Identification and preliminary functional analysis of alternative splicing of Siah1 in Xenopus laevis Luan Wen a,b , Jiantao Liu a,c , Yonglong Chen a , Donghai Wu a, * a Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Road, Guangzhou Science Park, 510530 Guangzhou, China b Graduate University of Chinese Academy of Sciences, 100049 Beijing, China c Laboratory of Veterinary Pharmacology, College of Veterinary Medicine South China Agricultural University, Guangzhou, China article info Article history: Received 11 April 2010 Available online 22 April 2010 Keywords: Xenopus xSiah1a xSiah1b abstract Siah proteins are vertebrate homologs of the Drosophila ‘seven in absentia’ gene. In this study, we char- acterized two splicing forms, Siah1a and Siah1b, of the Xenopus seven in absentia homolog 1 gene (Siah1). Overexpression of xSiah1a led to severe suppression of embryo cleavage, while that of xSiah1b was not effective even at a high dose. Competition analysis demonstrated that co-expression of xSiah1a and 1b generated the same phenotype as overexpression of xSiah1a alone, suggesting that xSiah1b does not interfere with the function of xSiah1a. Since xSiah1b has an additional 31 amino acids in the N-terminus compared to xSiah1a, progressive truncation of xSiah1b from the N-terminus showed that inability of xSiah1b to affect embryo cleavage was associated with the length of the N-terminal extension of extra amino acids. The possible implication of this finding is discussed. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction The seven in absentia homolog (Siah) proteins are homologs of the Drosophila seven in absentia (sina) gene. This gene codes for a protein downstream from the Sevenless tyrosine kinase receptor that degrades the transcriptional repressor Tramtrack and is re- quired for R7 cell fate specification during Drosophila eye develop- ment [1–3]. Humans have two unlinked Siah genes, SIAH1 and SIAH2 [4], but mice have two Siah1 (Siah1a and Siah1b) and single Siah2 genes [5]. The Siah1 protein contains an N-terminal RING fin- ger domain, required for interaction with E2 ubiquitin conjugating enzymes [6], as well as a coiled coil domain to form homo- and heterodimers [7]. As an essential component of E3 ubiquitin ligase complexes, Siah1 is responsible for the ubiquitin proteosome deg- radation that terminates cellular signaling and also for turnover of many key proteins, such as DCC [8], b-catenin [9], Kid [10], c-Myb [11], OBF-1 [12,13], Numb [14], Synaptophysin [15], TIEG-1 [16], Synphilin-1 [17], CtIP [18], T-STAR [19], Polycystin [20], Af4 [21], PHD1 and PHD3 [22], HIPK2 [23], FIH [24,25], PLCepsilon [26] and TRB3 [27]. Siah1a knockout mice exhibit severe growth retardation and male sterility due to a blockade in spermatogenesis [28]. Several splicing variants of SIAH1 appear to play important regulatory roles; for example, SIAH1S, an alternative splicing form of SIAH1, acts as a dominant negative inhibitor of SIAH1 in the regulation of b-catenin activity [29]. SIAH1L, another splicing variant of SIAH1, is induced in response to p53 and plays a key role in the regulation of b-catenin activity [9,30,31]. In Xenopus laevis, overexpression of xSiah2 led to small eyes [32] due to degradation of PHD2 [33], indicating its critical role in retinal development. However, the function of Siah1 in Xenopus embryogenesis still remains largely unknown. In the present study, we report for the first time the identification of two splicing forms of Siah1 in X. laevis and their distinct functional roles. 2. Materials and methods 2.1. Isolation of xSiah1a and xSiah1b in X. laevis Two Xenopus Siah1protein sequences were found based on a Blastp search using the human Siah1 coding sequence; one was from X. laevis (NP_001085438), the other was from the Xenopus tropicalis (NP_001015836). The predicted open reading frame (ORF) of the X. laevis gene was longer than that of X. tropicalis. Com- parison of the genomic organization of the two ORFs using Blat re- vealed that the X. laevis Siah1 has an alternative exon in the UTR (Fig. 2A). The full X. laevis Siah1a sequence (GU377277) was cloned by RT-PCR using the forward primer 5 0 -TCCGGTGTCTTTCTATG- GAAGCGGT-3 0 and the reverse primer 5 0 -CATGTACACACCCAG- CTGGGCATCTTTTGTA-3 0 . The correct clones were verified by sequencing. 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.04.107 * Corresponding author. E-mail address: wu_donghai@gibh.ac.cn (D. Wu). Biochemical and Biophysical Research Communications 396 (2010) 419–424 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc