2086 Nucleic Acids Research, 2005, Vol. 33, No. 7 Figure 7. Antagonistic effect of the RRM-lacking isoform of Fxh on UGCAUG-dependent N30 inclusion in butyrate-treated Y79 cells. The indi- cated minigenes (see Figure 4A for diagram) were co-transfected with increas- ing amounts (0.7, 2 and 6 mg, indicated by right triangles) of the expression construct for the indicated protein or with an empty vector in Y79 cells. The cells were treated with butyrate to induce differentiation of Y79 cells. The minigene mRNAs were analyzed by RT–PCR. Dose-dependent protein expres- sion was verified by immunoblot analysis using an anti-myc antibody. nuclei efﬁciently, this also raises the possibility that the RRM-defective isoforms may have an inhibitory function on the N30 splicing in muscle cells. DISCUSSION Two major ﬁndings are described in this report. First, Fxh and A2BP1 facilitate neural cell-speciﬁc inclusion of the cassette- type exon via binding to the speciﬁc intronic sequence UGCAUG. In addition to a minigene model system, Fxh and A2BP1 are capable of facilitating N30 inclusion of the endogenous pre-mRNA. This result provides an important demonstration of physiological relevance and supports the notion that the NMHC-B pre-mRNA is likely to be the true target for Fxh or A2BP1-mediated regulation. However, whether the endogenous Fxh or A2BP1 regulates endogenous NMHC-B pre-mRNA splicing needs to be determined in a future study. In vertebrates, small interfering RNAs and gene targeting strategies have recently been used successfully to address the roles of endogenous splicing regulators in alternative splicing of endogenous target pre-mRNAs (8,32–36). A second and more novel ﬁnding is the identiﬁca- tion of tissue-speciﬁc isoforms of Fxh and A2BP1 with dif- ferent splicing activities as well as different subcellular localizations. This ﬁnding raises the possibility that the pro- ducts of the Fxh and A2BP1 genes can contribute to a mech- anism as to how tissue speciﬁcity of alternative splicing is achieved. Many splicing factors are detected not only in the nuclei, but also in the cytoplasm (37). They are shuttling between the nucleus and the cytoplasm and, in some instances, extracel- lular stimuli trigger changes in subcellular distribution of these proteins. Such translocations have been reported for hnRNPA1 and PTB (38,39). Moreover, a number of RNA-binding pro- teins have been demonstrated to play a role in multiple steps during gene expression in different subcellular compartments, such as pre-mRNA processing in nuclei, mRNA export from nuclei to cytoplasm and mRNA localization, stability and translation in cytoplasm (37,40). Therefore, not surprisingly, Fxh and A2BP1 isoforms were found to be distributed in both the nuclei and the cytoplasm in HeLa and Y79 cells. However, the relative ratios of proteins distributed between the two subcellular compartments at steady-state differ among the isoforms. In agreement with Jin et al. (21), substantial amounts of the brain isoform A016 are detected in nuclei. Other A2BP1 isoforms, the brain isoform A030 and the muscle isoforms A713 and A715, are only poorly detected in nuclei. This observation is consistent with the reports where endogenous A2BP1 in cerebellar Purkinje cells, hippocampus neurons and cardiac myocytes were shown to be localized essentially to the cytoplasm (23,24). Thus, inclusion and exclusion of A53 and differences in the very N-terminal sequences results in A2BP1 isoforms with a distinct subcellular localization. It is likely that A2BP1 proteins have multiple roles, involving both nuclear and cytoplasmic events. In contrast, all three Fxh isoforms predominantly localized to the nuclei. Therefore, in terms of their localization, Fxh proteins are better candidates for regulators of the pre-mRNA splicing that takes place in nuclei. Of note, however, our preliminary results of 50 RACE, as well as the EST database, detect multiple 50 end sequences for both Fxh and A2BP1 mRNAs, which are presumably generated by alternative promoters and alternative splicing. The diversity of the 50 end cDNA sequences leads to the generation of a number of unique N-terminal amino acid sequences. Therefore, this study does not exclude the possible existence of other isoforms with different subcellular localizations for both Fxh and A2BP1. Our study also does not exclude the possibility that some of the isoforms translocate between the nucleus and the cytoplasm following stimuli. The main aim of this study is to determine the relative activities of tissue-dependent isoforms of Fxh and A2BP1 in neural cell-speciﬁc and UGCAUG element-dependent alternative splicing. To obtain an indication of the relative speciﬁc activity of each isoform in transfected cells, the same amounts of the expressed proteins should be available for the splicing reaction in the nuclei. For this reason, an exogenous NLS was included in the expressed proteins. Essen- tially, all of the expressed proteins with the exogenous NLS localized to nuclei. Thus, the amounts of the expressed pro- teins determined by immunoblots represent the nuclear con- centrations. The analysis using the proteins expressed with the exogenous NLS allowed us to compare directly the splicing activities of these proteins. Furthermore, this analysis also allowed us to deﬁne the critical regions of the proteins for splicing activation. As shown in Figure 5, the splicing activities of the various isoforms of Fxh and A2BP1 are intrinsically different, regard- less of the subcellular localization properties of the wild-type proteins. Among the isoforms tested in this study, F011 and A016, which include B40, are found to have higher activities in promoting N30 inclusion. When the primary amino acid sequences, outside of the RRM, of these two proteins are compared, the C-terminal regions (amino acids 190–377 of F011) show a higher homology with 71% identity, whereas the N-terminal regions (amino acids 1–112 of F011) show only 53% identity. The C-terminal region includes four subregions of nearly identical stretches of amino acids (Figure 8, I–IV). One subregion (II) includes 13 amino acids encoded by exon B40. Substitution of this subregion with exon M43 in Fxh causes substantial changes in amino acid sequences resulting in only a 21% identity in this region between F011 and F411. Another subregion (IV) is located at the C-terminal end and A030 lacks this homologous region by the inclusion of exon