Asynchronous regulation of splicing events within protein 4.1 pre-mRNA during erythroid differentiation

Protein 4.1 is an 80-kD structural component of the red blood cell (RBC) cytoskeleton. It is critical for the formation of the spectrin/actin/protein 4.1 junctional complex, the integrity of which is important for the horizontal strength and elasticity of RBCs. We and others have previously shown that multiple protein 4.1 mRNA isoforms are generated from a single genomic locus by several alternative mRNA splicing events, leading to the insertion or skipping of discrete internal sequence motifs. The physiologic significance of these motifs: (1) an upstream 17-nucleotide sequence located at the 5' end of exon 2 that contains an in-frame ATG initiation codon, the inclusion of which by use of an alternative splice acceptor site in exon 2 allows the production of a 135-kD high-molecular-weight isoform present in nonerythroid cells; (2) exon 16, which encodes a 21-amino acid (21aa) segment located in the 10-kD "spectrin/actin binding domain" (SAB), the presence of which is required for junctional complex stability in RBCs. Previous studies by our group and others suggested that, among blood cells, this exon was retained only in mature mRNA in the erythroid lineage. Exon 16 is one of a series of three closely linked alternatively spliced exons, generating eight possible mRNA products with unique configurations of the SAB. In this communication, we report studies of the expression of both the translation initiation region and the SAB region during induced erythroid maturation in mouse erythroleukemia (MEL) cells. We have found that only two of eight possible combinatorial patterns of exon splicing at the SAB region are encountered: the isoform lacking all three exons, present in predifferentiated cells, and the isoform containing only exon 16, which increases in amount during erythroid differentiation. The protein isoform containing the 21aa segment encoded by exon 16 efficiently and exclusively incorporates into the membrane, whereas the isoform lacking this 21aa segment remains in the cytoplasm, as well as the membrane. In contrast with exon 16, the erythroid pattern of exon 2 splicing, i.e., skipping of the 17-base sequence at the 5' end, was found to be already established in the uninduced MEL cells, suggesting strongly that this regulated splicing event occurs at an earlier stage of differentiation. Our results demonstrate asynchronous regulation of two key mRNA splicing events during erythroid cell maturation. These findings also show that the splicing of exon 16 alters the intracellular localization of protein 4.1 in MEL cells, and appears to be essential for its targeting to the plasmalemma.     

Regulated Fox-2 isoform expression mediates protein 4.1R splicing during erythroid differentiation

A regulated splicing event in protein 4.1R pre-mRNA-the inclusion of exon 16-encoding peptides for spectrin-actin binding-occurs in late erythroid differentiation. We defined the functional significance of an intronic splicing enhancer, UGCAUG, and its cognate splicing factor, mFox2A, on exon 16 splicing during differentiation. UGCAUG displays cell-type-specific splicing regulation in a test neutral reporter and has a dose-dependent enhancing effect. Erythroid cells express 2 UGCAUG-binding mFox-2 isoforms, an erythroid differentiation-inducible mFox-2A and a commonly expressed mFox-2F. When overexpressed, both enhanced internal exon splicing in an UGCAUG-dependent manner, with mFox-2A exerting a much stronger effect than mFox-2F. A significant reciprocal increase in mFox-2A and decrease in mFox-2F occurred during erythroid differentiation and correlated with exon 16 inclusion. Furthermore, isoform-specific expression reduction reversed mFox-2A-enhancing activity, but not that of mFox-2F on exon 16 inclusion. Our results suggest that an erythroid differentiation-inducible mFox-2A isoform is a critical regulator of the differentiation-specific exon 16 splicing switch, and that its up-regulation in late erythroid differentiation is vital for exon 16 splicing.     

Evolutionarily conserved alternative pre-mRNA splicing regulates structure and function of the spectrin-actin binding domain of erythroid protein 4.1

A developmental alternative splicing switch, involving exon 16 of protein 4.1 pre-mRNA, occurs during mammalian erythropoiesis. By controlling expression of a 21-amino acid peptide required for high- affinity interaction of protein 4.1 with spectrin and actin, this switch helps to regulate erythrocyte membrane mechanical stability. Here we show that key aspects of protein 4.1 structure and function are conserved in nucleated erythroid cells of the amphibian Xenopus laevis. Analysis of protein 4.1 cDNA sequences cloned from Xenopus erythrocytes and oocytes showed that tissue-specific alternative splicing of exon 16 also occurs in frogs. Importantly, functional studies with recombinant Xenopus erythroid 4.1 demonstrated specific binding to and mechanical stabilization of 4.1-deficient human erythrocyte membranes. Phylogenetic sequence comparison showed two evolutionarily conserved peptides that represent candidate spectrin-actin binding sites. Finally, in situ hybridization of early embryos showed high expression of 4.1 mRNA in ventral blood islands and in developing brain structures. These results demonstrate that regulated expression of structurally and functionally distinct protein 4.1 isoforms, mediated by tissue-specific alternative splicing, has been highly evolutionarily conserved. Moreover, both nucleated amphibian erythrocytes and their enucleated mammalian counterparts express 4.1 isoforms functionally competent for spectrin-actin binding.     

A splicing alteration of 4.1R pre-mRNA generates 2 protein isoforms with distinct assembly to spindle poles in mitotic cells

The C-terminal region of erythroid cytoskeletal protein 4.1R, encoded by exons 20 and 21, contains a binding site for nuclear mitotic apparatus protein (NuMA), a protein needed for the formation and stabilization of the mitotic spindle. We have previously described a splicing mutation of 4.1R that yields 2 isoforms: One, CO.1, lacks most of exon 20-encoded peptide and carries a missense C-terminal sequence. The other, CO.2, lacks exon 20-encoded C-terminal sequence, but retains the normal exon 21-encoded C-terminal sequence. Knowing that both shortened proteins are expressed in red cells and assemble to the membrane skeleton, we asked whether they would ensure 4.1R mitotic function in dividing cells. We show here that CO.2, but not CO.1, assembles to spindle poles, and colocalizes with NuMA in erythroid and lymphoid mutated cells, but none of these isoforms interact with NuMA in vitro. In microtubule-destabilizing conditions, again only CO.2 localizes to the centrosomes. These data suggest that the stability of 4.1R association with centrosomes requires an intact C-terminal end, either for a proper conformation of the protein, for a direct binding to an unknown centrosome-cytoskeletal network, or for both. We also found that 4.1G, a ubiquitous homolog of 4.1R, is present in mutated as well as control cells and that its C-terminal region binds efficiently to NuMA, suggesting that in fact mitotic spindles host a mixture of the two 4.1 family members. These findings led to the postulate that the coexpression at the spindle poles of 2 related proteins, 4.1R and 4.1G, might reflect a functional redundancy in mitotic cells.     

Expression of cytoskeletal protein 4.1 during avian erythroid cellular maturation.

We have isolated a cDNA clone encoding part of protein 4.1, an integral component of the erythrocyte cytoskeleton. The recombinant was isolated by immunological screening of a chicken erythroid lambda gt11 cDNA library using a monoclonal antibody directed against protein 4.1. DNA blot analysis shows that the gene is present as a single copy per haploid chicken genome, while RNA blot analysis reveals the presence of a single mRNA of 7 kilobases in reticulocytes. Message of the same size (in reduced amounts) is also present in an erythroleukemic cell line transformed by avian erythroblastosis virus and is also present in vastly reduced quantities in nonerythroid hemopoietic cells. Immunoblotting and immunofluorescence experiments show that a subset of the chicken 4.1 variant proteins is preferentially expressed during in vitro differentiation of chicken erythroleukemic cells. These data indicate that the gene is both actively transcribed and translated during early erythroid cellular maturation.     

Mechanism for cryptic splice site activation during pre-mRNA splicing.

The 5' splice site of a pre-mRNA is recognized by U1 small nuclear ribonucleoprotein particles (snRNP) through base pairing with the 5' end of U1 small nuclear RNA (snRNA). Single-base substitutions within a 9-nucleotide 5'-splice-site sequence can abolish or attenuate use of that site and, in higher eukaryotes, can also activate nearby "cryptic" 5' splice sites. Here we show that the effects of single-base substitutions within a 5' splice site can be completely or partially suppressed by cis mutations that improve the overall complementarity of the site to U1 snRNA. We further show that in the presence of the normal 5' splice site, a cryptic 5' splice site can be activated by increasing its complementarity to U1 snRNA. U1 snRNP binding experiments confirm that cryptic 5' splice sites are activated when their affinity for U1 snRNP approaches that of the authentic 5' splice site. Based upon these results, we propose a spliceosome competition model for 5'-splice-site selection and cryptic 5'-splice-site activation. We discuss our results with regard to the factors involved in 5'-splice-site recognition.     

Integrin expression profiles during erythroid differentiation.

To study the expression of integrins at the erythroid progenitor level we isolated selected populations of cells from human fetal liver after immunoadherence to anti-beta 2 integrin (CD18) coated plates. These CD18 adherent cells (CD18-Ad), in contrast to CD18 nonadherent cells (CD18-NAd), have a blastlike cell morphology and are highly enriched in all progenitor types (14% to 37% progenitors). By several criteria progenitor cells present in CD18-Ad cells appear to have a higher proliferative potential and diversity than the ones found in CD18-NAd, which were mostly later erythroid progenitors. Positivity of CD18-Ad cells with the common beta 2 integrin (CD18) is largely attributable to expression of alpha L (CD11a) chain, rather than alpha M (CD11b). CD11a is present in all types of progenitors, but it is selectively lost at later stages of erythroid differentiation/maturation. By contrast, CD11b appears to be virtually absent from all progenitors but it has an enhanced expression during granulomonocytic differentiation/maturation. In addition to beta 2 integrins, CD18-Ad cells express several other cytoadhesion molecules (VLA-4, VLA-5, I-CAM, H-CAM) as well as other progenitor cell antigens (CD34, HLA-DR, CD38). Cells expressing all these antigens were selectively enriched in CD18-Ad cells. Our data add new information on the regulation of CD11a and CD11b molecules in hematopoiesis and on the composite profile of integrin expression at several stages of erythroid differentiation.     

An exonic splicing enhancer in human IGF-I pre-mRNA mediates recognition of alternative exon 5 by the serine-arginine protein splicing factor-2/alternative splicing factor.

The human IGF-I gene has six exons, four of which are alternatively spliced. Variations in splicing involving exon 5 may occur, depending on the tissue type and hormonal environment. To study the regulation of splicing to IGF-I exon 5, we established an in vitro splicing assay, using a model pre-mRNA containing IGF-I exons 4 and 5 and part of the intervening intron. Using a series of deletion mutants, we identified an 18-nucleotide purine-rich splicing enhancer in exon 5 that increases the splicing efficiency of the upstream intron from 6 to 35%. We show that the serine-arginine protein splicing factor-2/alternative splicing factor specifically promotes splicing in cultured cells and in vitro and is recruited to the spliceosome in an enhancer-specific manner. Our findings are consistent with a role for splicing factor-2/alternative splicing factor in the regulation of splicing of IGF-I alternative exon 5 via a purine-rich exonic splicing enhancer.     

Multiple Rh messenger RNA isoforms are produced by alternative splicing.

Three Rh-related cDNAs have been isolated from a human bone marrow cDNA library and by polymerase chain reaction (PCR) amplification of human bone marrow and erythroblast mRNAs. They potentially encode a family of Rh protein isoforms that exhibit several unexpected structural properties as compared with the Rh polypeptide encoded by the cDNA clone identified previously. These modifications include several peptide deletions, the predicted alteration of Rh protein topology within the cell membrane, variations in the number and surface exposition of cysteine residues, and the generation of new C-terminal polypeptide segments caused by frameshift mutations. The four Rh mRNAs now described correspond to different splicing isoforms transcribed from the same Rh gene, and all exist in the same cell lineage (erythroid). Moreover, PCR experiments indicated that at least three of these RNA species exist in reticulocytes from donors with different commonly expressed Rh phenotypes. Although the translated proteins have not yet been characterized, these results suggest that the two genes at the RH locus may direct the synthesis of several protein species possibly corresponding to different Rh antigenic variants.     

Caspase-2 pre-mRNA alternative splicing: Identification of an intronic element containing a decoy 3' acceptor site

We have established a model system using the caspase-2 pre-mRNA and initiated a study on the role of alternative splicing in regulation of programmed cell death. A caspase-2 minigene construct has been made that can be alternatively spliced in transfected cells and in nuclear extracts. Using this system, we have identified a 100-nt region in downstream intron 9 that inhibits the inclusion of the 61-bp alternative exon. This element (In100) can facilitate exon skipping in the context of competing 3' or 5' splice sites, but not in single-intron splicing units. The In100 element is also active in certain heterologous pre-mRNAs, although in a highly context-dependent manner. Interestingly, we found that In100 contains a sequence that highly resembles a bona fide 3' splice site. We provide evidence that this sequence acts as a "decoy" acceptor site that engages in U2 snRNP-dependent but nonproductive splicing complexes with the 5' splice site of exon 9, hence conferring competitive advantage to the exon-skipping splicing event (E8-E10). These results reveal a mechanism of action for a negative intronic regulatory element and uncover a role for U2 snRNP in the regulation of alternative splicing.     

Metabolic adaptation during erythropoietin-mediated terminal differentiation of mouse erythroid cells.

Metabolic development was examined in erythroid precursor cells, which were isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus (FVA cells). FVA cells undergo differentiation in vitro from the proerythroblast stage through the reticulocyte stage over a 48-hour period in the presence of erythropoietin. Concomitant with marked decreases in cellular size and energy demand, metabolic capacities of both glycolysis and oxygen consumption diminish after 48 hours in culture by 7- and 18-fold, respectively. Because the oxidative capacity decreases more than glycolytic ability does, the metabolic machinery increasingly shifts toward anaerobic metabolism. During the 48-hour period of differentiation, the 2,3-diphosphoglyceric acid (DPG) content per cell and 2,3-DPG mutase activity per cell increased eightfold and threefold, respectively. Freshly harvested FVA cells have adenosine triphosphate (ATP) levels of 7.23 +/- 2.52 mumol/10(10) cells or 3.76 +/- 1.31 mumol/mL cell water which are 12- or 2.3-fold higher, respectively, than the ATP levels of mature red blood cells. In the course of FVA cell differentiation, ATP content per cell decreases by fourfold, but ATP concentration in cell water remains unchanged because of a corresponding decrease in cellular size and water content during differentiation. These studies show that in the face of dramatic decreases in cell size and cellular energy demand, terminally differentiating erythroid cells maintain a constant ATP level by undergoing an involution of their glycolytic machinery as well as by losing their aerobic metabolic capacity.     

Tissue-specific alternative splicing of protein 4.1 inserts an exon necessary for formation of the ternary complex with erythrocyte spectrin and F-actin

Erythrocyte protein 4.1 is an 78- to 80-Kd peripheral membrane protein that promotes the interaction of spectrin with actin protofilaments and links the resulting interlocking network to the integral membrane proteins. There are several isoforms of protein 4.1 that appear to be expressed in a restricted group of tissues. These arise from alternative mRNA splicing events that lead to the combinational insertion or deletion of at least 10 blocks of nucleotides (motifs) within the mature mRNA. One of these, motif I, consists of 63 nucleotides encoding 21 amino acids in the N-terminal region of the putative spectrin/actin-binding domain. The expression of the motif U- containing isoform occurs late in erythroid maturation. We generated recombinant isoforms of protein 4.1 and of the putative 10-Kd spectrin/actin-binding fragment that contain or lack this 21 amino acid sequence and examined their ability to form a ternary complex with erythrocyte spectrin and F-actin. The isoforms of the complete protein and of the 10-Kd fragment that contain the sequence encoded by motif I efficiently form the ternary complex. Isoforms that lack this sequence, but are otherwise identical, do not participate in the formation of the ternary complex. These results, in conjunction with the expression of motif I during late erythroid maturation, suggest that interaction with actin and the erythroid form of spectrin is a specialized property of the erythrocyte form of protein 4.1. Alternative mRNA splicing in developing red blood cells thus plays a key adaptive role in the formation of the highly specialized erythrocyte membrane.