One of the intronic cis-elements, which are involved in tissue- speciﬁc or differentiation stage-dependent regulation of altern- ative splicing, is the hexanucleotide UGCAUG. The importance of this element was originally recognized in ﬁbronectin pre- mRNA by Huh and Hynes (13). Since then, alternative splicing speciﬁc to a variety of tissues or cell types, including neural cells, muscles, epithelial cells and erythrocytes, has been shown to be modulated via this element (14–21). Recently, Jin et al. (21) discovered that a zebraﬁsh homolog of Caenorhabditis elegans Fox-1 (22) could bind speciﬁcally to the pentanuc- leotide GCAUG by in vitro selection from randomized RNA sequences. This pentanucleotide is almost identical to the hexa- nucleotide UGCAUG except for the ﬁrst U. Moreover, the zeb- raﬁsh Fox-1 homolog, as well as the mouse Fox-1 homolog, are capable of repressing the inclusion of an alternative cassette exon of the ATP synthase F1g pre-mRNA via binding to GCAUG, which mimics muscle-speciﬁc exclusion of this exon. This mouse homolog is identical to the ataxin-2 binding protein 1 (A2BP1), which has been previously cloned in humans and mice as the cDNA encoding a protein, which interacts with ataxin-2, the product of the causative gene for spinocerebellar ataxia type 2 (23,24). In addition to A2BP1/Fox-1, another mouse homolog of C.elegans Fox-1, Fxh, has been independ- ently cloned as a cDNA, which is induced by androgen in motor neurons (25). Of note is that A2BP1/Fox-1 and Fxh share an identical RNA recognition motif (RRM) at the amino acid level. Therefore, two genes in the mouse genome encode homo- logs of nematode Fox-1. According to the names given by the ﬁrst cDNA cloning, we used the nomenclature of A2BP1 and Fxh in this report. A2BP1 and Fxh have been named A2BP1 and Rbm9, respectively, in the human and mouse genomes. We have been studying regulatory mechanisms of neural cell-speciﬁc alternative splicing using the non-muscle myosin heavy chain II-B (NMHC-B) gene as a model system (14,26). NMHC-B mRNA is expressed ubiquitously. However, an alternative exon, N30, which encodes a 30 nt coding sequence, is included in the mRNAs from some neural cells, but is skipped in those from all other cells in mammals and birds (27,28). In cultured cells, a switch in N30 splicing from exclusion to inclu- sion can be seen in neural retinoblastoma Y79 cells during the post-mitotic and differentiated stages triggered by butyrate treatment. We have previously deﬁned an intronic distal down- stream enhancer (IDDE), which confers neural cell speciﬁcity on N30 inclusion, using this cell line (14). The IDDE includes two copies of UGCAUG. Mutation of these hexanucleotides results in N30 skipping in post-mitotic differentiated Y79 cells. In this study, we investigated the possible involvement of A2BP1 and Fxh in the regulation of N30 splicing. To this end, we have isolated cDNA clones for A2BP1 and Fxh from brain and muscles. cDNA cloning revealed the existence of tissue-speciﬁc (enriched) isoforms of both A2BP1 and Fxh. Of importance, different isoforms of A2BP1 and Fxh show different activities with respect to N30 splicing as well as different subcellular localizations. MATERIALS AND METHODS Database disposition The sequences reported in this paper have been deposited in the GenBank database with accession numbers AY659951 (F011), Nucleic Acids Research, 2005, Vol. 33, No. 7 2079 AY659952 (F411), AY659953 (F402), AY659954 (A016), AY659955 (A030), AY659956 (A713), AY659957 (A715) and AY659958 (A704). RNA preparation and RT–PCR Total RNAs were isolated from mouse tissues and cultured cells using an RNA isolation kit (Stratagene) or an RNeasy mini kit (Qiagen). To obtain the full-length coding regions of cDNAs for Fxh and A2BP1, RT–PCRs were performed using Superscript II RNase H reverse transcriptase (Invitrogen) and Pfu Turbo DNA polymerase (Stratagene). The PCR primers used to obtain all Fxh cDNAs were 50-ctcaggcctccactagttATG- GAGAAAAAGAAAATGGTAACTC-30 and 50-ctcaggcctcct- ctagaaGTAGGGGGCAAATCGGCTGTA-30. The upstream primers for the brain (A016 and A030) and muscle (A713, A715 and A704) A2BP1 cDNAs were 50-ctcaggcctccactagtg- ATGAATTGTGAAAGAGAGCAGCT-30 and 50-ctcaggcctc- cactagtcATGTTGGCGTCGCAAGGAGTCC-30, respectively, and the downstream primer for all A2BP1 cDNAs was 50-ctcaggcctcctctagagATATGGAGCAAAACGGTTGTATCC -30. Lower case letters represent adapter sequences including restriction enzyme sites. 50 Rapid ampliﬁcation of cDNA ends (RACE) was performed using Marathon-Ready cDNA (BD Biosciences Clontech). For the analysis of minigene and NMHC-B mRNAs, RT–PCRs were performed as described previously (14,26). Sequences of primers P1–P9 shown in Figures 1, 4 and 6 are as follows: P1, 50-AATTCACCCAG- CAACCAGAAT-30; P2, 50-TAGAGGGATGTAAGTGTTG- ATGCC-30; P3, 50-CAGAGGGCGGACAGTGTATGGT-30; P4, 50-GGCGGCAGGGGCGAGGGCAT-30; P5, 50-CCGTG- GTCGCACCGTGTACAAC-30; P6, 50-CAGCGGCAGTGG- CAGGGGTG-30; P7, 50-AGGAAGAAAGGACCATAATAT- TCC-30; P8, 50-CCTCCACCCAGCTCCAGTTGT-30; and P9, 50-CCTGTAGTTATTAAATCCTTCAAG-30. Preparation of expression constructs and minigenes The cDNAs of Fxh and A2BP1 were introduced into a plasmid pCS3 + MT, which contains a myc-epitope, and its modiﬁed version pCS3 + MT + NLS, which in addition contains the nuclear localization signal (NLS) of the SV40 large T antigen (26). Minigenes G, J without the IDDE, and H with the wild- type IDDE are the same as minigenes W, D4 and Cm0, respectively in ref. (14). The 201 nt IDDEs with mutations were generated by recombinant PCR using the appropriate primers, which included mutated sequences. The hexanuc- leotide TGCATG sequences at the 50 and 30 sides were chan- ged to GTTACT and ACCTAC, respectively. Electrophoresis mobility shift assay Template DNAs for in vitro RNA transcription were prepared by PCR using the wild-type and mutant IDDEs in the mini- genes as templates and an upstream primer that included the T7 promoter sequence at the 50 end. The probe and com- petitor RNAs were transcribed by T7 RNA polymerase in the presence of [a-32P]UTP and a trace amount of [35S]UTPaS, respectively, using a MAXIscript kit (Ambion). Mole concentrations of synthesized RNAs were estimated by radioactivities. Fxh and A2BP1 proteins with a myc tag were synthesized in vitro by using a TNT quick-coupled