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

Abstract

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.

The splicing regulator TIA-1 interacts with U1-C to promote U1 snRNP recruitment to 5' splice sites

“…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 μl of nuclear extracts complemented with buffer D to a total volume of 25 μl for 30 min on ice. After addition of 60 μl 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.…”

“…for 1 min, the pellet was resuspended in SDS loading buffer, fractionated together with the aliquot of supernatant by electrophoresis on a 10–15% SDS–polyacrylamide gel and transferred onto nitrocellulose (Schleicher & Schuell). The blots were probed with anti‐TIA‐1, anti‐U1A, anti‐U1A/U2B″, anti‐U1‐70k, anti‐U1C (Dumortier et al ., 1998; Hoet et al ., 1998), anti‐Sm (Y12; Pisetsky and Lerner, 1982) or anti‐GST antibodies. HRP‐conjugated anti‐mouse or anti‐rabbit antibodies were used as secondary reagents, and visualized by enhanced chemoluminescence (ECL; Amersham).…”

Understanding eukaryotic linear motifs and their role in cell signaling and regulation

“…They are used primarily because they can detect the surface exposed motifs. Typically this involves raising an antibody to a peptide that expresses the motif and then using this antibody to test the surface exposure or accessibility of the motif (for an example of this technique see (123)). The precise structure, or lack thereof, of bound peptides and motifs to domains or antibodies is not known in the majority of cases.…”

Purification and crystallization of spliceosomal snRNPs

“…The U1-C protein contains a C 2 H 2 -type zinc finger motif and does not bind to the snRNA but contacts U1-70k and B/B'. The interaction to U1-70k is mediated by the N-terminal zinc finger, which is not accessible to antibodies when U1-C is bound to U1 snRNP (Dumortier et al, 1998). Furthermore, U1-C has been implicated in 5' splice site binding by UV crosslinking assays (Nelissen et al, 1994(Nelissen et al, , 1991aRossi et al, 1996;Du and Rosbash, 2002).…”

“…The U1-C zinc finger comprises around 30-40 residues and is too small to be visualized on the SDS gel. However it has been shown that the N-terminus of U1-C, comprising the Zn-finger, is buried in the U1 snRNP particle (Dumortier et al, 1998). Depending on whether the U1-70k binding site was truncated or not, an N-terminal fragment of U1-C was presumably part of a degraded U1 snRNP.…”