Pre-mRNA splicing serves as an essential source of mRNA diversity in higher eukaryotes by joining discontinuous regions of gene transcripts in alternative patterns (Wang et al.
). The fidelity of pre-mRNA splice-site choice is critical, since an error of even a single nucleotide can be lethal or lead to human genetic diseases (reviewed in Wang & Cooper, 2007
). A dynamic ribonucleoprotein complex called the spliceosome assembles at consensus pre-mRNA sequences, where it removes the introns and ligates the exons into mature mRNA by two successive transesterification reactions (Weiner, 1993
; Michel & Ferat, 1995
). A branch-point sequence (BPS) located near the 3′ splice site contains an adenosine that ultimately serves as the nucleophile in the splicing reaction (Padgett et al.
). The BPS usually accompanies a polypyrimidine tract (Irimia & Roy, 2008
) and the proximity of the two sequences is important for splice-site identification (Reed, 1989
Splicing factor 1 (SF1) recognizes the BPS in the early stages of pre-mRNA splicing. A tight complex between SF1 and U2 auxiliary factor 65 kDa subunit (U2AF65
) undergoes conformational changes that couple BPS with polypyrimidine-tract recognition (Gupta et al.
). In turn, the SF1/U2AF65
/pre-mRNA complex facilitates stable association of the small nuclear ribonucleoprotein particles to form the active spliceosome (Wahl et al.
). The importance of SF1 in early spliceosomal assembly is emphasised by its requirement for embryonic development in mice and viability in human cells and yeast (Shitashige et al.
; Tanackovic & Krämer, 2005
; Abovich & Rosbash, 1997
). Depletion of SF1 from yeast or human extracts decreases the efficiency of spliceosome assembly on traditional splicing substrates (Guth & Valcárcel, 2000
; Rutz & Séraphin, 1999
). More recently, SF1 has been shown to be required for alternative splicing of transcripts encoding fibroblast growth factor receptor 1 oncogene partner, TNFAIP3-interacting protein 1, procollagen-lysine 1, 2-oxoglutarate 5-dioxygenase 2 and UPF3 regulator of nonsense transcript homologue A (Corioni et al.
). SF1 also plays roles in pre-mRNA retention in yeast (Rutz & Séraphin, 2000
) and transcription activation and elongation in humans (Zhang et al.
; Goldstrohm et al.
The majority of SF1 domains with known functions in mediating protein–protein or protein–RNA interactions during pre-mRNA splicing belong to well characterized fold families (Fig. 1
). A short N-terminal SF1 region termed a ‘U2AF ligand motif’ (ULM) inserts a conserved tryptophan into a hydrophobic pocket of a C-terminal ‘U2AF homology motif’ (UHM) of U2AF65
(Selenko et al.
; Kielkopf et al.
). A central hnRNP K homology motif and the adjoining quaking 2 (KH-QUA2) region specifically contact the BPS, in particular the key branch-site adenosine (Berglund et al.
; Liu et al.
). A poorly conserved domain composed of zinc knuckles contributes to the RNA affinity of non-vertebrate SF1 homologues, but is [;dispensable for RNA binding by vertebrate SF1 homologues (Berglund et al.
; Garrey et al.
). At the C-terminus, a proline-rich domain of SF1 interacts with WW-domain-containing splicing factors (Abovich & Rosbash, 1997
; Goldstrohm et al.
; Bedford et al.
Figure 1 (a) Domain boundaries of human SF1. ULM, U2AF ligand motif; KH-QUA2, hnRNP K homology and quaking 2 motif; Zn, zinc knuckle; Pro-rich, proline-rich domain. (b) Sequence alignment of human (NCBI RefSeq NP_004621) and S. cerevisiae (NCBI RefSeq NP_013217) (more ...)
In contrast, the structure and function of an approximately 100-amino-acid region of SF1 between the ULM and the KH-QUA2 domains (residues 26–132) are poorly defined at present. Accordingly, we call this region the SF1 ‘mystery domain’ (SF1md). The SF1md is the most highly conserved region of the protein (48% sequence identity between Saccharomyces cerevisiae
and human SF1md, compared with 35% identity between the full-length sequences; Fig. 1
). In human cells, serines 80 and 82 of an SF1 ‘SPSP’ motif are phosphorylated by U2AF homology motif kinase 1 (UHMK1; Manceau et al.
). Although no clear UHMK1 homologues are evident in S. cerevisiae
, the SPSP motif of yeast SF1 is conserved and an adjoining PPx
Y motif has been suggested to mediate interactions with the WW domain of the splicing factor Prp40p (Abovich & Rosbash, 1997
). Despite these functional implications, the SF1md lacks detectable homology with known structures. As a step towards determining this potentially novel structure, we report the expression, purification, circular-dichroism (CD) spectrum, crystallization and preliminary X-ray analysis of human SF1md.