The Xenopus laevis hTrCP gene: genomic organization, alternative splicing, 5Vand 3Vregion characterization and comparison of its structure with that of human hTrCP genes $ Monica Ballarino, Marcella Marchioni, Francesca Carnevali * Consiglio Nazionale delle Ricerche, Centro di Studio per gli Acidi Nucleici, Rome, Italy Received 26 February 2002; received in revised form 27 May 2002; accepted 4 June 2002 Abstract hTrCP plays a relevant role in the control of stability of several key protein factors. In Xenopus, hTrCP acts as an inhibitor of Wnt signaling and dorsal axis formation. We determined the primary structure of the frog hTrCP gene, which consists of 14 exons and 13 introns, spanning over 34 kb. Isoforms of x-hTrCP have been found, which show differences in the NH 2 and COOH regions. NH 2 isoforms differ for the presence or absence of a 30 aa sequence, coded by exon III. In COOH isoforms, 19 C-terminal amino acids are replaced by three different amino acids. Occurrence of two 5V splice donor sites for splicing of intron XIII provides an explanation for these isoforms, based on alternative splicing. The DNA region of the putative hTrCP promoter contains several TATA elements, one GCCAAT box, and putative binding sites for Ets, Tcf/Lef and NF-nB transcription factors. Two transcription initiation sites have been mapped downstream of TATA boxes proximal to ATG for start of translation. Comparison of the Xenopus and human hTrCP genes indicates high conservation of exon nucleotide and amino acid sequences, size and organization; differences are limited to exons coding for N- and C-terminal regions. D 2002 Elsevier Science B.V. All rights reserved. Keywords: hTrCP gene; Alternative-splicing; Isoform; h-catenin; Carcinogenesis 1. Introduction Ubiquitin-mediated proteolysis has a central role in controlling intracellular levels of several important regula- tory molecules. Substrate specificity in ubiquitination reac- tions is in large part mediated by specific association of E3- ubiquitin ligases with their substrates [1]. One of the best characterized enzyme complexes that catalyzes attachment of ubiquitin to proteins is SCF [2], consisting of Skp1 [3],a Cullin family member [4], Hrt1p [5], and an F-box protein [3]. Among the subunits of the SCF complex, F-box proteins serve as receptors for specific substrates. hTrCP is the receptor for h-catenin, InBa, ATF4 and Vpu [6–11]. hTrCP consists of two essential modular domains: F-box and seven WD40 repeats [12]. F-box is required for binding to Skp1. A deletion mutant, lacking the F-box domain, acts as a dominant negative form and causes stabilization of the substrate, an effect opposite from that triggered by the full- length protein [6,13 – 15]. WD40 repeats are necessary for substrate interaction. Although all seven repeats are required for optimal binding, the first WD motif seems to be particularly important for interaction with target proteins [6]. In addition, a third domain, named domain D, located upstream of the F-box, has been shown to be responsible for protein dimerization [16]. No function has been assigned to N- and C-terminal ends, although direct and indirect evi- dences show that N- [8,17] or C-terminal [6] deletion mutations affect hTrCP activity. Phosphorylation of serine residues in the DSGXXS consensus sequence of substrate is necessary for binding by hTrCP and hence for subsequent ubiquitination and proteolysis processes [18–20]. The accumulation of un- phosphorylated substrates is often associated with delete- rious effects. In the case of h-catenin, phosphorylation in 0167-4781/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S0167-4781(02)00416-5 $ Nucleotide sequences have been submitted to the EMBL Data Libraries and assigned the accession numbers AJ428930 to AJ428940. * Corresponding author. Centro di Studio per gli Acidi Nucleici, CNR c/o Dipartimento di Genetica e Biologia Molecolare, Universita ` di Roma La Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy. Tel.: +39-6-4991- 2225; fax: +39-6-4991-2500. E-mail address: francesca.carnevali@uniroma1.it (F. Carnevali). www.bba-direct.com Biochimica et Biophysica Acta 1577 (2002) 81 – 92