2084 Nucleic Acids Research, 2005, Vol. 33, No. 7 of either one of the two copies of UGCAUG (ma, mb) abolishes the inclusion of N30 (Figure 4C, upper panel, lanes 8–10, 13–15). In the context of minigene H, which contains shorter introns, the larger extent of N30 inclusion is induced by either F011 or A030 overexpression (Figure 4C, middle panel, lanes 7 and 12). The N30 inclusion of the mini- gene H mRNAs also depends on the presence of the IDDE (Figure 4C, middle panel, lanes 6 and 11). Mutation of both copies of UGCAUG (mc) results in a complete loss of N30 inclusion (Figure 4C, middle panel, lanes 10 and 15). The mut- ant ma shows stronger inhibition of N30 inclusion compared with mb (Figure 4C, middle panel, lanes 8 and 9). This obser- vation is consistent with the competition experiments of the EMSA shown in Figure 4B, indicating that the 50 hexanuc- leotide is more important than the 30 hexanucleotide for binding of Fxh or A2BP1 to the IDDE as well as an activation of N30 splicing. Comparable amounts of F011 and A030 are expressed in each transfection as veriﬁed by immunoblots using an anti- myc antibody (Figure 4C, lower panel, lanes 6–17). Since minigenes J and H lack a portion of the intron between exons N30 and E6 and, therefore, the IDDE is located 100 nt downstream of N30, instead of 1.5 kb as in the native gene, we also analyzed the effects of Fxh and A2BP1 on the N30 spli- cing of the wild-type minigene G, which includes full-length introns among E5, N30 and E6 (Figure 4A). As shown in Figure 4C (right panel), both F011 and A030 are capable of promoting N30 inclusion, with F011 showing a higher activity (Figure 4C, lanes 16 and 17). Although A030 can promote N30 inclusion as efﬁciently as F011 in the contexts of the minigene J and H transcripts, it can do so less efﬁciently than F011 in the context of the minigene G transcript. Interpretation of this observation will be discussed below. Taken together, Fxh and A2BP1 can activate N30 inclusion in an IDDE-dependent manner. The hexanucleotide motif UGCAUG is indispensable for this activation. Differential activities of alternatively spliced isoforms of Fxh and A2BP1 in promoting N30 inclusion Both Fxh and A2BP1 mRNAs are expressed in brain and A2BP1 is also expressed in striated muscles and Fxh is expressed in an even wider variety of tissues. As demonstrated above, however, both Fxh and A2BP1 transcripts undergo tissue-dependent alternative splicing, producing muscle- speciﬁc and brain-enriched isoforms. Therefore, the relative activity of individual isoform of Fxh and A2BP1 in promoting N30 inclusion was compared using minigene G. First, in order to evaluate the relative speciﬁc activity of each isoform in the splicing reaction separately from its ability to localize to nuclei, an exogenous NLS was included in the expressed protein to equalize the nuclear concentration of the expressed protein in these experiments. Essentially, all of the expressed proteins with the exogenous NLS are local- ized to the nucleus (data not shown). Therefore, the relative nuclear concentrations of the expressed proteins can be estim- ated easily by immunoblots. As shown in immunoblots in Figure 5A, similar quantities of proteins are expressed in a dose-dependent manner. Expression of the brain isoform F011 causes a dose-dependent increase in N30 inclusion and the extent of N30 inclusion reaches a plateau with 85% of the mRNAs including N30. In contrast, inclusion of N30 Figure 5. Differential activities of Fxh and A2BP1 isoforms in N30 splicing. (A) The increasing amounts (0.05, 0.1, 0.2, 0.5, 1 and 2 mg, indicated by right triangles) of the expression constructs for the indicated proteins, which include a myc-epitope, were co-transfected in Y79 cells with minigene G as shown in Figure 4A. The mRNAs derived from the minigene were analyzed by RT–PCR. Dose-dependent protein expression was verified by immunoblot analysis using an anti-myc antibody. (B) The various expression constructs encoding the myc- tagged proteins indicated were co-transfected with minigene G and pEGFP. RT–PCR of the minigene mRNAs and immunoblots using anti-myc and anti-GFP antibodies are shown. GFP serves as an indicator for transfection efficiency. promoted by F411, the predominant isoform from skeletal muscles, reaches a plateau with only 40% of the mRNAs. With respect to A2BP1, the brain isoform A030 shows higher activity in N30 inclusion than the muscle isoform A715, which shows almost no activation, as shown in Figure 5A. Including additional isoforms, the activities of individual isoforms with an exogenous NLS in promoting N30 inclusion are shown in Figure 5B (lanes 8–13) and are also summarized in Figure 1A (nls). Special care was taken to ensure that a similar quantity of each protein was expressed and, if not, different amounts were tested to obtain comparable expres- sion. In the presence of the exogenous NLS (nls), F011 and A016 show the highest activities among all isoforms tested. A30 shows a considerably lower activity than A016. Each of the muscle isoforms (F411, A713 and A715) has a lower activity than that of their brain counterparts (F011, A016 and A030, respectively). As expected, isoforms lacking a part or all of the RRM (F402 and A704) have no activity for N30 inclusion.