The Structure and Biochemical Properties of the Human Spliceosomal Protein U1C Yutaka Muto 1,2 †, Daniel Pomeranz Krummel 1 †, Chris Oubridge 1 † Helena Hernandez 3 , Carol V. Robinson 3 , David Neuhaus 1 and Kiyoshi Nagai 1 * 1 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK 2 Protein Research Group RIKEN Genomic Sciences Center, Suehiro-cho Tsurumi-ku, Yokohama Kanagawa 230-0045, Japan 3 Cambridge University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW UK The spliceosomal U1C protein is critical to the initiation and regulation of precursor messenger RNA (pre-mRNA) splicing, as part of the U1 small nuclear ribonucleoprotein particle (snRNP). We have produced full-length and 61 residue constructs of human U1C in soluble form in Escherichia coli. Atomic absorption spectroscopy and mass spectrometry show that both constructs contain one Zn atom and are monomeric. Gelmobility-shift assays showed that one molecule of recombinant U1C, either full-length or 61 residue construct, can be incorporated into the U1 snRNP core domain in the presence of U1 70k. This result is in perfect agreement with the previous experiment with U1C isolated from the HeLa U1 snRNP showing that the recombinant U1C is functionally active. We have determined the solution structure of the N-terminal 61 residue con- struct of U1C by NMR. A Cys 2 His 2 -type zinc finger, distinct from the TFIIIA-type, is extended at its C terminus by two additional helices. The two Zn-coordinating histidine residues are separated by a five residue loop. The conserved basic residues in the first two helices and the inter- vening loop may be involved in RNA binding. The opposite b-sheet face with two surface-exposed Tyr residues may be involved in protein con- tacts. Both the full-length and 61 residue constructs of human U1C fail to bind RNA containing the 5 0 splice site sequence, in contrast to what has been reported for the Saccharomyces cerevisiae orthologue. q 2004 Elsevier Ltd. All rights reserved. Keywords: mass spectrometry; NMR; pre-mRNA splicing; U1C; zinc-finger *Corresponding author Introduction Excision of introns and splicing together of exons from pre-mRNA occurs in the nucleus through two successive trans-esterification reactions within a large and highly dynamic macromolecular assem- bly termed the spliceosome. 1 The spliceosome is composed of four (U1, U2, U5 and U4/U6) small nuclear ribonucleoprotein particles (snRNPs) that assemble onto pre-mRNA together with accessory proteins that often have a regulatory role. 2 The critical initial event in pre-mRNA splicing is the recognition of the 5 0 exon–intron junction (the 5 0 splice site) by U1 snRNP, an ATP-independent assembly step that is commonly referred to as for- mation of the commitment complex. 3–7 U1 snRNP is the best characterized of the spli- ceosomal snRNPs, both biochemically and func- tionally. Human U1 snRNP contains a single RNA and ten protein subunits. 1 The RNA subunit of U1 snRNP is 165 nucleotides long, has a trimethyl- guanosine cap at its 5 0 end, and folds into a second- ary structure consisting of four stem-loop motifs (Figure 1A). The single-stranded 5 0 end of U1 snRNA is complementary to the conserved sequence at the 5 0 splice site and hence plays an important role in 5 0 splice site recognition via base- pairing. 3–7 Seven Sm proteins (B or B 0 , D1, D2, D3, E, F and G) assemble to form a heptameric ring known as the core domain around the U1 snRNA Sm site, 8,9 a single-stranded region located between 0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. Supplementary data associated with this article can be found at doi: 10.1016/j.jmb.2004.04.078 † Y.M., D.P.K. and C.O.contributed equally. E-mail address of the corresponding author: kn@mrc-lmb.cam.ac.uk Abbreviations used: snRNP, small nuclear ribonucleoprotein particle; snRNA, small nuclear RNA; RBD, RNA-binding domain. doi:10.1016/j.jmb.2004.04.078 J. Mol. Biol. (2004) 341, 185–198