Previous studies have shown that the (U)GCAUG element functions as both a silencer and enhancer of alternative splicing (9
). The evolutionarily conserved (U)GCAUG element is widely associated with neuron-specific and muscle-specific exons (28
). Recently, Fox proteins have been shown to regulate splicing via binding to (U)GCAUG elements (5
). The mouse Fox-1 and Fox-2 are both specifically expressed in the brain in addition to the muscle and heart (30
). In the brain, these proteins are specifically expressed in neurons and not glia (44
). We report here that Fox-1- and Fox-2-mediated splicing regulation also plays an important role in controlling tissue-specific alternative RNA processing of human calcitonin/CGRP pre-mRNA. We demonstrate that two UGCAUG elements, one located 34 nucleotides upstream of and the other 45 nucleotides downstream of the beginning of exon 4, function as splicing silencers for exon 4 inclusion. Furthermore, Fox-1 and Fox-2 proteins specifically bind these silencers and negatively regulate exon 4 inclusion. These results indicate that Fox-1 and Fox-2 are important for the neuron-specific CGRP-specific pathway, and they function by blocking the calcitonin-specific exon 4 inclusion. These results appear to be contradictory to a previous study on the role of the (U)GCAUG elements in regulating the rat calcitonin/CGRP alternative RNA processing (12
). The previous study concluded that these elements function as enhancer elements for the calcitonin-specific exon 4 inclusion (12
). However, in that study, the results appear to be very complex when deletion mutants and mutants with inserted multimerized repeats were tested. For example, an intronic deletion removing two (U)GCAUG repeats did cause a slight increase in exon 4 inclusion in a CGRP-specific cell line. In addition, when multimerized UGCAUG elements were inserted upstream of exon 4, calcitonin-specific splicing was substantially reduced (12
). The discrepancy between the two studies may also reflect differences between the rat and human calcitonin/CGRP gene.
It has been shown that many factors can bind to the calcitonin/CGRP pre-mRNA and regulate exon 4 inclusion. U1 snRNP, SRp20, PTB, and TIAR bind to the intronic enhancer elements located downstream of exon 4 and promote exon 4 inclusion (21
). Tra2β and SRp55 are recruited to ESE A and B and activate exon 4 inclusion (42
). However, all of these regulators are expressed in many tissues and are unlikely to be responsible for neuron-specific regulation of calcitonin/CGRP alternative processing. Therefore, how the neuronal pattern of processing in the calcitonin/CGRP transcript is achieved remained poorly understood. Recently, our group showed that the neuron-specific Hu proteins can bind to the U-tract of the intronic enhancer element located downstream of exon 4, thereby blocking access of TIAR and as a result, promoting the CGRP-specific pathway (H. Zhu, R. A. Hasman, and H. Lou, unpublished data). Interestingly, Hu proteins are not sufficient to block exon 4 inclusion because overexpression of Hu proteins in HeLa cells did not change the ratio of RNA with exon 4 included (Zhu et al., unpublished). Studies in the present report identify Fox-1 and Fox-2 proteins as major contributors to neuron-specific exon 4 exclusion for calcitonin/CGRP pre-mRNA. However, it is also clear that Fox-1 and Fox-2 are not the only neuron-specific regulators responsible for the CGRP pathway. For example, as shown in Fig. , when both ESE and UGCAUG silencer elements are mutated, exon 4 inclusion is consistently lower in CA77 cells than in HeLa cells. In this case, Hu proteins may be responsible for the reduced exon 4 inclusion.
Hints that sequences located near the 3′ splice site of exon 4 contain neuron-specific splicing silencers came from a few previous studies. Analyzing a number of deletion and substitution mutant surrounding the 3′ splice site of exon 4 in the rat calcitonin/CGRP reporter constructs, Emeson et al. suggested that a cis
-acting element at the calcitonin-specific 3′ splice junction plays an important role in regulating tissue-specific processing of the calcitonin/CGRP pre-mRNA (10
). Consistent with this idea, two calcitonin/CGRP splice regulator proteins (CSRs) were suggested to function as neuron-specific negative regulators in alternative calcitonin/CGRP splicing (8
). These CSRs were purified through biochemical approaches from rat brain nuclear extracts based on their ability to bind an RNA substrate containing a sequence of 380 nucleotides surrounding the calcitonin-specific exon 4 (8
). However, to date, the identity of these two CSR proteins has not been reported. It is likely that Fox-1 and Fox-2 proteins are the two CSR proteins based on the following considerations. First, the two CSR proteins show approximate molecular masses of 43 and 41 kDa, respectively, similar to Fox-1 and Fox-2. Second, previous studies demonstrated that both proteins have the ability to form a weak complex with a 21-nucleotide target sequence containing the −34 UGCAUG silencer elements (33
). Finally, Fox-1 and Fox-2 proteins are expressed in the brain, specifically in neurons (44
Although it is clear that Fox-1 and Fox-2 proteins regulate alternative splicing of a handful of pre-mRNAs through binding to (U)GCAUG elements, the mechanism of such regulation has not been explored. Our results demonstrate for the first time that the Fox-1 and Fox-2 proteins reduce U2AF65
binding to the polypyrimidine tract of the 3′ splice site of the calcitonin/CGRP exon 4 in a UGCAUG element-dependent manner (Fig. ). U2AF plays an essential role in promoting the ATP-dependent binding of U2 snRNP to the pre-mRNA branchpoint (36
). It has been shown that a number of splicing regulatory factors affect U2AF binding to the polypyrimidine tract upstream from the 3′ splice site, thereby regulating splicing. For example, in female fruit flies, SXL protein binds to the polypyrimidine tract of the male-specific 3′ splice site of the transformer pre-mRNA to block access of U2AF, thereby promoting usage of the downstream female-specific 3′ splice site (45
). Repression of splicing by PTB can also occur by competing with U2AF65
). It has also been shown that ESE binding proteins such as SR proteins promote splicing by increasing U2AF65
). It remains to be determined how Fox-1 and Fox-2 interfere with U2AF65
binding. Since the UGCAUG at −34 is only six nucleotides upstream from the branchpoint sequence, it is possible that Fox-1 or Fox-2 bound at this element physically interferes with U2AF or U2 snRNP interaction with their cognate binding sites. It is also possible that Fox-1 or Fox 2 interacts with a component(s) of the splicing complex, thereby affecting its assembly at the 3′ splice site upstream of exon 4, which may explain why the small increase in U2AF binding does not appear to correlate with the large decrease in Fox binding in the mutant. Additional experiments are necessary to distinguish these possibilities.
The exonic UGCAUG silencer element at+45 position on exon 4 is very close to the ESEs mediated by Tra2β and SRp55 protein (18 nucleotides from ESE A and 62 nucleotides from ESE B) (Fig. ). Thus, repression of splicing by Fox-1 and Fox-2 can also occur by interfering with Tra2β and SRp55 proteins binding to these ESEs. Our results indicate that the +45 UGACUG silencer element functions to suppress ESE-dependent calcitonin-specific exon 4 splicing. When the ESEs are mutated, the second mutation at the +45 UGCAUG silencer did not increase exon 4 inclusion to the same extent as it did with the 5′ss mutation-containing reporter (Fig. and ). A number of studies have demonstrated that the ESE-dependent 3′ splice site activation by SR proteins involves the recruitment of U2AF65
to weak polypyrimidine tracts (16
). It is therefore possible that Fox-1 and Fox-2 antagonize the binding of Tra2β and SRp55 to ESEs through interacting with the +45 UGCAUG silencer element. As a result, the Fox proteins inhibit U2AF65
binding, leading to repression of the exon 4 inclusion.
Our results also indicate an interesting coordinated regulation of exon 4 inclusion. In HeLa cells, the mutant reporter minigene containing a mutation at the intronic pseudo 5′ splice site produced little exon 4 inclusion (15%). Intriguingly, combining the pseudo 5′ splice site mutation with the −34+45 silencer mutation resulted in 63% exon 4 inclusion, which is very close to the 70% exon 4 inclusion for the wild-type reporter minigene (Fig. ). This result indicates that a delicate balance between the hexanucleotide silencer and intronic enhancer elements has evolved to control exon 4 definition. The silencer elements and intronic enhancer elements contribute individually to exon 4 recognition. However, the intronic enhancer element is only required when the UGCAUG silencer element is present. When the same combined mutant reporter was introduced into CA77 cells, the level of exon 4 inclusion approaches that in HeLa cells, indicating that this mutant combination results in a loss of cell-specific regulation of exon 4 inclusion. This result suggests that neuron-specific regulatory elements for calcitonin/CGRP pre-mRNA are located within the silencer elements and/or intronic enhancer elements, which is consistent with the identification of Fox-1/Fox-2 and Hu proteins as neuron-specific regulators of the calcitonin/CGRP system. A number of alternatively included exons have been demonstrated to undergo complex control through a regulatory network involving both positive and negative factors (11
Like many other alternatively included exons, the calcitonin-specific exon 4 is flanked by suboptimal splicing signals, which is typified by the presence of a uridine (cytosine in the rat calcitonin/CGRP pre-mRNA) instead of the canonical adenosine branchpoint. It was demonstrated previously that exon 4 inclusion is significantly increased when the noncanonical branchpoint is mutated to adenosine in a cell type-independent fashion (47
). Our results indicate that Fox-mediated repression of exon 4 inclusion can override the presence of a strong branchpoint. Addition of Fox proteins decreased exon 4 inclusion of the reporter that contains the U-A branchpoint mutation (Fig. ). This result is consistent with the fact that exon 4 inclusion increases from 31 to 57% (compare to 88% in HeLa cells) when the branchpoint is changed from U to A in CA77 cells (Fig. ), suggesting that the repressors in these cells such as Fox proteins are still at work. Therefore, the suboptimal uridine branchpoint and UGCAUG silencer elements may coregulate exon 4 inclusion in an additive but not synergistic manner.
Based on our results, a model for how tissue-specific regulation of calcitonin/CGRP is achieved is depicted in Fig. . Identification of a novel neuron-specific regulatory factor in this system represents a significant step forward toward fully understanding the regulatory mechanism controlling this specific alternative splicing event. It will be important to decipher how Fox proteins interact with the spliceosome to repress splicing of specific exons.
FIG. 9. Models to explain how nonneuronal and neuron-specific alternative RNA processing of the human calcitonin/CGRP pre-mRNA are achieved. In nonneuronal cells, a number of trans-acting factors promote exon 4 inclusion at both ends of this exon. In neuronal (more ...)