FIRMA: a method for detection of alternative splicing from exon array data.

MOTIVATION: Analyses of EST data show that alternative splicing is much more widespread than once thought. The advent of exon and tiling microarrays means that researchers now have the capacity to experimentally measure alternative splicing on a genome wide level. New methods are needed to analyze the data from these arrays. RESULTS: We present a method, finding isoforms using robust multichip analysis (FIRMA), for detecting differential alternative splicing in exon array data. FIRMA has been developed for Affymetrix exon arrays, but could in principle be extended to other exon arrays, tiling arrays or splice junction arrays. We have evaluated the method using simulated data, and have also applied it to two datasets: a panel of 11 human tissues and a set of 10 pairs of matched normal and tumor colon tissue. FIRMA is able to detect exons in several genes confirmed by reverse transcriptase PCR. AVAILABILITY: R code implementing our methods is contributed to the package aroma.affymetrix.

Computational analysis of candidate intron regulatory elements for tissue-specific alternative pre-mRNA splicing.

Alternative pre-mRNA splicing is a major cellular process by which functionally diverse proteins can be generated from the primary transcript of a single gene, often in tissue-specific patterns. The current study investigates the hypothesis that splicing of tissue-specific alternative exons is regulated in part by control sequences in adjacent introns and that such elements may be recognized via computational analysis of exons sharing a highly specific expression pattern. We have identified 25 brain-specific alternative cassette exons, compiled a dataset of genomic sequences encompassing these exons and their adjacent introns and used word contrast algorithms to analyze key features of these nucleotide sequences. By comparison to a control group of constitutive exons, brain-specific exons were often found to possess the following: divergent 5' splice sites; highly pyrimidine-rich upstream introns; a paucity of GGG motifs in the downstream intron; a highly statistically significant over-representation of the hexanucleotide UGCAUG in the proximal downstream intron. UGCAUG was also found at a high frequency downstream of a smaller group of muscle-specific exons. Intriguingly, UGCAUG has been identified previously in a few intron splicing enhancers. Our results indicate that this element plays a much wider role than previously appreciated in the regulated tissue-specific splicing of many alternative exons.

Regulation of alternative pre-mRNA splicing during erythroid differentiation.

Although the mature enucleated erythrocyte is no longer active in nuclear processes such as pre-mRNA splicing, the function of many of its major structural proteins is dependent on alternative splicing choices made during the earlier stages of erythropoiesis. These splicing decisions fundamentally regulate many aspects of protein structure and function by governing the inclusion or exclusion of exons that encode protein interaction domains, regulatory signals, or translation initiation or termination sites. Alternative splicing events may be partially or entirely erythroid-specific, ie, distinct from the splicing patterns imposed on the same transcripts in nonerythroid cells. Moreover, differentiation stage-specific splicing "switches" may alter the structure and function of erythroid proteins in physiologically important ways as the cell is morphologically and functionally remodeled during normal differentiation. Derangements in the splicing of individual mutated pre-mRNAs can produce synthesis of truncated or unstable proteins that are responsible for numerous erythrocyte disorders. This review will summarize the salient features of regulated alternative splicing in general, review existing information concerning the widespread extent of alternative splicing among erythroid genes, and describe recent studies that are beginning to uncover the mechanisms that regulate an erythroid splicing switch in the protein 4.1R gene.

Regulation of protein 4.1R, p55, and glycophorin C ternary complex in human erythrocyte membrane.

Three binary protein-protein interactions, glycophorin C (GPC)-4.1R, GPC-p55, and p55-4.1R, constitute the GPC-4.1R-p55 ternary complex in the erythrocyte membrane. Little is known regarding the molecular basis for the interaction of 4.1R with either GPC or p55 and regarding the role of 4.1R in regulating the various protein-protein interactions that constitute the GPC-4.1R-p55 ternary complex. In the present study, we present evidence that sequences in the 30-kDa domain encoded by exon 8 and exon 10 of 4.1R constitute the binding interfaces for GPC and p55, respectively. We further show that 4.1R increases the affinity of p55 binding to GPC by an order of magnitude, implying that 4.1R modulates the interaction between p55 and GPC. Finally, we document that binding of calmodulin to 4.1R decreases the affinity of 4.1R interactions with both p55 and GPC in a Ca(2+)-dependent manner, implying that the GPC-4.1R-p55 ternary protein complex can undergo dynamic regulation in the erythrocyte membrane. Taken together, these findings have enabled us to identify an important role for 4.1R in regulating the GPC-4.1R-p55 ternary complex in the erythrocyte membrane.

Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins.

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.

Molecular and functional characterization of protein 4.1B, a novel member of the protein 4.1 family with high level, focal expression in brain.

Brain-enriched isoforms of skeletal proteins in the spectrin and ankyrin gene families have been described. Here we characterize protein 4.1B, a novel homolog of erythrocyte protein 4.1R that is encoded by a distinct gene. In situ hybridization revealed high level, focal expression of 4.1B mRNA in select neuronal populations within the mouse brain, including Purkinje cells of the cerebellum, pyramidal cells in hippocampal regions CA1-3, thalamic nuclei, and olfactory bulb. Expression was also detected in adrenal gland, kidney, testis, and heart. 4.1B protein exhibits high homology to the membrane binding, spectrin-actin binding, and C-terminal domains of 4.1R, including motifs for interaction with NuMA and FKBP13. cDNA characterization and Western blot analysis revealed multiple spliceoforms of protein 4.1B, with functionally relevant heterogeneity in the spectrin-actin and NuMA binding domains. Regulated alternative splicing events led to expression of unique 4. 1B isoforms in brain and muscle; only the latter possessed a functional spectrin-actin binding domain. By immunofluorescence, 4. 1B was localized specifically at the plasma membrane in regions of cell-cell contact. Together these results indicate that 4.1B transcription is selectively regulated among neuronal populations and that alternative splicing regulates expression of 4.1B isoforms possessing critical functional domains typical of other protein 4.1 family members.

Alternative splicing of protein 4.1R exon 16: ordered excision of flanking introns ensures proper splice site choice.

Alternative splicing plays a major role in regulating tissue-specific expression of cytoskeletal protein 4.1R isoforms. In particular, expression of the protein's functionally critical spectrin-actin binding domain, essential for maintenance of red cell membrane mechanical properties, is governed by a developmentally regulated splicing switch involving alternative exon 16. Using a model 3-exon 4.1R pre-messenger RNA (pre-mRNA), we explored the sequence requirements for excision of the introns flanking exon 16. These studies revealed that splicing of this alternative exon occurs preferentially in an ordered fashion. The first step is excision of the downstream intron to join exons 16 and 17, followed by excision of the upstream intron. Constructs designed to test the converse pathway were spliced less efficiently and with less fidelity, in part due to activation of a cryptic 5' splice site in exon 16. This downstream-first model for ordered splicing is consistent with the hypothesis that regulated alternative splicing requires cooperation between multiple exonic and/or intronic regulatory elements whose spatial organization is critical for recruitment of appropriate splicing factors. Our results predict that exon 16 splicing is regulated at the first step-excision of the downstream intron-and that cells unable to catalyze this step will exhibit exon 16 skipping. In cells that include exon 16, adherence to an ordered pathway is important for efficient and accurate production of mature 4.1R mRNA encoding an intact spectrin-actin binding domain. (Blood. 2000;95:692-699)

A novel neuron-enriched homolog of the erythrocyte membrane cytoskeletal protein 4.1.

We report the molecular cloning and characterization of 4.1N, a novel neuronal homolog of the erythrocyte membrane cytoskeletal protein 4.1 (4.1R). The 879 amino acid protein shares 70, 36, and 46% identity with 4.1R in the defined membrane-binding, spectrin-actin-binding, and C-terminal domains, respectively. 4.1N is expressed in almost all central and peripheral neurons of the body and is detected in embryonic neurons at the earliest stage of postmitotic differentiation. Like 4.1R, 4.1N has multiple splice forms as evidenced by PCR and Western analysis. Whereas the predominant 4.1N isoform identified in brain is approximately 135 kDa, a smaller 100 kDa isoform is enriched in peripheral tissues. Immunohistochemical studies using a polyclonal 4.1N antibody revealed several patterns of neuronal staining, with localizations in the neuronal cell body, dendrites, and axons. In certain neuronal locations, including the granule cell layers of the cerebellum and dentate gyrus, a distinct punctate-staining pattern was observed consistent with a synaptic localization. In primary hippocampal cultures, mouse 4.1N is enriched at the discrete sites of synaptic contact, colocalizing with the postsynaptic density protein of 95 kDa (a postsynaptic marker) and glutamate receptor type 1 (an excitatory postsynaptic marker). By analogy with the roles of 4.1R in red blood cells, 4.1N may function to confer stability and plasticity to the neuronal membrane via interactions with multiple binding partners, including the spectrin-actin-based cytoskeleton, integral membrane channels and receptors, and membrane-associated guanylate kinases.

Deciphering the nuclear import pathway for the cytoskeletal red cell protein 4.1R.

The erythroid membrane cytoskeletal protein 4.1 is the prototypical member of a genetically and topologically complex family that is generated by combinatorial alternative splicing pathways and is localized at diverse intracellular sites including the nucleus. To explore the molecular determinants for nuclear localization, we transfected COS-7 cells with epitope-tagged versions of natural red cell protein 4.1 (4.1R) isoforms as well as mutagenized and truncated derivatives. Two distant topological sorting signals were required for efficient nuclear import of the 4.1R80 isoform: a basic peptide, KKKRER, encoded by alternative exon 16 and acting as a weak core nuclear localization signal (4.1R NLS), and an acidic peptide, EED, encoded by alternative exon 5. 4.1R80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R80 was efficiently imported in the nuclei of digitonin-permeabilized COS-7 cells in the presence of recombinant Rch1 (human importin alpha2), importin beta, and GTPase Ran. Quantitative analysis of protein-protein interactions using a resonant mirror detection technique showed that 4.1R80 bound to Rch1 in vitro with high affinity (KD = 30 nM). The affinity decreased at least 7- and 20-fold, respectively, if the EED motif in exon 5 or if 4.1R NLS in exon 16 was lacking or mutated, confirming that both motifs were required for efficient importin-mediated nuclear import of 4.1R80.

Protein 4.1R-deficient mice are viable but have erythroid membrane skeleton abnormalities.

A diverse family of protein 4.1R isoforms is encoded by a complex gene on human chromosome 1. Although the prototypical 80-kDa 4.1R in mature erythrocytes is a key component of the erythroid membrane skeleton that regulates erythrocyte morphology and mechanical stability, little is known about 4.1R function in nucleated cells. Using gene knockout technology, we have generated mice with complete deficiency of all 4.1R protein isoforms. These 4.1R-null mice were viable, with moderate hemolytic anemia but no gross abnormalities. Erythrocytes from these mice exhibited abnormal morphology, lowered membrane stability, and reduced expression of other skeletal proteins including spectrin and ankyrin, suggesting that loss of 4. 1R compromises membrane skeleton assembly in erythroid progenitors. Platelet morphology and function were essentially normal, indicating that 4.1R deficiency may have less impact on other hematopoietic lineages. Nonerythroid 4.1R expression patterns, viewed using histochemical staining for lacZ reporter activity incorporated into the targeted gene, revealed focal expression in specific neurons in the brain and in select cells of other major organs, challenging the view that 4.1R expression is widespread among nonerythroid cells. The 4.1R knockout mice represent a valuable animal model for exploring 4.1R function in nonerythroid cells and for determining pathophysiological sequelae to 4.1R deficiency.

The role of alternative pre-mRNA splicing in regulating the structure and function of skeletal protein 4.1.

Alternative pre-mRNA splicing plays a major role in regulating cell type-specific expression of the protein 4.1 family of skeletal proteins. The biological importance of alternative splicing as a mechanism for 4.1 gene regulation is underscored by studies of the prototypical 4.1R gene in erythroid cells: activation of exon 16 inclusion in mRna at the erythroblast stage greatly enhances the ability of newly synthesized 4.1R protein to bind spectrin and actin, and thus assemble into a stable membrane skeleton. This gain-of- function has profound effects on the biophysical properties of deformability and membrane strength that are critical to red cell survival in the circulation. Another example of developmentally regulated splicing occurs in differentiating mammary epithelial cells in culture, where cell morphogenesis is accompanied by a splicing switch that reversibly activates inclusion of alternative exon muscle. Few other genes are known to be so richly endowed with regulated switches in pre-mRna splicing making the 4.1R gene an interesting paradigm for the role of alternative splicing as a mediator of cell function. Recent evidence that other members of the 4.1 gene family are also regulated by alternative splicing suggests, moreover, that this phenomenon is of general importance in regulating the structure of this class of skeletal proteins.

Characterization of multiple isoforms of protein 4.1R expressed during erythroid terminal differentiation.

In erythrocytes, 80-kD protein 4.1R regulates critical membrane properties of deformability and mechanical strength. However, previously obtained data suggest that multiple isoforms of protein 4. 1, generated by alternative pre-mRNA splicing, are expressed during erythroid differentiation. Erythroid precursors use two splice acceptor sites at the 5' end of exon 2, thereby generating two populations of 4.1 RNA: one that includes an upstream AUG-1 in exon 2' and encodes high molecular weight isoforms, and another that skips AUG-1 in exon 2' and encodes 4.1 by initiation at a downstream AUG-2 in exon 4. To begin an analysis of the complex picture of protein 4.1R expression and function during erythropoiesis, we determined the number and primary structure of 4.1R isoforms expressed in erythroblasts. We used reverse-transcription polymerase chain reaction to amplify and clone full-length coding domains from the population of 4.1R cDNA containing AUG-1 and the population excluding AUG-1. We observed an impressive repertoire of 4.1R isoforms that included 7 major and 11 minor splice variants, thus providing the first definitive characterization of 4.1R primary structures in a single-cell lineage. 4.1R isoforms, transfected into COS-7 cells, distributed to the nucleus, cytoplasm, plasma membrane, and apparent centrosome. We confirmed previous studies showing that inclusion of exon 16 was essential for efficient nuclear localization. Unexpectedly, immunochemical analysis of COS-7 cells transfected with an isoform lacking both AUG-1 and AUG-2 documented that a previously unidentified downstream translation initiation codon located in exon 8 can regulate expression of 4.1R. We speculate that the repertoire of primary structure of 4.1R dictates its distinct binding partners and functions during erythropoiesis.

Four paralogous protein 4.1 genes map to distinct chromosomes in mouse and human.

Four highly conserved members of the skeletal protein 4.1 gene family encode a diverse array of protein isoforms via tissue-specific transcription and developmentally regulated alternative pre-mRNA splicing. In addition to the prototypical red blood cell 4.1R (human gene symbol EPB41,) these include two homologues that are strongly expressed in the brain (4.1N, EPB41L1; and 4.1B, EPB41L3) and another that is widely expressed in many tissues (4.1G, EPB41L2). As part of a study on the structure and evolution of the 4.1 genes in human and mouse, we have now completed the chromosomal mapping of their respective loci by reporting the localization of mouse 4.1N, 4.1G, and 4.1B, as well as human 4.1B. For the mouse 4.1 genes, Southern blot analysis of RFLPs in The Jackson Laboratory BSS interspecific backcross yielded the following assignments: 4.1N (Epb4.1l1,) chromosome 2; 4.1G (Epb4.1l2,) chromosome 10; and 4.1B (Epb4.1l3,) mouse chromosome 17. Human 4.1B was physically mapped to chromosome 18p11 using fluorescence in situ hybridization. All of the mouse genes mapped within or adjacent to regions of conserved synteny with corresponding human chromosomes. We conclude that a set of four paralogous 4.1 genes has been evolutionarily conserved in rodents and primates.

Neurobehavioral deficits in mice lacking the erythrocyte membrane cytoskeletal protein 4.1.

The erythrocyte membrane cytoskeletal protein 4.1 (4.1R) is a structural protein that confers stability and flexibility to erythrocytes via interactions with the cytoskeletal proteins spectrin and F-actin and with the band 3 and glycophorin C membrane proteins. Mutations in 4.1R can cause hereditary elliptocytosis, a disease characterized by a loss of the normal discoid morphology of erythrocytes, resulting in hemolytic anemia [1]. Different isoforms of the 4.1 protein have been identified in a wide variety of nonerythroid tissues by immunological methods [2-5]. The variation in molecular weight of these different 4.1 isoforms, which range from 30 to 210 kDa [6], has been attributed to complex alternative splicing of the 4.1R gene [7]. We recently identified two new 4.1 genes: one is generally expressed throughout the body (4. 1G) [8] and the other is expressed in central and peripheral neurons (4.1N) [9]. Here, we examined 4.1R expression by in situ hybridization analysis and found that 4.1R was selectively expressed in hematopoietic tissues and in specific neuronal populations. In the brain, high levels of 4.1R were discretely localized to granule cells in the cerebellum and dentate gyrus. We generated mice that lacked 4.1R expression; these mice had deficits in movement, coordination, balance and learning, in addition to the predicted hematological abnormalities. The neurobehavioral findings are consistent with the distribution of 4.1R in the brain, suggesting that 4.1R performs specific functions in the central nervous system.

Cloning and characterization of 4.1G (EPB41L2), a new member of the skeletal protein 4.1 (EPB41) gene family.

The prototypical erythrocyte membrane skeletal protein 4.1 (HGMW-approved symbol EPB41), here designated 4.1R, is encoded by a large, complexly spliced gene located on human chromosome 1p32-p33. In this paper we report evidence for a second 4.1 gene, 4.1G (HGMW-approved symbol EPB41L2), which maps to human chromosome 6q23 and is widely expressed among human tissues. The complete nucleotide sequence of 4.1G cDNA predicts a 113-kDa protein that exhibits three regions of high homology to 4.1R, including the membrane binding domain, the spectrinactin binding domain, and the C-terminal domain. Interspersed among the shared domains are unique sequences that may define functional differences between 4.1R and 4.1G. Specific isoforms of 4.1R and 4.1G exhibit differential subcellular localizations. These results expand the 4.1 gene superfamily and demonstrate that the diverse cellular complement of 4.1 isoforms results from both alternative splicing and expression of distinct genes.

The 13-kD FK506 binding protein, FKBP13, interacts with a novel homologue of the erythrocyte membrane cytoskeletal protein 4.1.

We have identified a novel generally expressed homologue of the erythrocyte membrane cytoskeletal protein 4.1, named 4.1G, based on the interaction of its COOH-terminal domain (CTD) with the immunophilin FKBP13. The 129-amino acid peptide, designated 4.1G-CTD, is the first known physiologic binding target of FKBP13. FKBP13 is a 13-kD protein originally identified by its high affinity binding to the immunosuppressant drugs FK506 and rapamycin (Jin, Y., M.W. Albers, W.S. Lane, B.E. Bierer, and S.J. Burakoff. 1991. Proc. Natl. Acad. Sci. USA. 88:6677- 6681); it is a membrane-associated protein thought to function as an ER chaperone (Bush, K.T., B.A. Henrickson, and S.K. Nigam. 1994. Biochem. J. [Tokyo]. 303:705-708). We report the specific association of FKBP13 with 4.1G-CTD based on yeast two-hybrid, in vitro binding and coimmunoprecipitation experiments. The histidyl-proline moiety of 4.1G-CTD is required for FKBP13 binding, as indicated by yeast experiments with truncated and mutated 4.1G-CTD constructs. In situ hybridization studies reveal cellular colocalizations for FKBP13 and 4.1G-CTD throughout the body during development, supporting a physiologic role for the interaction. Interestingly, FKBP13 cofractionates with the red blood cell homologue of 4.1 (4.1R) in ghosts, inside-out vesicles, and Triton shell preparations. The identification of FKBP13 in erythrocytes, which lack ER, suggests that FKBP13 may additionally function as a component of membrane cytoskeletal scaffolds.

Cloning of mDEAH9, a putative RNA helicase and mammalian homologue of Saccharomyces cerevisiae splicing factor Prp43.

Yeast splicing factor Prp43, a DEAH box protein of the putative RNA helicase/RNA-dependent NTPase family, is a splicing factor that functions late in the pre-mRNA splicing pathway to facilitate spliceosome disassembly. In this paper we report cDNA cloning and characterization of mDEAH9, an apparent mammalian homologue of Prp43. Amino acid sequence comparison revealed that the two proteins are approximately 65% identical over a 500-aa region spanning the central helicase domain and the C-terminal region. Expression of mDEAH9 in S. cerevisiae bearing a temperature-sensitive mutation in prp43 was sufficient to restore growth at the nonpermissive temperature. This functional complementation was specific, as mouse mDEAH9 failed to complement mutations in related splicing factor genes prp16 or prp22. Finally, double label immunofluorescence experiments performed with mammalian cells revealed colocalization of mDEAH9 and splicing factor SC35 in punctate nuclear speckles. Thus, the hypothesis that mDEAH9 represents the mammalian homologue of yeast Prp43 is supported by its high sequence homology, functional complementation, and colocalization with a known splicing factor in the nucleus. Our results provide additional support for the hypothesis that the spliceosomal machinery that mediates regulated, dynamic changes in conformation of pre-mRNA and snRNP RNAs has been highly conserved through evolution.

Cell shape-dependent regulation of protein 4.1 alternative pre-mRNA splicing in mammary epithelial cells.

Expression of the complex gene encoding multiple isoforms of structural protein 4.1 is regulated by alternative pre-mRNA splicing. During erythropoiesis, developmental stage-specific inclusion of exon 16 generates protein 4.1 isoforms having a fully functional spectrin-actin binding domain. Here we show that human mammary epithelial cells (HMEC), coincident with the dramatic morphological changes induced by altered culture conditions, exhibit a novel pre-mRNA splicing switch involving a new exon (exon 17B, 450 nucleotides) in the COOH-terminal coding region. 4.1 RNA expressed in proliferating HMEC adherent to culture dishes mostly excluded exon 17B, whereas 4.1 transcripts processed in nondividing suspension cultures of HMEC strongly included this exon. This pre-mRNA splicing switch was reversible: cells transferred from poly(2-hydroxyethyl methacrylate) back to plastic resumed cell division and down-regulated exon 17B expression. More detailed studies revealed complex tissue-specific alternative splicing of exon 17B and another new exon 17A (51 nucleotides). These results predict the existence of multiple 4.1 protein isoforms with diverse COOH termini. Moreover, they strongly suggest that regulation of gene expression during differentiation of epithelial cells is mediated not only by transcriptional mechanisms, but also by post-transcriptional processes such as alternative pre-mRNA splicing.