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
). A second and more novel finding is the identification of tissue-specific isoforms of Fxh and A2BP1 with different splicing activities as well as different subcellular localizations. This finding raises the possibility that the products of the Fxh
genes can contribute to a mechanism 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, extracellular stimuli trigger changes in subcellular distribution of these proteins. Such translocations have been reported for hnRNPA1 and PTB (38
). Moreover, a number of RNA-binding proteins 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
). 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
), 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
). 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 5′ RACE, as well as the EST database, detect multiple 5′ end sequences for both Fxh and A2BP1 mRNAs, which are presumably generated by alternative promoters and alternative splicing. The diversity of the 5′ 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. Essentially, all of the expressed proteins with the exogenous NLS localized to nuclei. Thus, the amounts of the expressed proteins determined by immunoblots represent the nuclear concentrations. 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 , the splicing activities of the various isoforms of Fxh and A2BP1 are intrinsically different, regardless 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 (, 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 A53, which results in a frame shift. Since F411 and A030 show poor splicing activation compared with F011 and A016, respectively, these two subregions of F011 and A016 appear to serve as activation domains, presumably by interacting with other proteins. This notion is supported by the finding that the RRM-lacking isoform F402, which includes the same subregions II and IV as F011, functions apparently as a dominant-negative mutant to the wild-type Fxh, consistent with the interpretation that the mutant and wild-type are competing to interact with other protein(s). To date, Fyn tyrosine kinase and estrogen receptor-α have been reported to interact with Fxh, and ataxin-2 with A2BP1 (23
). Whether these proteins participate in the regulation of pre-mRNA splicing is currently unknown. Of interest, A030 with the exogenous NLS activates N30 splicing as efficiently as F011 in the pre-mRNAs derived from minigenes J and H, which contain the shorter intron, whereas this isoform poorly activates N30 splicing in the pre-mRNA from minigene G, which contains the full-length intron. This observation implies that the interactions of A030 with different factors are required in the different pre-mRNA contexts. Therefore, the isoforms of Fxh and A2BP1 described here may have different effects on other UGCAUG-regulated alternative splicing.
The involvement of the hexanucleotide UGCAUG in regulated alternative splicing has been experimentally demonstrated in a number of neural cell-specific, as well as other tissue-specific, model systems (13
). This hexanucleotide element plays a role, in most cases, as an enhancer in regulating alternative splicing of cassette-type exons as well as mutually exclusive exons. Furthermore, computational analysis has revealed that UGCAUG is over-represented in the introns in which splicing is regulated, compared with the constitutively spliced introns (43
). This analysis also pointed to the UGCAUG element as playing a role in the regulation of tissue-specific alternative splicing in a wide range of tissues, but not in specific tissues.
To date, KH-type splicing regulatory protein (KSRP) (44
), A2BP1 (21
) and Fxh (this study) are known to be capable of binding to UGCAUG. KSRP is expressed ubiquitously and tissue-specific variants of this gene have not been described so far. In this study, we have described the existence of tissue-dependent isoforms of Fxh and A2BP1, which, while not identical in some areas of the molecule, may contain the same RRM. The physiological relevance of these isoforms is that they have different splicing activities and different subcellular localizations. The brain isoforms promote N30 inclusion more efficiently than the muscle isoforms of both Fxh and A2BP1. The isoforms lacking the RRM are normally expressed to a significant extent in skeletal muscles. This isoform is incapable of activating N30 splicing and, moreover, can inhibit N30 inclusion. The properties of these isoforms are consistent with Fxh and A2BP1 acting as regulators for N30 splicing, since N30 is included in neuronal cells, but excluded in muscles. Therefore, despite the tissue-independent occurrence of UGCAUG as a regulatory element, given the tissue-dependent isoforms of the UGCAUG-binding proteins (Fxh and A2BP1) with different activities, the hexanucleotide UGCAUG could confer tissue specificity on regulated splicing. One of the major problems in understanding the mechanisms responsible for alternative pre-mRNA splicing is the manner in which tissue specificity is determined. In vertebrates, to date, only a few tissue-specific proteins have been identified as splicing regulators (8
). Here, we have shown that the tissue-dependent isoforms of the sequence-specific RNA-binding proteins, which themselves are generated by alternative splicing, have different activities in tissue-specific alternative splicing of target pre-mRNA. Therefore, these isoforms play a role in the determination of tissue specificity of target pre-mRNA splicing. The discovery of these tissue-dependent isoforms of the UGCAUG-binding proteins with different splicing activities adds an important new dimension to the molecular mechanisms responsible for regulating tissue-dependent alternative splicing mediated via UGCAUG.