Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins to silencer elements in the exon and that down-regulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This article demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.Alternative splicing of pre-mRNA leads to the synthesis of multiple protein isoforms from a single gene. It is an important mechanism for regulating gene expression and may be utilized by 40 -60% of human genes (1-4). Thus, the estimated 25,000 to 30,000 genes of the human genome can generate a much larger number of proteins. Regulation of alternative splicing occurs in both a tissue-and development-specific manner, resulting in alterations in the structure and function of critical proteins. Altered splicing regulation can also be of widespread importance in the etiology of human disease (5-7).The protein 4.1 gene family serves as an excellent model for investigating the regulation of alternative splicing. The four genes that comprise the family (4 .1R, 4.1G, 4.1B, and 4.1N) display a remarkable array of highly regulated, tissue-specific splicing events. These alternative splicing events facilitate expression of distinct isoforms of 4.1 protein in cells of erythroid, epithelial, neural, and muscle origin (8 -14); thus, they provide opportunities for understanding the mechanisms that regulate alternative splicing in several different cell types. To date, mechanistic studies have focused predominantly on erythroid cells, in which 4.1R protein is a structural component of the erythrocyte plasma membrane and is important for structural integrity and stability of the membrane skeleton. In differentiating erythroid progenitor cells, a dramatic switch in pre-mRNA splicing result...
In the human pathogen Staphylococcus aureus, there exists an enormous diversity of proteins containing DUFs (domains of unknown function). In the present study, we characterized the family of conserved staphylococcal antigens (Csa) classified as DUF576 and taxonomically restricted to Staphylococci. The 18 Csa paralogues in S. aureus Newman are highly similar at the sequence level, yet were found to be expressed in multiple cellular locations. Extracellular Csa1A was shown to be post-translationally processed and released. Molecular interaction studies revealed that Csa1A interacts with other Csa paralogues, suggesting that these proteins are involved in the same cellular process. The structures of Csa1A and Csa1B were determined by X-ray crystallography, unveiling a peculiar structure with limited structural similarity to other known proteins. Our results provide the first detailed biological characterization of this family and confirm the uniqueness of this family also at the structural level. We also provide evidence that Csa family members elicit protective immunity in in vivo animal models of staphylococcal infections, indicating a possible important role for these proteins in S. aureus biology and pathogenesis. These findings identify the Csa family as new potential vaccine candidates, and underline the importance of mining the bacterial unknown proteome to identify new targets for preventive vaccines.
A switch in the alternative splicing pattern in protein 4.1R pre-mRNA, involving activation of exon 16 splicing, occurs in late erythropoiesis. Because the inclusion of exon 16 leads to synthesis of protein isoforms with high affinity for spectrin and actin, and mechanical strengthening of the membrane, this switch is critical for normal erythyroid differentiation. Previous studies have shown that Fox-2 protein is a splicing regulator that binds to intronic enhancer sequences downstream of exon 16, and that this binding stimulates the inclusion of exon 16. Fox binding sites are also located downstream of other tissue-specific alternative exons, some of which exhibit variable splicing efficiencies, leading us to investigate whether splicing switch strength is influenced by variations in the sequence of the Fox binding-site motif (UGCAUG) and by the number of these protein binding sites. A series of minigenes was created in which protein 4.1R E16 splicing efficiency was measured in transfection studies. Mutation of the first U residue of the Fox binding site resulted in a significantly weaker, but still Fox dependent activation of splicing, whereas mutation of the terminal G residue dramatically reduced enhancer activity. Insertion of two wild-type UGCAUG elements enhanced splicing substantially in a Fox protein concentration-dependent manner, and four elements gave even stronger inclusion. To test whether the reduced splicing efficiency observed for minigenes containing mutated enhancer elements was due to a reduction in Fox binding, surface plasmon resonance (SPR) was employed. Our binding studies indicate that full-length mouse Fox-2a bound to the UGCAUG sequence with high affinity (KD ∼100nM). Furthermore, in agreement with our functional splicing assays, Fox binding affinity for the hexameric sequence was reduced with mutations in position one, and almost eliminated with mutations in position six. There was an excellent correlation between binding affinity of Fox protein for the enhancer motif, and strength of enhancer activity measured in functional splicing assays. Additional studies demonstrated that Fox enhancer activity can be reduced by the presence of a closely linked binding site for hnRNP A1, a known splicing inhibitor. Taken together, these studies demonstrate that the composition and number of binding sites for Fox can significantly affect the efficiency of splicing for E16. It is likely that through these combinatorial mechanisms, Fox splicing factors are able to modulate the efficiency with which tissue specific exons in the erythroid alternative splicing program can be spliced into mature mRNA.
An erythroid differentiation stage-specific alternative splicing switch involving activation of protein 4.1R exon 16 splicing is critical for the mechanical stability of the erythrocyte plasma membrane. We have previously shown that inclusion of E16 can be negatively regulated by binding of hnRNP A/B proteins to splicing silencer element(s) in E16 and that strongly decreased expression of hnRNP A/B proteins is temporally correlated with exon 16 activation. Moreover, our earlier unpublished data showed that Fox-2 is a candidate activator protein for this splicing switch, based on observations that Fox-2 binds to an intron enhancer containing three copies of UGCAUG located 96–144nt downstream of exon 16; that Fox-2 enhances exon 16 splicing in HeLa cell co-transfection assays; that mutations blocking Fox-2 binding abrogate its stimulation of exon 16 splicing; and that Fox-2 is expressed in erythroblasts. New experiments reinforce these findings by showing that knockdown of Fox-2 expression, using two different siRNA sequences, strongly inhibits exon 16 splicing efficiency. Together these results indicate that A1 and Fox-2 have antagonistic splicing activities on exon 16. To test whether antagonism involves competitive binding to the intron enhancer region, in vitro binding studies were performed using a biotinylated 39-mer RNA containing two UGCAUG elements from the intron 16 enhancer. This RNA bound strongly to in vitro-synthesized Fox-2 protein, as shown by pull down assays followed by Western blot analysis. hnRNP A1 was also bound to this intronic enhancer region. UGCAUG mutations eliminated Fox-2 binding to the RNA, but did not block A1 binding; such mutants exhibited significantly lower exon 16 splicing efficiency. Most importantly, a competitive binding experiment showed that Fox-2 protein reduces A1 binding to the enhancer RNA in a concentration-dependent manner. Together with previous findings by ourselves and others, these results suggest that exon 16 splicing is governed by two pairs of antagonist interactions: (1) in exon 16, the silencer protein hnRNP A1 antagonizes SF2/ASF activity; and (2) in the downstream intron, hnRNP A1 antagonizes the Fox-2 activator protein. Exon 16 is known to be expressed in brain and muscle in addition to late erythroid cells. We propose that this regulatory network may provide independent mechanisms for exon 16 activation in different cell types by altering the relative abundance of these activators and inhibitors, and thereby the relative efficiency of spliceosome recruitment as the first step in the exon inclusion pathway. To test the mechanism of Fox-2 activity, we are attempting to isolate physiologically relevant co-factors that interact with Fox-2. A recombinant Fox-2 protein containing a biotinylation tag has been expressed in HeLa cells. In transfected cells, the biotin-tagged Fox-2 is properly localized to the nucleus, and retains the ability to enhance 4.1 exon 16 splicing in standard splicing assays as judged by RT/PCR analysis of mRNA products that include or exclude exon 16. Streptavidin pull-down assays should facilitate isolation of Fox-2 complexes and ultimately provide novel insights into the mechanism of this critical splicing switch during erythroid differentiation.
During erythroid differentiation, stage-specific activation of protein 4.1R exon 16 splicing is critical for the mechanical stability of the erythrocyte plasma membrane. The molecular mechanism of this erythroid splicing switch involves multiple factors including stimulation by Fox proteins acting at splicing enhancers in the proximal downstream intron, and inhibition by hnRNPA1 protein acting at silencer elements in exon 16. To explore how the dynamic interplay among these and other factors can fine tune splicing efficiency, we created a series of exon 16-containing minigenes in which splicing efficiency was measured as a function of variation in exon and intron regulatory elements. In the context of wild type exon 16 with intact hnRNP A1 silencer elements and a weak 5′ splice site, an enhancerless construct with no Fox binding sites exhibited little or no exon 16 inclusion in transfected HeLa cells, and over-expression of Fox-2 did not significantly promote inclusion. Insertion of two wild type UGCAUG elements enhanced splicing substantially in a Fox-2-dependent manner and four elements gave even stronger inclusion. Since another study identified the pentamer GCAUG as the Fox binding site, we tested binding site sequence as a potential source of variation in splicing efficiency. Mutation of the first U residue in UGCAUG yielded weaker, but still Fox-2 dependent, activation of splicing, whereas mutation of the terminal G residue dramatically reduced enhancer activity. To investigate whether enhancer activity of Fox binding sites can be modulated by adjacent sequence motifs, we compared exon 16 splicing efficiency in constructs having Fox sites flanked either by neutral sequence or by an A1 silencer element UAGGG. Introduction of the A1 binding site led to a dramatically reduced enhancer activity including its responsiveness to Fox-2 overexpression. These results indicated that efficiency of splicing of Fox-regulated exons is strongly influenced by the number and sequence of intron enhancer elements and by the presence of adjacent antagonistic elements. In further experiments, we demonstrated that the efficiency of splicing is also strongly dependent on exon 16 and its splice sites. Constructs lacking the major exon 16 silencer element for hnRNP A1 binding exhibited partial exon 16 inclusion in the absence, and very strong inclusion in the presence, of a Fox intron enhancer. Finally, strengthening the weak 5′ splice site of exon 16 abrogated many of these regulatory effects and led to strong inclusion of exon 16 independent of other variables. These findings are consistent with previous data showing antagonism between A1 and Fox in their effects on exon 16 splicing, and suggest that Fox proteins primarily function to overcome the weak 5′ splice site and its repression by hnRNP A1 bound at nearby exonic site(s). We propose that the erythroid alternative splicing program can activate splicing a number of alternative exons with variable efficiency based on each exon’s individual complement of exonic and intronic splicing regulatory elements. Modulation of splicing factor expression, typified by the stage-specific down-regulation of hnRNP A1 during erythroblast differentiation, can further alter splicing efficiency of these exons in a selective, motif-dependent manner. Future experiments with exon microarrays will be aimed at identifying some of the alternative exons that are regulated by that program, and determine its importance to the erythroid differentiation process.
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