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Genomics 86 (2005) 701 – 707 www.elsevier.com/locate/ygeno

Evolutionarily conserved coupling of transcription and alternative splicing in the EPB41 ( 4.1R) and EPB41L3 (protein 4.1B)

Jeff S. Tan a, Narla Mohandas b, John G. Conboy a,*

a Life Sciences Division and Genomics Department, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA b Red Cell Physiology Group, New York Blood Center, New York, NY 10021, USA

Received 20 April 2005; accepted 11 August 2005 Available online 20 October 2005

Abstract

Recent studies have shown that transcription and alternative splicing can be mechanistically coupled. In the EPB41 (protein 4.1R) and EPB41L3 (protein 4.1B) genes, we showed previously that promoter/alternative first exon choice is coupled to downstream splicing events in exon 2. Here we demonstrate that this coupling is conserved among several vertebrate classes from fish to mammals. The EPB41 and EPB41L3 genes from fish, bird, amphibian, and mammal genomes exhibit shared features including alternative first exons and differential splice acceptors in exon 2. In all cases, the 5V-most exon (exon 1A) splices exclusively to a weaker internal acceptor site in exon 2, skipping a fragment designated as exon 2V. Conversely, alternative first exons 1B and 1C always splice to the stronger first acceptor site, retaining exon 2V. These correlations are independent of cell type or species of origin. Since exon 2V contains a translation initiation site, splice variants generate protein isoforms with distinct N-termini. We propose that these genes represent a physiologically relevant model system for mechanistic analysis of transcription- coupled alternative splicing. D 2005 Elsevier Inc. All rights reserved.

Keywords: Protein 4.1R; Alternative first exons; Alternative mRNA splicing; Protein 4.1B; Transcription and splicing coupling

Gene expression within an organism is orchestrated and the balance between splicing enhancers and silencers is through a variety of cellular mechanisms, a major one being thought to play a key role in regulation. However, the manner alternative splicing of pre-mRNAs. It is estimated that as in which these elements are spatially and temporally many as 60% of human products undergo differential integrated with other RNA processing events in the nucleus splicing, with many transcripts having multiple splicing is not well understood. An abundance of data supports the patterns and some having thousands [1–3].ThisRNA general hypothesis that downstream processes such as RNA processing can lead to differences in exon composition at splicing and polyadenylation are mechanistically coupled to the 5V end, within the internal coding regions, or at the 3V end upstream transcriptional events [7–10]. Recent studies have [4,5]. Since this regulated inclusion or exclusion of selected demonstrated, moreover, that transcription can also be exons allows the synthesis of multiple protein isoforms that coupled to alternative pre-mRNA splicing; i.e., the nature of possess unique biological properties, polypeptide variants the transcriptional promoter can influence the ability of from the same gene can engage in vastly different cellular known splicing factors to enhance the efficiency of inclusion processes. of specific alternative exons [11,12]. In addition, coactivator Many cis-acting regulatory sequences that strongly influ- that enhance steroid hormone receptor transcription ence splicing efficiency have been identified in the RNA and alter its splicing have recently been reported, further within or near regulated alternative exons [6]. In addition, a reinforcing this concept [13]. number of trans-acting splicing factors that interact at these In previous studies, we presented evidence of coupling sites to control splicing decisions have been characterized, between transcription and alternative splicing in the EPB41 and EPB41L3 genes (also known as protein 4.1R and 4.1B, * Corresponding author. Fax: +1 510 486 6746. respectively) of human and mouse [14,15]. Specifically, both E-mail address: [email protected] (J.G. Conboy). EPB41 and EPB41L3 possess alternative first exons that are

0888-7543/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2005.08.005 702 J.S. Tan et al. / Genomics 86 (2005) 701–707 mutually exclusive and that can splice to different splice observed in the human. These results indicate that the coupling acceptor sites in downstream exon 2. For example, EPB41 between upstream promoters and downstream alternative gene transcription is driven from three different promoters splicing has been conserved for at least 500 million years and produces three classes of transcripts with different first and suggest that the 5V complexity of the EPB41/EPB41L3 exons. These exons, termed 1A, 1B, and 1C, are distin- genes plays a critical but underappreciated role in regulating guished by their distant location from the coding exons (72– expression of these genes encoding essential cytoskeletal 100 kb), as well as their specificity in splicing to exon 2. adaptor proteins. These genes may offer a physiologically Whereas exon 1A splices to exon 2 to exclude a small portion relevant model system for studying mechanisms of coupling at the 5V end (termed exon 2V), transcripts with 1B and 1C between transcription and alternative splicing. include the small exon 2V region along with the main body of exon 2. As there is a start codon located in exon 2V, this Results alternative splicing effects a switch in translation initiation, which results in different protein products: 80 kDa (1A) vs Complex 5V gene structure is phylogenetically conserved in the 135 kDa (1B and 1C) [14]. A precisely analogous pattern is EPB41/EPB41L3 genes across several vertebrate classes also observed for EPB41L3, involving multiple far-upstream promoters/first exons and their differential splicing to Previous studies have shown that the mouse Epb4.1 and alternative splice acceptor sites in exon 2 [15]. Epb4.113 genes, like those in human, also possess alternative The goal of the current study was to explore the first exons [14,15]. In this study, we analyzed genetic databases evolutionary conservation of putative transcription-coupled and recent genome assembly data to examine phylogenetic splicing in the protein 4.1 genes among various vertebrate conservation of EPB41 and EPB41L3 gene organization classes. Here, we report that the N-terminus complexity of among other vertebrate classes. For mammals, comparative EPB41 and EPB41L3 genes has been preserved across genomics revealed strong orthologs of EPB41 exons 1A and vertebrate classes from fish to mammals. Moreover, the first 1C and EPB41L3 exons 1A and 1B in the dog (Fig. 1), as well exons in all of these organisms follow strictly defined splicing as in the chimp, rat, and cow (data not shown). The authenticity patterns to alternative acceptor sites in exon 2, identical to that of these first exons is supported by their high homology to the

Fig. 1. Multiple first exons are present in the EPB41/EPB41L3 genes in several vertebrate classes. (A) 5V gene organization of EPB41 for the various species. At least two alternative first exons have been identified in the dog, frog, and chicken: one each from the 1A and 1B/C type. In zebrafish a good candidate 1B/C is indicated; a predicted exon 1A has also been found in a zebrafish transcript. However, since the zebrafish genome assembly is currently incomplete, its identity is uncertain at this time. Note that the alternative first exons are distinguished by their large separation from the downstream exon 2. (B) First exon organization for EPB41L3. Multiple first exons were also found for EPB41L3 from human to fish, except for chicken. The small numbers between the exons indicate distance in kilobases. 1A, 1B, and 1C refer to distinct types of exons based on their splicing pattern to exon 2/2V. AUG1 represents the start codon located in exon 2V found in all species examined. J.S. Tan et al. / Genomics 86 (2005) 701–707 703 validated human exons (¨70–90% identity), their location category, involving the most upstream exon (1A), splices to upstream of exon 2, and in many cases their representation in an internal acceptor site in exon 2 and excludes a small bona fide cDNAs from the various species (see below). portion at the 5V end (exon 2V). Since there is a start codon in For other vertebrate classes such as birds, amphibians, and exon 2V, this skipping leads to translation initiation much fish, the orthologous first exons could not be identified directly farther downstream in exon 4, using an AUG2 that is by genome comparison, presumably due to sequence diver- conserved in all species examined. In contrast, first exons gence among these noncoding exons. Therefore, we used a of the 1B/C type splice to the first acceptor site in exon 2 so different strategy for identifying their alternative first exons. as to include exon 2V. As it leads to alternative start codon The alternative approach for finding first exons involved usage, this differential splicing creates protein isoforms. retrieving species-specific EPB41 and EPB41L3 transcripts To determine whether the strict coupling between first exon from the genetic databases, screening for clones that contain choice and downstream exon 2 splicing is also conserved sequences extending upstream of exon 2, and then mapping the among vertebrate species other than human and mouse, we 5V-end sequences of these transcripts back to the cognate undertook a survey of EPB41 and EPB41L3 transcripts in the genome assemblies. Using this method, we were able to GenBank database. Representative transcripts were analyzed identify alternative exons for both genes in nearly all of the not only from dog, chicken, frog, and zebrafish genomes (Figs. nonmammalian vertebrates considered in this study, including 2B and 2C), but also from orangutan, pig, rat, cow, and horse EPB41 orthologs of chicken, frog, and zebrafish (1B/C only), (not shown). This analysis demonstrated unequivocally that, as well as EPB41L3 orthologs of frog and zebrafish (Fig. 1). A for all of these organisms, the 5V-most exon (1A) always splices candidate zebrafish EPB41 exon 1A has also been identified, to the internal (distal) acceptor site in exon 2, while the but this assignment is tentative due to the fact that its sequence succeeding first exon(s) of the 1B/1C type always splices to the is not yet represented in the zebrafish genome assembly. more proximal site. This is a very robust observation, since a Based on these data, the overall organization of the 4.1 combined total of 35 cDNAs of type 1A and 30 cDNAs of type genes has been conserved for at least 500 million years back to 1B/C always obeyed this coupling rule, independent of species the emergence of fish. An intriguing feature of this structure is or tissue type. the very long 5V intronic distances that have been evolutionarily Although both the proximal and the distal splice sites are preserved. Although the zebrafish genome assembly is not yet used, these acceptors are very disparate in quality. Using an complete for these large 4.1 genes, a consistent pattern emerges algorithm designed to estimate splice site strength (see from other vertebrate classes in which intron length increases Materials and methods), we found that the pyrimidine-rich with time up to mammals. For example, whereas the distance proximal acceptor site, without exception, is the much from EPB41 exon 1A to exon 2 is approximately 49 kb in the stronger one for both EPB41 and EPB41L3 in all species frog and 56 kb in the chicken, in the these tested (Fig. 3). The internal (distal) splice sites are consis- exons are separated by ¨100 kb. Similarly, EPB41L3 exon 1A tently more purine-rich and score much lower for predicted is 85 kb upstream of exon 2 in the frog, but ¨141 kb distant acceptor site strength. For instance, the proximal site of from exon 2 in human. EPB41 from human to fish is on average 86.6 T 1.78, Since in the dog, chicken, frog, and zebrafish we could compared to the distal site_s average value of 70.9 T 1.79. not find both 1B and 1C orthologs for either EPB41 or Discrepancy between the two sites is even more pronounced EPB41L3, we organized the first exons of the vertebrates in for EPB41L3. For this gene, the proximal site has an average this study into two distinct groups: the 1A type and the 1B/C splice value of 92.7 T 1.82, while for the distal site, the value type. The 1A type represents the farthest upstream (most 5V) is 67.88 T 1.21 (excluding the zebrafish datum, which is first exon, and the 1B/C type represents the remaining first unusually low). The conclusion from these data is that cells exon(s). This positional classification was also accompanied must possess an active regulatory mechanism to facilitate the by functional correlates: exons 1A consistently exhibited a consistent and selective splicing of 1A-type exons across splicing pattern that was distinct from all exons 1B and 1C large introns to the weaker distal acceptor site in exon 2. (see also below). Conservation of exon 2V primary sequences Alternative first exons splice to different acceptor sites in exon 2 Although the EPB41 and EPB41L3 first exons do not exhibit primary sequence conservation across vertebrate From earlier studies of EPB41 and EPB41L3 alternative classes, in contrast the short exon 2V region is highly first exons in the human and mouse, it was shown that these conserved from human to zebrafish (Table 1). For EPB41, exons possess their own transcriptional promoters and that the length of exon 2V from human to fish is essentially fixed they splice differently to downstream acceptor sites in exon 2 (17 T 1 nt), whereas exon 2V in EPB41L3 varies from 21 nt [14,15]. This unique splicing paradigm suggested coupling (chicken and frog), to 27 nt (mouse, rat, cow, zebrafish), to 33 between upstream transcription events and downstream nt (human and dog). Sequence alignments revealed significant alternative splicing events (Fig. 2A). For both EPB41 and identity of exon 2V between EPB41 and EPB41L3 among the EPB41L3, the alternative first exons can be classified into species, especially in an 11-nt region spanning the translation two distinct types based on their splicing to exon 2. One start site. Here, there is a motif AYCATGACRAC that is 704 J.S. Tan et al. / Genomics 86 (2005) 701–707

Fig. 2. The multiple first exons splice to different acceptor sites in downstream exon 2. (A) Alternative first exons of the EPB41 and EPB41L3 genes can be classified into two distinct categories based on their splicing pattern to exon 2. The exon farthest upstream, designated type 1A, splices to an internal acceptor site in exon 2; the more downstream first exons, denoted type 1B/C, splice to exon 2 to include exon 2V. This leads to alternative use of start codons (denoted by AUG1/ AUG2 and asterisks). (B) The accession IDs of the various EPB41 cDNA clones found for each species. The first exons of these clones can be classified without exception into either the 1A or the 1B/C type. (C) The accession IDs of the cDNA clones found for EPB41L3 in the various species are listed here. Again, the 5V ends can be categorized into either the 1A or the 1B/C type based on their splicing pattern to exon 2/2V. .More than one clone available. *Exon deduced by homology to human exon ortholog. present in all of the species examined, with only very minor Discussion deviations. Functionally, this conserved sequence is required for translation initiation at ATG1; it could also potentially This study demonstrates that complex 5V gene structure is function in the selection of the distal splice site for exons 1A. phylogenetically conserved across several vertebrate classes for In the mouse and human genes for EPB41 and EPB41L3, the genes encoding two members of the protein 4.1 family of skipping of exon 2V prevents translation of exon 2 sequences, cytoskeletal proteins: EPB41 and EPB41L3. For these genes leading to translation initiation at ATG2 downstream in exon 4. from fish to mammals, genome and transcript analyses reveal This results in synthesis of protein isoforms with shorter N- multiple alternative first exons that map far upstream of the terminal domains [14,15]. Since the ATG2 in exon 4 is present coding regions, as well as unique splicing patterns for these and conserved in all the species examined (data not shown), exons to specific splice acceptor sites in exon 2 that result in this supports the notion that the same mechanism operates in differential translation initiation. The most remarkable finding is the 4.1 genes of avians, amphibians, and fish. that, despite the long intronic sequences that separate these first J.S. Tan et al. / Genomics 86 (2005) 701–707 705

vertebrate species revealed no evidence of any exons upstream of those designated here as first exons. An intriguing issue surrounding these first exons is their ability to discriminate, from a position tens of kilobases upstream of exon 2, between splice acceptor sites that are only 17–33 nt apart. Computational analyses indicate that the proximal and distal acceptor sites exhibit dramatic differences in intrinsic splice site ‘‘strength’’—with the former always stronger than the latter—suggesting that the first acceptor would likely be the default splice site. Future mechanistic studies will need to explain how exon 1A can selectively bypass the stronger acceptor to choose the apparently weaker site just downstream. One attractive hypothesis is that there is a physical and functional coupling between transcription and alternative splicing [11–13,18]. Since the long CTD repeat domain of the RNA Pol II large subunit has been proposed as a platform for assembly of protein complexes required for downstream RNA processing events, it may also serve a similar function for proteins that mediate alternative splicing decisions [19– 23]. A number of interesting proteins that can interact with both CTD and RNA have been identified as potential candidates for coupling of transcription and splicing. Among these are SAF-B, SCAFs (SR-like CTD-associated factors), TLS/FUS-related proteins, p54nrb/PSF, and U2AF65-related proteins CAPERa and CAPERh [24–30,13,18]. But how might promoter structure influence alternative splicing? If loading of splicing cofactors onto the CTD is influenced by Fig. 3. The splice sites in exons 2/2V have unequal strength. (A) Schematic promoter-specific regulatory elements, then it is easy to showing the alternative splice sites at the exon 2 and 2V junction. The first splice speculate that the promoters associated with each alternative acceptor has been designated the proximal site and the internal acceptor as the distal site, based on their relative proximity to the upstream exons. (B) The first exon might assemble different cofactors that determine estimated splice strengths of alternative 3V splice acceptors of EPB41 exon 2V the splicing pattern at exon 2V/2. The observed ability of for the species examined in this study. The data indicate a clear discrepancy in CAPERa and CAPERh to modulate downstream alternative splice site quality. The difference is even more dramatic for that of EPB41L3, splicing specifically from steroid hormone-responsive, but not shown in C. nonresponsive, promoters represents a precedent for this type of model [13]. exons and their downstream splice acceptors, there is a strictly Alternatively, the promoter might influence downstream obeyed paradigm: transcripts that initiate at exon 1A always skip splicing events in a couple of other ways. The promoter may exon 2V, while transcripts that start at exons 1B/C always include recruit molecules that can phosphorylate the CTD. Since exon 2V (Fig. 4). This specific splicing is observed across many phosphorylated CTD has been shown to be much stronger in tissue sources and in diverse vertebrate classes ranging from fish activating the spliceosomal assembly than its nonphosphory- to mammals (this study, [14]). lated form, molecules that alter the level of CTD phosphor- Since exons 1A and 1B/C have been associated with ylation can have a potent impact in splicing regulation [31]. independent transcriptional promoters, it is assumed that the Finally, promoters might also influence splicing events by splicing correlations of 4.1 transcripts are due to coupling recruiting factors that can manipulate transcription kinetics. It between transcription and alternative splicing. Promoter has been reported that introduction of transcriptional pause activity has been demonstrated for flanking, upstream sites downstream of a weak splice site can improve its sequences of human EPB41 exons 1A and 1B, as well as efficiency of recognition by the spliceosomal machinery EPB41L3 exons 1A and 1B, using luciferase as a reporter [32,33]. However, this particular model is difficult to [14,15]. Since other mammalian exons in this study share reconcile for the regulated splicing events at EPB41 and significant sequence identity with the human orthologs, this EPB41L3 exon 2V. Since the stronger splice acceptor site is strongly suggests their authenticity as being true first exons. transcribed before the weaker competing site, it is not For the nonmammalian vertebrates, the strong similarity in apparent how transcriptional pausing could impart a kinetic genomic organization and splicing patterns to exon 2V/2 advantage for the weaker site. suggests these exons as being functionally homologous first Regardless of its exact mechanism, the differential exons with associated promoter activity. In addition, analysis splicing of alternative exons to acceptor sites in their of 5V sequences in dozens of 4.1 cDNAs from multiple downstream exon 2 has important functional ramifications 706 J.S. Tan et al. / Genomics 86 (2005) 701–707

Table 1 Conservation of exon 2V in EPB41 and EPB41L3

The nucleotide sequences of exon 2V in both (A) EPB41 and (B) EPB41L3 for all the species examined are shown here. There is an 11-nt sequence, marked by the motif AYCATGACRAC (highlighted box), which is conserved for both EPB41 and EPB41L3 among all the species. This stretch of sequence may play an important role in the selection of the distal splice site for exon type 1A. The start codon in exon 2V is highlighted in red. of the encoded protein 4.1 polypeptides. By regulating different splicing efficiencies when transcriptionally activated expression of the translation initiation site in exon 2V, by different promoters [11,12]. An analogous situation could upstream transcriptional events ultimately regulate the apply to natural endogenous genes by assembly of functionally synthesis of protein isoforms with very different N-terminal distinct transcription complexes, involving alternative enhancer domains. Since the N-terminal domain in the longer isoform cofactors, without overt changes in the 5V end of the transcribed of EPB41 (135 kDa) can significantly alter the binding of RNA. In such cases the transcription-alternative splicing EPB41 to membrane proteins such as band 3 and connection would be impossible to discern from mRNA , transcription-splicing regulation may be an analysis alone. Future studies will be needed to investigate this essential determinant of important biological properties of hypothesis. the protein [34]. The observation that this mechanism has been extant for many millions of years of evolution further Materials and methods supports its biological importance as a method of gene regulation and expression. Identification of alternative EPB41/EPB41L3 5V exons in various Finally, we speculate that coupling between transcription vertebrate classes and alternative splicing may be far more widespread than is Alternative 5V exons of the human and mouse EPB41/EPB41L3 genes were currently appreciated. The clear manifestation of this phenom- identified in earlier studies through genetic database searches (GenBank and the enon in the 4.1 genes is due to involvement of independent first UCSC genomics Web site, http://www.genome.ucsc.edu) and RACE experi- exons with completely distinct nucleotide sequences repre- ments [14,15]. To identify alternative 5V exons in the EPB41/EPB41L3 sented in the mature mRNAs. It is entirely possible that a orthologs of the dog (Canis familiaris), chicken (Gallus gallus), frog (Xenopus similar coupling occurs in many genes in which no such tropicalis), and zebrafish (Danio rerio), the exon 2 sequences of human EPB41 and EPB41L3 were first used in BLAT searches in the UCSC genomics ‘‘sequence tag’’ remains at the 5V end of the mRNA. Precedence database to identify their orthologs in these species. Once the orthologs were for such a model has been reported using synthetic constructs to identified, alternative 5V sequences for this gene were analyzed through show that an alternative exon-containing minigene can exhibit identification of existing, known clones. In cases for which no submitted data were available, putative first exons were deduced through homology to the human first exon (1A, 1B, or 1C) sequences; sequences that showed significant identity to the human exons and that mapped upstream of exon 2 in the expected locations were accepted as orthologs. This approach led to the identification of multiple first exons for both EPB41 and EPB41L3 in almost all of the species analyzed. Note that the zebrafish EPB41 clone extends the previously reported 5V sequence so as to include the bona fide translation initiation site AUG1, which encodes an N-terminal tetrapeptide MTTD, highly homologous to the mammalian MTTE N-terminal motif [16]. Fig. 4. Summary of first exon splicing to downstream acceptor sites in exons 2/ 2V. Transcription initiated from the most upstream exon (1A) leads to the Analysis of splice site strength in exon 2/2V of EPB41/EPB41L3 selection of the weaker, more distal splice site in exon 2, while transcription from more downstream first exons (1B/C) allows the use of the stronger, more Splice site strengths of both the proximal and the distal acceptors in exon proximal splice acceptor. In this study, this paradigm is followed without 2/2V were computed using a special program (http://www.genet.sickkids.on.ca/ exception among all the exons examined. The use of the much weaker splice ~ali/splicesitescore.html) [17]. To compute the acceptor scores, a 15-bp site for the 1A class of first exons strongly suggests a special mechanism sequence was inputted into the program; this sequence consisted of 14 bp coupling transcription to splicing. before and 1 bp after the splice junction. Although this program is designed J.S. Tan et al. / Genomics 86 (2005) 701–707 707 for human genes, it was used to assess the splice acceptors for all the species Zon, S. 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