Next, the splicing activities of the wild-type proteins without an exogenous NLS were examined. Representative data are shown in Figure 5B (lanes 2–7) and the relative activities are summarized in Figure 1A (wt). The protein amounts detected by immunoblots in these experiments rep- resent the total amounts of the proteins distributed to both the nuclei and the cytoplasm. The splicing activities of the wild- type proteins are consistent with the activities that combine the splicing activities of the proteins with the exogenous NLS and the activities of the native proteins to localize to nuclei. In the absence of the exogenous NLS (wt), the brain isoforms F011, and A016 to a lesser extent, are still capable of activating N30 inclusion efﬁciently. The muscle isoforms for both Fxh and A2BP1 (F411, A713 and A715), as well as the brain isoform A030, show only minimal activities. Therefore, F011 and A016 appear to have the most physiological relevance to N30 splicing activation. Overexpression of Fxh and A2BP1 activates N30 inclusion of endogenous NMHC-B mRNAs The human NMHC-B gene consists of 41 constitutive exons and 3 alternative exons. Its pre-mRNA is 156 kb in length and it is much more complex than the pre-mRNA from the minigenes. In addition, the minigene pre-mRNAs are driven by a heterologous promoter. Therefore, we next examined if Fxh and A2BP1 were capable of promoting N30 inclusion of the endogenous transcript. Y79 cells were stably transfected with the expression construct for F011 or A016. Both F011 and A016 are enriched in the brain and show higher activation of N30 inclusion in the minigene transcripts. mRNAs encoding endogenous NMHC-B were analyzed by using RT–PCR. As shown in Figure 6, the inclusion of exon N30 with and without another alternative exon, R18, in the endogenous mRNAs is markedly increased in the clones which were stably transfec- ted with the construct for F011 or A016 containing an exo- genous NLS (Figure 6, lanes 4 and 5). Although to a lesser Figure 6. Increase in N30 inclusion of the endogenous NMHC-B mRNA by exogenous expression of the brain-enriched isoforms of Fxh and A2BP1 in Y79 cells. Y79 cells were stably transfected with expression constructs for the myc- tagged proteins indicated. Endogenous NMHC-B mRNAs were analyzed by RT–PCR. The 364 bp product includes another alternative exon R18 as well as N30. The bottom diagram indicates the exon–intron organization of the NMHC-B gene and location of the PCR primers (P7 and P9). Immunoblots using an anti-myc antibody verify the expression of the myc-tagged proteins. Nucleic Acids Research, 2005, Vol. 33, No. 7 2085 extent, transfection of the wild-type constructs without an exogenous NLS also results in a signiﬁcant increase in N30 inclusion (Figure 6, lanes 2 and 3). Thus, exogenously expressed Fxh and A2BP1 are capable of activating N30 inclu- sion not only in the transcripts from the minigenes, but also in those from the native NMHC-B gene in Y79 cells. Fxh may cooperate with other factor(s) to promote N30 inclusion To address the role of endogenous Fxh and its potential inter- action with other proteins in promoting N30 inclusion, we made use of an isoform of Fxh, F402, which lacks the RRM. The mutant proteins lacking an RRM for other RNA-binding proteins have previously been reported to func- tion in a dominant-negative fashion and inhibit the activities of the wild-type proteins (26,31). Therefore, the effects of F402 on the N30 inclusion of minigene H mRNAs were examined in the context of the Y79 cells treated with butyrate. Upon butyrate treatment, as reported previously, Y79 cells enter in a post-mitotic and differentiated stage, and importantly, the endogenous as well as the minigene mRNAs in those cells include N30 to a large extent, unlike those in the untreated cells that predominantly exclude N30 (14). As shown in Figure 7, in the absence of exogenous expression of F402, large quantities of the mRNAs derived from minigene H-wt, which contains the wild-type IDDE, include N30 (Figure 7, upper panel, lane 4). In contrast, only small quantities of N30 inclusion are detected in the mRNAs from minigene H-mc, which has mutations in both copies of UGCAUG in the IDDE (Figure 7, upper panel, lane 8). Therefore, the butyrate-treated Y79 cells contain factor(s), which are capable of activating the UGCAUG-dependent N30 inclusion. Co-transfection of the wild-type minigene H-wt with the F402 expression construct causes a dose-dependent inhibition of N30 inclusion (Figure 7, lanes 1–3), indicating that F402 has an antagonistic effect on the endogenous factors with respect to N30 inclusion. Notably, the UGCAUG-independent N30 inclusion seen in the mutant minigene H-mc is not affected by the co-expression of F402 (Figure 7, lanes 5–7), indicating that the inhibitory activity of F402 depends on the UGCAUG element. The parallel experi- ment with the F011 expression construct does not show a signiﬁcant effect on N30 splicing in either the wild-type or mutant minigene (Figure 7, lanes 9–16). This may be due to the fact that N30 inclusion of the wild-type minigene mRNAs has already reached a maximal level and is consistent with the idea that butyrate-treated Y79 cells contain sufﬁcient amounts of protein(s) functionally equivalent to F011, which can pro- mote N30 inclusion in a UGCAUG-dependent manner. Since F402 does not bind to the UGCAUG element, and has a UGCAUG-dependent antagonistic effect on N30 splicing, it most probably disrupts protein–protein interactions of the endogenous proteins that are required for the UGCAUG- dependent activation of N30 splicing. Endogenous Fxh (and/or A2BP1) would be a good candidate whose function could be antagonized by exogenously expressed F402. Thus, this observation suggests that the endogenous Fxh has an effect on the activation of N30 splicing and that other proteins cooperate with Fxh for N30 activation. Since muscle cells express the RRM-defective isoforms to a signiﬁcant extent (Figure 2C) and the wild-type F402 localize to