Primary antibodies used were mouse anti-myc (Invitrogen), rabbit anti-NMHC-B (29). Secondary antibodies were Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes). DAPI was used for DNA staining. Specimens were mounted in ProLong antifed kit (Molecular Probe). The images were collected using Leica SP confocal microscopy (Leica). RESULTS Tissue-dependent isoforms of Fxh and A2BP1 and their subcellular distribution The A2BP1 mRNAs are detected almost exclusively in brain and striated muscles (heart and skeletal muscles) in adult mice, whereas the Fxh mRNAs are expressed in a wide vari- ety of tissues with the highest expression in brain and heart [(21,25) and S. Kawamoto, unpublished data]. These expres- sion proﬁles prompted us to characterize the mRNAs of A2BP1 and Fxh in brain and muscles. We have cloned cDNAs for two gene transcripts from brain, heart and skeletal muscles by RT–PCR and 50 RACE. The isolated cDNAs are schematically presented with genomic structures in Figure 1 (for amino acid sequences see also Figure 8). Both gene transcripts are found to undergo tissue-speciﬁc alternative splicing. In both cases, brain and striated muscles express unique splice variants generated by mutually exclusive spli- cing of exons B40 and M43, respectively, which provide different coding sequences in the middle of the carboxyl half of the molecules. Southern blot analysis of the RT– PCR products of the Fxh mRNAs from the different tissues probed by oligonucleotides corresponding to B40 and M43 shows that M43 is exclusively used in heart and skeletal muscles, while B40 is predominantly used in brain (Figure 2A). Digestion of the RT–PCR products of the A2BP1 mRNAs with restriction enzymes unique to B40 and M43 demonstrates that B40 and M43 are used almost exclusively in brain and muscles, respectively (Figure 2B). A2BP1 contains an additional cassette type alternative exon A53, consisting of 53 nt. Inclusion and exclusion of exon A53 results in two different amino acid sequences at the C- terminal region owing to a frame shift. In the case of the A2BP1 gene, moreover, it appears that brain and striated muscle utilize alternative promoters, resulting in different amino acid sequences at the N-terminus. The 50 RACE of skeletal muscle mRNAs yielded essentially a single species of sequence with 29 unique N-terminal amino acids. The 50 RACE using brain mRNAs; however, yielded multiple pro- ducts with multiple deduced amino acid sequences, all of which differ from the muscle amino acid sequence at the very N-terminus. Here, we focus our analysis on a clone containing nine unique amino acids at the N-terminus, which was obtained most frequently. Exon–intron organiza- tion of Fxh and A2BP1 shows remarkable similarities and a RRM is encoded in four exons (Figure 1B). Of note is that the signiﬁcant amounts of Fxh and A2BP1 mRNAs from skeletal muscles are missing a part of the RRM by exon skipping. Typically, as shown in Figure 2C, they lack the 93 nt exon that encodes RNP1, one of the two most critical motifs of the RRM (30). Some of them lack almost the entire RRM (e.g. F402 in Figure 1A). Nucleic Acids Research, 2005, Vol. 33, No. 7 2081 Figure 2. Tissue-dependent alternatively spliced isoforms of Fxh and A2BP1. (A) Tissue-dependent mutually exclusive usage of exons B40 and M43 in the Fxh mRNAs. mRNAs isolated from the indicated tissues were subjected to RT– PCR using primers P3 and P4 as indicated in Figure 1A. Ethidium bromide staining (Etd.) of agarose gel electrophoresis of the PCR products and Southern blots probed by the oligonucleotides specific to the B40 and M43 are shown. Sk. M, skeletal muscle. (B) Tissue-dependent mutually exclusive usage of exons B40 and M43 in A2BP1 mRNAs. The RT–PCR products generated by using primers P5 and P6 indicated in Figure 1A were digested with the restriction enzymes (RE), BsgI (B) and PflMI (P), which uniquely cut B40 and M43, respectively, or were undigested ( ). (C) Expression of the RRM-defective Fxh mRNA in skeletal muscles. RT–PCR was carried out using primers P1 and P2 as indicated in Figure 1A. The 531 bp products contain the complete RRM sequence. The 438 bp products lack the 93 nt exon, which encodes a part of RRM. To examine the subcellular distribution of each isoform of Fxh and A2BP1, myc-tagged proteins were transiently expressed in cultured cells and immunostained with an anti- myc antibody. Initially, HeLa cells were used to investigate subcellular localization, since these cells are more suited for these studies. Representative confocal images are shown in Figure 3A. DAPI and anti-NMHC-B antibodies serve as mark- ers for nuclei and cytoplasm, respectively. As noted, the ratio of protein distributed between nuclei and cytoplasm differs among the proteins. All isoforms of Fxh have a predominant nuclear localization. The brain isoform of A2BP1 without the A53 exon (A016) localizes to both nuclei and cytoplasm, whereas the other brain isoform with the A53 exon (A030) localizes predominantly to cytoplasm with only a minimum being in the nuclei. The relative amounts of both muscle iso- forms of A2BP1 (A713 and A715) are somewhat between the amounts of the two brain isoforms. These data are summarized in Figure 1A. The subcellular distribution of representative isoforms (F011, A016 and A030) was also examined in ret- inoblastoma Y79 cells, which were used as host cells for the transfection experiments in order to characterize splicing activities of Fxh and A2BP proteins (see below). Although Y79 cells have a spherical shape and have only thin cytoplasm,