2078–2089 Nucleic Acids Research, 2005, Vol. 33, No. 7 doi:10.1093/nar/gki338 Tissue-dependent isoforms of mammalian Fox-1 homologs are associated with tissue-specific splicing activities Shingo Nakahata and Sachiyo Kawamoto* Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA Received as resubmission February 14, 2005; Accepted March 16, 2005 DDBJ/EMBL/GenBank accession nos AY659951–AY659958 ABSTRACT An intronic hexanucleotide UGCAUG has been shown to play a critical role in the regulation of tissue- specific alternative splicing of pre-mRNAs in a wide range of tissues. Vertebrate Fox-1 has been shown to bind to this element, in a highly sequence-specific manner, through its RNA recognition motif (RRM). In mammals, there are at least two Fox-1-related genes, ataxin-2 binding protein 1 (A2BP1)/Fox-1 and Fxh/Rbm9, which encode an identical RRM. Here, we demonstrate that both mouse Fxh and A2BP1 transcripts undergo tissue-specific alternative spli- cing, generating protein isoforms specific to brain and muscle. These tissue-specific isoforms are char- acterized for their abilities to regulate neural cell- specific alternative splicing of a cassette exon, N30, in the non-muscle myosin heavy chain II-B pre-mRNA, previously shown to be regulated through an intronic distal downstream enhancer (IDDE). All Fxh and A2BP1 isoforms with the RRM are capable of binding to the IDDE in vitro through the UGCAUG elements. Each isoform, however, shows quantitative differ- ences in splicing activity and nuclear distribution in transfected cells. All Fxh isoforms and a brain isoform of A2BP1 show a predominant nuclear localization. Brain isoforms of both Fxh and A2BP1 promote N30 splicing much more efficiently than do the muscle- specific isoforms. Skeletal muscles express addi- tional isoforms that lack a part of the RRM. These isoforms are incapable of activating neural cell- specific splicing and, moreover, can inhibit UGCAUG-dependent N30 splicing. These findings suggest that tissue-specific isoforms of Fxh and A2BP1 play an important role in determining tissue specificity of UGCAUG-mediated alternative splicing. INTRODUCTION Alternative splicing of pre-mRNA is one of the fundamental mechanisms for the regulation of gene expression in higher eukaryotes (1,2). Developmentally regulated, cell type- or tissue-speciﬁc, and signal-induced alternative splicing of pre-mRNAs takes place in multicellular organisms throughout their lifetimes. Misregulation or abnormalities in pre-mRNA splicing, in some instances, leads to cellular dysfunctions found in human and animal diseases (3–5). Using various model systems of regulated alternative splicing, a number of pre-mRNA features that inﬂuence alternative splice site selection have been deﬁned (1,2). These include enhancer and repressor RNA sequences located in exons and introns. Identiﬁcation of RNA-binding proteins targeting these cis- regulatory elements is currently in progress. In vertebrates, participation of the SR family proteins and hnRNP proteins, such as PTB and hnRNPA1, in alternative splicing regulation via binding to the cis-regulatory elements have been shown in many tissue-speciﬁc splicing models (6,7). Although these RNA-binding proteins are ubiquitously expressed, their dif- ferent abundance in different cells, differences in their post- translational modiﬁcations in different cellular contexts and their different abilities to assemble multiprotein complexes in different pre-mRNA contexts are thought to contribute to the determination of cell type-speciﬁc patterns of alternative splicing. For the last few years, tissue-speciﬁc and tissue- enriched RNA-binding proteins have begun to be identiﬁed as splicing regulators. These include brain-speciﬁc (or enriched) Nova-1, nPTB (brPTB) and some of the CELF fam- ily proteins (8–12). Discovery of these proteins has had a great impact on studies aimed at understanding the molecular mech- anisms of alternative splicing regulation. *To whom correspondence should be addressed at National Institutes of Health, Building 10, Room 8N202, 10 Center Dr MSC 1762, Bethesda, MD 20892-1762, USA. Tel: +1 301 496 1912; Fax: +1 301 402 1542; Email: KawamotS@MAIL.NIH.GOV ª The Author 2005. Published by Oxford University Press. All rights reserved. The online version of this article has been published under an open access model. 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