👤 W J van Venrooij

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. Show less

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. Show less

Genes for the snRNP proteins U1-70K, U1-A, Sm-B'/B, Sm-D1 and Sm-E have been isolated from various metazoan species. The genes for Sm-D1 and Sm-E, which were isolated from a murine and human source respectively, appear to belong to a multigene family. It has been suggested that also for the mammalian U1-C protein such a multigene family exists. With the human U1-C cDNA as a probe, two genes containing sequences homologous to the probe sequence were isolated from a mouse genomic library. Simultaneously, a murine U1-C cDNA was isolated from a mouse cDNA library. This 0.74 kb cDNA contains an open reading frame (ORF) of 477 bp encoding a polypeptide of 159 amino acids (aa) which differs at only one position (position 65) from the human U1-C protein. One of the isolated U1-C genes contains an ORF as well and shares 92% nucleotide sequence identity with the mouse U1-C cDNA. The features of this gene, in particular the absence of introns, the acquisition of a 3' poly(A) tail and flanking direct repeats, indicate that it represents a processed pseudogene. At the predicted aa sequence level, substitutions of conserved residues at functionally important positions are observed, strongly suggesting that expression of this gene would not lead to a functional polypeptide. The second U1-C gene appeared to be a pseudogene as well because it is also intronless and contains a frameshift mutation compared to the ORF in the mouse U1-C cDNA. The characterization of these two pseudogenes points to the existence of a U1-C multigene family in mice. Furthermore, comparison of aa sequences of the murine, human and Xenopus U1-C shows that the protein is highly conserved through evolution. Since the Xenopus U1-C differs from the two mammalian counterparts solely at a number of positions in the C-terminal region, it can be concluded that aa changes are less well tolerated in the N-terminal region of U1-C than in the rest of the protein. Show less