snRNP) binds to the pre-mRNA 5' splice site (ss) at early stages of spliceosome assembly. Recruitment of U1 to a class of weak 5' ss is promoted by binding of the protein TIA-1 to uridine-rich sequences immediately downstream from the 5' ss. Here we describe a molecular dissection of the activities of TIA-1. RNA recognition motifs (RRMs) 2 and 3 are necessary and sufficient for binding to the pre-mRNA. The non- consensus RRM1 and the C-terminal glutamine-rich (Q) domain are required for association with U1 snRNP and to facilitate its recruitment to 5' ss. Co-precipitation experiments revealed a specific and direct interaction involving the N-terminal region of the U1 protein U1-C and the Q-rich domain of TIA-1, an interaction enhanced by RRM1. The results argue that binding of TIA-1 in the vicinity of a 5' ss helps to stabilize U1 snRNP recruitment, at least in part, via a direct interaction with U1-C, thus providing one molecular mechanism for the function of this splicing regulator.
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    ABSTRACT: To study the localization and function of the U1snRNP associated U1C protein, so far only human sera from systemic lupus erythematosus (SLE) overlap syndrome patients have been used. Here we report for the first time the isolation of human monoclonal anti-UIC autoantibody fragments from IgG derived combinatorial and semi-synthetic human antibody libraries. Two classes of human monoclonal anti-UIC (auto)antibodies were found: specific anti-U1C autoantibodies, recognizing U1C only, and cross-reactive antibodies which also react with U1A and Sm-B/B'proteins. The heavy chains (V(H)genes) of all five antibodies from the semi-synthetic libraries and two of the three U1C-specific patient derived autoantibody fragments are encoded by V(H)3 genes, in which V(H) 3-30 (DP-49) was overrepresented. The heavy chain of the two cross-reactive autoantibodies are derived from the 3-07 (DP-54) gene. Three epitope regions on the U1C protein are targeted by these antibodies. (1) Four U1C specific antibodies recognize an N-terminal region of U1C in which amino acids 30-63 are essential for recognition, (2) two antibodies recognize only the complete U1C protein, and (3) two cross-reactive and one U1C specific antibody recognize the C-terminal domain in which amino acids 98-126 are critical for recognition. The two cross-reactive antibodies (K 11 and K 15) recognize the proline-rich region of the U1C protein (amino acids 98 126) and cross-react with proline-rich regions in Sm-B/B' (amino acids 163-184) and U1A (amino acids 187-204). All 10 antibody fragments are able to immunoprecipitate the native U1snRNP particle. The two cross-reactive antibodies immunoprecipitate the other Sm containing snRNPs as well. Using confocal immunofluorescence microscopy we could show that the major part of the U1C protein is localized within the coiled body structure.
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