Regulation of of CD45 by Antagonistic Effects of SR Protein Splicing Factors1

Gerdy B. ten Dam,2* Christian F. Zilch,‡ Diana Wallace,§ Be´Wieringa,* Peter C. L. Beverley,§ Lambert G. Poels,† and Gavin R. Screaton¶

CD45 is a transmembrane glycoprotein possessing tyrosine phosphatase activity, which is involved in cell signaling. CD45 is expressed on the surface of most leukocytes and can be alternatively spliced by the inclusion or skipping of three variable (4, 5, and 6 or A, B, and C) to produce up to eight isoforms. In T cells, the splicing pattern of CD45 isoforms changes after activation; naive cells express high m.w. isoforms of CD45 which predominantly express A (CD45RA), whereas activated cells lose expression of exon A to form low m.w. isoforms of CD45 including CD45RO. Little is known about the specific factors controlling the switch in CD45 splicing which occurs on activation. In this study, we examined the influence of the SR family of splicing factors, which, like CD45, are expressed in tissue-specific patterns and have been shown to modulate the alternative splicing of a variety of transcripts. We show that specific SR proteins have antagonistic effects on CD45 splicing, leading either to exon inclusion or skipping. Furthermore, we were able to demonstrate specific changes in the SR protein expression pattern during T cell activation. The Journal of Immunology, 2000, 164: 5287–5295.

ntron removal is an essential step during eukaryotic gene 12, 14, 26, 27). SR proteins are expressed in tissue-specific pat- expression. This process requires recognition of the 5Ј and 3Ј terns (8, 10, 14, 26) and can be regulated by phosphorylation (28). I splice sites by the : a multicomponent ribonucle- The effects of the SR proteins SF2/ASF can be antagonized by the oprotein complex. The major components of the spliceosome are heterogeneous ribonucleoproteins A1 and A2B1 (29, 30). In addi- the small ribonucleoprotein particles (snRNPs)3 U1, U2, and tion, a further level of control of SR protein function may be sub- U4/U6 and the non-snRNP proteins including the family of SR served by nucleocytoplasmic shuttling (31, 32). proteins (reviewed by Green (3) and Kramer (4)). This family of CD45 is a transmembrane glycoprotein expressed on leukocytes closely related, highly conserved proteins is characterized by the (reviewed by Thomas (1) and Trowbridge and Thomas (2)). Al- presence of one or two N-terminal RNA recognition motifs ternative splicing of three variable exons (4, 5, and 6 or A, B, and (RRMs) and a C-terminal domain rich in arginines and serines (RS C) allows the production of eight possible isoforms. In rodent domain). SR proteins are essential for constitutive splicing (5–10) cells, all of these isoforms have been isolated (33–35), whereas in

by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. and alternative splicing (10–17). The family is highly conserved humans only five have been identified (36, 37). The splicing is between diverse species and have been shown to be essential for cell-type specific and activation dependent. B cells express mainly cell survival (18, 19). SR proteins function at multiple steps during the high m.w. isoform (ABC), whereas T cells express a panel of the splicing reaction. In early steps, they facilitate U1 snRNP bind- different isoform ranging form the smallest isoform (null), found ing to the 5Ј splice site (20, 21). Furthermore, they stabilize com- also on thymocytes, to the largest isoform (ABC). T cell activation plex assembly at the 3Ј splice site by assisting the binding of U2AF leads to a programmed shift from high to the low m.w. isoforms, (22) and by forming bridges to connect 5Ј and 3Ј splice sites i.e., down-regulation of CD45RA expression and concomitantly (23–25). up-regulation of CD45RO expression. It is this change in cell sur- Substrate-specific effects of individual SR proteins have been face that is used to differentiate naive from memory T http://classic.jimmunol.org demonstrated in constitutive as well as alternative splicing (7, 11, cells (38). During this activation, there is also a decrease in expression of CD45RC which is higher than the small decrease Departments of *Cell Biology and †Anatomy, Faculty of Medical Sciences, Univer- observed for CD45RB but less prominent than the CD45RA sity of Nijmegen, Nijmegen, The Netherlands; ‡Imperial Research Fund Tu- decrease (39). mour Immunology Unit, University College London Medical School, London, United In this study, we sought to identify factors involved in the reg- Kingdom; §The Edward Jenner Institute for Vaccine Research, Compton, Newbury, United Kingdom; and ¶Institute of Molecular Medicine, John Radcliffe Hospital, ulation of alternative splicing of CD45. Members of the SR protein

Downloaded from Headington, Oxford, United Kingdom family were analyzed for their influence on splice site selection by Received for publication November 1, 1999. Accepted for publication March 1, 2000. cotransfection experiments with a CD45 in COS-1 cells The costs of publication of this article were defrayed in part by the payment of page in vitro (40). Furthermore, we analyzed the SR protein profile in T charges. This article must therefore be hereby marked advertisement in accordance cells before and after stimulation. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a European Molecular Biology Organization short-term fellowship (to G.B.t.D.), a Deutscher Akademischer Austauschdienstgrant (to C.F.Z.), Materials and Methods the Medical Research Council and the Wellcome Trust (to G.R.S.), and the Imperial DNA cloning Cancer Research Fund. 2 Address correspondence and reprint requests to Dr. Gerdy B. ten Dam at her current The human CD45 minigene pSEC-S-LCA1-7 was constructed in the pSEC address: Department of Biochemistry, Trigon Building, Faculty of Medical Sciences, expression plasmid (41) and contains 1) the 5Ј CD45 structural gene region University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. (exons 1 and 2) comprising the ATG codon and the signal peptide; 2) the E-mail address:[email protected] genomic CD45 sequence of exon 3 to exon 7 with the alternative exons 4, 3 Abbreviations used in this paper: SR, serine(S)-arginine(R)-rich; RRM, RNA rec- 5, and 6; and 3) the decay accelerating factor (DAF) GPI anchor attachment ognition motif; DAF, decay accelerating factor. signal to ensure membrane expression of the expressed proteins (Fig. 1A).

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 5288 REGULATION OF CD45 SPLICING

FIGURE 1. CD45 alternative splicing. A, Diagram- matic representation of the CD45 minigene. Tran- scription is driven by the SV40 promoter, are shown as lines, numbered boxes are CD45 exons. DAF GPI attachment site. The position of the oligo- nucleotide primers LCA2 and LCA7 used for PCR are shown as arrows above exons 2 and 7, respectively. B, Diagram of the eight possible alternatively spliced CD45 transcripts and the predicted sizes of the PCR products after amplification with the LCA2 and LCA7 primers. The CD45 isoform expression pattern in the human and mouse is taken from Thomas (1). by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd.

SR protein cDNAs were cloned in the vector pCGT7 which carries an denaturing polyacrylamide gel and detection and quantitation were con- N-terminal tag from the bacteriophage T7 gene (31, 42). SR protein prod- ducted by autoradiography and PhosphoImage analysis (Kodak X-OMAT ucts can therefore be recognized by the T7 tag mAb (Novagen, Madison, S1, Molecular Analyst Bio-Rad, Hercules, CA), respectively. WI). SRp75 was cloned into the parental vector pCG, which lacks the RNA from PHA-stimulated ROϩ and RAϩ T cells was reversed tran- T7 tag. scribed with the LCA9 primer (5Ј-GTAATCCACAGTGATGTTTGC-3Ј) The SR protein chimeras and deletion mutants are listed in Fig. 5. RRM and amplified using LCA2 and LCA7 primers. PCR products were run on or RS domain swaps or deletions were all constructed by PFU-PCR am- a 2% agarose gel. plification of the desired fragments with specifically designed primers and For immunofluorescence analysis, COS-1 cells were seeded on cover- subcloning of the fragments in the pCGT7 expression vector. (Nomencla- slips in 6-well plates and transfected with the CD45 minigene. Cells were http://classic.jimmunol.org ture of the constructs is illustrated for SF2/ASF (30aRRM1–30aRRM2– stained 48 h after transfection at 4°C with the CD45 exon B-specific Ab 30aRS) and SC35 (30bRRM1–30bRS), e.g., substitution of the RS domain PD7/26 (Dakopatts, Copenhagen, Denmark). of SF2/ASF by the RS domain of SC35 is called 30aRRM1–30aRRM2– 30bRS, deletion of a domain, e.g., RS domain of SC35 is indicated by ⌬ SR proteins and immunoblotting or ␦, 30bRRM1-⌬30bRS). To generate the SRp20-SC35 chimera’s PCR products of SRp20-RRM (aa 1–88), SRp20-RS (aa 88–164), SC35-RRM To analyze SR protein products (SR proteins, chimeric constructs, and (aa 1–115), and SC35-RS (aa 116–221) were subcloned in pCGT7 in the deletion mutants) expressed by the pCGT7 vector, transfected COS-1 cells indicated combinations. The SF2-ASF-SRp40 chimeras and SF2/ASF de- were lysed in 5% SDS in 50 mM Tris-HCl (pH 6.8) and 20 mM EDTA, Downloaded from letion mutants were described by Caceres et al. (31, 32). sonicated, and resolved by 12% SDS-polyacrylamide gels. Proteins were electroblotted and probed with the T7 tag Ab (Novagen) or the 104 mAb (43). In vivo analysis of alternative CD45 splicing For SR protein detection in lymphocytes, human T cells were isolated from fresh buffy coats and separated into CD45RAϩ and CD45ROϩ pop- COS-1 cells were cultured in DMEM (Life Technologies, Rockville, MD) ulations by selection with the UCHL1 (CD45RO) and the SN130 supplemented with 10% FCS (Life Technologies) in 6-well plates. At a cell (CD45RA) Abs. Both populations were stimulated with PHA-P for 0, 12, density of 30–40%, cells were (co)transfected with 1 ␮g of minigene DNA 36, 72, and 144 h and analyzed at indicated time points for CD45RA/RO and the indicated amount of SR protein construct using Lipofectin reagents expression by flow cytometry to observe changes in RA/RO expression. according to the manufacturer (Life Technologies). Subsequently, equal amounts of cells were lysed in 5% SDS lysis buffer Transfected cells were harvested 48–72 h after transfection and RNA and sonicated twice for 20 s. Proteins were resolved on 12% SDS-poly- was isolated with the RNAzol B (Cinna/Biotecx Laboratories, Friends- acrylamide gels, electroblotted, and probed with the 104 mAb. wood, TX) method and analyzed by RT-PCR. First-strand specifically primed (DAF reverse primer, 5Ј-CCTAAATGAAGAGCACAATTGCA- Cell separation and stimulation of CD45RAϩ and CD45ROϩ T 3Ј) cDNA synthesized with superscript reverse Transcriptase (Life Tech- cells nologies) from 200 ng of RNA was amplified for 22 cycles using a 5Ј end-labeled forward primer (LCA2, 5Ј-ATTGGATCCGCTGACTTCCA PBMC were isolated from buffy coats (North East London Blood Trans- GATATGACC-3Ј)andanonlabeledreverseprimer(LCA7,5Ј-CCGAGATC fusion Service, London, U.K.) by Ficoll-Paque density gradient centrifu- TTCAGAGGCATTAAGGTAGGC-3Ј). PCR products were run on a 6% gation (Pharmacia, St Albans, U.K.). Monocytes and macrophages were The Journal of Immunology 5289

FIGURE 3. Role of SR proteins in regulation of CD45 alternative splic- ing. COS-1 cells were cotransfected with the CD45 minigene and the in- dicated SR protein constructs. Lane 1, CD45 marker (637, 295, and 154 bp); lane 2, control transfection with the CD45 minigene only; lane 3, control cotransfection with the pCGT7 lacking an insert; and lanes 4–11 cotransfection with the indicated SR proteins.

splicing of CD45 with a recently designed human CD45 minigene (pSEC-S-LCA1-7, Fig. 1A), which can be analyzed at both the RNA and protein levels (40). The minigene is driven by the SV40 promoter and contains exons 1–7 of CD45. A cDNA fragment of exons 1 and 2 is fused to a genomic fragment of exons 3–7. Exon 7 is then fused to the GPI anchor sequence of DAF, which allows the truncated CD45 sequences to be expressed at the cell surface. FIGURE 2. Alternative splicing of the CD45 minigene in COS-1 cells. COS-1 cells transfected with the CD45 minigene and stained with A, Immunofluorescence analysis of nonfixed transfected COS-1 cells a CD45 exon-specific Ab showed a granular membrane staining stained with the CD45RB Ab (PD7/26). B, Isoform expression pattern of

by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. pattern (Fig. 2A). Western blot analysis showed expression of the the CD45 minigene after transient transfection in COS-1 cells. The left CD45 isoforms ABC, BC, AB, B, and null with high expression panel shows radioactive-labeled RT-PCR products run on a denaturing gel. CD45 isoforms are indicated on the left and isoform size (bp) on the right. levels of the low m.w. isoforms (data not shown; Ref. 40). RT- M, CD45 marker (637, 295, and 154 bp). Phosphor image quantitation of PCR analysis of transfected COS-1 cells demonstrated that the the CD45 isoform expression (n ϭ 3). expression of the CD45 null isoform was the most abundant (50%), followed by an equal expression of the BC and B isoforms (20–25%) and a very low ABC expression (1–3%) (Fig. 2B). removed by adherence to plastic for1hat37°C and these were saved and used as APC after mitomycin C treatment where required. CD45RAϩ and SR proteins display antagonistic effects on the alternative ϩ http://classic.jimmunol.org CD45RO T cells were then negatively selected. Nonadherent cells were splicing of CD45 incubated at 4°C for 30 min with a mixture of mAbs, BU12 (anti-CD19, a The focus of this paper was to examine the role of SR proteins in gift from N. Ling, University of Birmingham Medical School, Birming- ham, U.K.), OKM1 (anti-CD11b; American Type Culture Collection), and CD45 alternative splicing. SR proteins are essential for constitu- R10 (antiglycophorin, a gift from P. Edwards, University of Cambridge, tive splicing (5–10) and alternative splicing (10–16) and have Cambridge, U.K.) along with SN130 (anti-CD45RA; Imperial Cancer Re- demonstrated substrate-specific effects on splicing (7, 11, 12, 14, ϩ search Fund, London, U.K.) for removal of CD45RA T cells or UCHL1 26, 27). The SR proteins are thus candidates to control CD45 (anti-CD45RO; Imperial Cancer Research Fund) for removal of CD45ROϩ Downloaded from T cells. After washing, the cells were incubated with sheep anti mouse splicing. IgG-coated magnetic beads at 4°C for 20 min (Dynal, Bromborough, In COS-1 cells, the CD45 minigene is processed to give a mix- U.K.); labeled cells were then removed with a Dynal magnet. After five ture of isoforms (Fig. 2B), of which the smallest is the most abun- rounds of magnetic bead separation, purity was Ͼ98% by FACS analysis. dant (CD45 null, which skips the alternative exons). The ability of ϫ 6 ϩ ϩ For stimulation, 2 10 CD45RA or CD45RO T cells were incu- SR proteins to modulate CD45 splicing was assessed by cotrans- bated in 24-well flat-bottom tissue culture plates (Becton Dickinson, Mountain View, CA) along with 5% mitomycin C-treated adherent cells fecting individual SR cDNAs with the CD45 minigene (Fig. 3). and 1 ␮g/ml PHA-P (Sigma, St. Louis, MO) for 0, 12, 36, 72, and 144 h. Three groups of SR proteins with different specificities were iden- Cells were analyzed by flow cytometry at the time points indicated for tified. In the first group, SF2/ASF, SC35, SRp30c, SRp40, and alterations in CD45RA and CD45RO expression. SRp75 overexpression resulted in down-regulation of the high m.w. isoforms and up-regulation of the CD45 null isoform with the Results CD45 B isoform still detectable (Fig. 3, lanes 6–9 and 11). The Alternative splicing of CD45 occurs by inclusion or skipping of second group, SRp20 and 9G8, showed the opposite (Fig. 3, lanes three alternative exons (4, 5, and 6 or A, B, and C) and results in 4 and 5), with elevated expression of the largest ABC isoform and a maximum of eight different isoforms, of which five are expressed a decrease in expression of the smallest CD45 null isoform. SRp20 in human hematopoietic cells (Fig. 1B). We studied alternative and 9G8 both have a single (canonical) RRM and the homology 5290 REGULATION OF CD45 SPLICING

FIGURE 4. Antagonistic effects of SR proteins SRp20 and 9G8 vs SRp30c and SRp40 on the alternative splicing of CD45. A, Increasing amounts (0.1–5 ␮g) of SRp20, 9G8, SRp30c, and SRp40 were cotransfected with a constant amount of the CD45 minigene. Results were analyzed by RT-PCR and radioactive products were resolved on denaturing polyacrylamide gels. B, Iso- forms were quantitated by phosphor image analysis and the amounts are shown as a percentage of the sum. The graph was extracted from the cotransfection experiments using 3 ␮g of the tested SR proteins shown in A. by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. http://classic.jimmunol.org

between the two proteins in this domain is high (79%) when com- These experiments were repeated several times in both COS-1 pared with the canonical RRM of the other SR proteins (35–45%) and HeLa cells and small differences in the levels of CD45 isoform (9). Finally, SRp55 showed no effect on the alternative splicing of expression in the control lanes were seen, but changes induced by CD45 (Fig. 3, lane 10). SR cotransfection were reproducible in all cases. SR protein ex-

Downloaded from The effects of the antagonistic SR proteins SRp30c and SRp40 pression (except SRp75) was analyzed and confirmed by Western (exon skipping) vs SRp20 and 9G8 (exon inclusion) were exam- blotting using the T7 epitope tag. Localization to nuclear speckles ined in more detail using incremental amounts of the SR construct was also verified by immunofluorescence analysis (data not cotransfected with 1 ␮g of the CD45 target (Fig. 4). The shift to shown). exon inclusion induced by SRp20 was almost complete with only small amounts of isoforms BC and B still detectable. The effect of Role of structural domains of SR proteins in splice site selection 9G8 was less dramatic, the ABC band increased to almost 20% of of the CD45 pre-mRNA the total sum, but the smallest isoform did not decrease and kept As detailed in the introduction, SR proteins consist of two or three steady at 50%. Interestingly, 9G8 promoted the appearance of the modular domains, i.e., the RS domain and either one or two N- AB isoform, which is hardly detectable in the other transfections. terminal RRMs. All SR proteins share the canonical RRM char- Overexpression of SRp30c and SRp40 resulted in a dramatic in- acterized by the conserved RNP-1 and RNP-2 submotifs. In addi- crease of the smallest isoform skipping exons A, B, and C. The tion, a subset of SR proteins also has a central atypical RRM which CD45 ABC and BC isoforms were hardly detectable anymore, lacks the conserved residues in the RNP submotifs (17, 44). To whereas the expression level of the B isoform only decreased mar- determine which of these domains is responsible for the switch in ginally or not at all. CD45 splicing noted above, we examined the function of a series The Journal of Immunology 5291

SRp20 with a switch to exon inclusion and production of higher m.w. isoforms (Fig. 6, B and D, lane 8). This is seen not only with the deletion of RRM2 in wild-type SF2/ASF but also in the chi- mera between the RS domain of SF2/ASF and the RRM of SC35 (Fig. 6B, lanes 5 and 8). Deletion of the RS domains of SF2 and SC35 has little effect on function, like the wild-type proteins, pro- moting exon skipping. (Fig. 6B, lane 6 and 6A, lane 6). All results of the experiments using chimeric SR proteins are tabulated in Fig. 5.

Changes in SR protein expression following the switch in CD45 splicing Upon stimulation, naive T cells switch from CD45RAϩ (exon in- clusion, expressing the CD45 isoforms ABC, AB, BC, and B) to CD45ROϩ (exon skipping, expressing the isoforms B and null), whereas mature memory CD45ROϩ cells remain ROϩ. Resting T cells were separated into ROϩ and RAϩ populations and activated by culture in the presence of PHA. After 6 days, 99% of the RAϩ T cells had lost expression of the exon A epitope and become ROϩ. RT-PCR revealed that at the RNA level in the CD45RAϩ population, the CD45 ABC, AB, and BC isoforms were down- regulated between 36 and 72 h and by 72 h there was a predom- inant expression of the CD45 null and B isoforms. The CD45ROϩ T cell population expressed mainly the CD45 null and B isoforms and remained CD45 null- and B-positive upon stimulation (Fig. 8A). mAb 104, which recognizes a phosphoepitope expressed by all SR proteins (43, 45), was used to assess SR protein expression in RAϩ and ROϩ cells over the time course of stimulation (Fig. 8B). Expression of SR proteins is low in resting cells and by 36–72 h, when cells were proliferating well, SR protein levels were in- creased. Besides this total increase of SR protein expression, sev- eral changes occur specifically in CD45RAϩ-stimulated T cells. FIGURE 5. Summary of the role of the SR proteins and their modular ϩ ϩ domains on the alternative splicing of CD45 in vivo. The SR protein con- SRp75 induction is greater and occurs later in RA -vsRO -

by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. ϩ structs, deletion mutants, and domain swap constructs used in this work are stimulated cells. Furthermore, in RA -stimulated T cells, a band shown schematically. Promotion of CD45 ABC or null splicing after over- appears in the 30-kDa cluster of SR proteins. This 30-kDa SR expression of each construct is indicated by ϩϩϩ or ϩϩ. No effect is protein increased markedly after 72 h of stimulation and judging indicated by Ϫ and a single ϩ indicates no promotion of ABC or null by its size it may represent SRp30c; the smallest SR protein in the splicing but formation of intermediates. Overexpression of 9G8 promotes 30-kDa group, however, direct identification is not possible, as a AB splicing in addition to ABC. specific mAb is not available. We have previously shown induc- tion of SRp30c at the RNA level following T cell activation. Also apparent from these blots are differences in the ratios of some of of domain deletion and domain swap constructs containing frag- the 30-kDa SR proteins. For instance, the third band possibly rep- http://classic.jimmunol.org ments of SRp20, SF2/ASF, SC35, and SRp40. Schematic drawings resenting SC35 is induced more in RA-stimulated cells whereas of all SR proteins, domain deletion, and domain swap constructs the fourth band, possibly 9G8, is not up-regulated in the RA-stim- are listed in Fig. 5. In each case, the expression and integrity of ulated cells. SRp55 and SRp20 were hardly detectable in these T these constructs was tested by Western blotting using the T7 tag cell populations. T cells from two individual were analyzed which Ab (data not shown). demonstrated comparable results after Western blot analysis with To evaluate whether the RRM or RS domain of SRp20 was the mAb 104.

Downloaded from responsible for the change in CD45 alternative splicing, we con- structed an RS domain deletion mutant (20 ␦ RS) and substituted

the RRM of SRp20 with the corresponding domain of SC35 (30b1- 20RS, Fig. 6, A and C). Deletion of the RS domain of SRp20 Discussion promoted skipping of all alternative exons (Fig. 6A, lane 5). When Alternative splicing of CD45 occurs by inclusion or exclusion of

the RRM of SRp20 was exchanged for the RRM of SC35 (30b1- three alternative exons (4, 5, and 6 or A, B, and C) in the N- 20RS), exon skipping was also seen giving a similar pattern to terminal part of the molecule, resulting in a maximum of eight wild-type SC35 (Fig. 6A, lanes 4 and 7), implying a role of the different isoforms. cis-Acting sequences and trans-acting factors RRM of SRp20 in the formation of the ABC isoform. have been postulated to influence the alternative splicing pattern of Domain swap chimeras between SF2/ASF and SRp40 are CD45. Linker scanning analysis revealed that in exon A three seg- shown in Fig. 7. Both the wild-type proteins promote exon skip- ments (positions 8–10, 40–91, and 127–137, total length 198 bp) ping, as do many of the mutants except those in which RRM2 of and in exon C one large segment (positions 16–137, total length SRp40 is fused to RRM1 of SF2/ASF (Fig. 7, lanes 6 and 7). 144 bp) were essential for tissue-specific alternative splicing (46, RRM2 of SF2/ASF seems to play a specific role in splice site 47). No such segments were found in exon 5, suggesting that splic- selection; when it is deleted, the splicing of SF2/ASF resembles ing of exon 5 is not regulated in a tissue-specific manner (46, 47). 5292 REGULATION OF CD45 SPLICING

FIGURE 6. Role of modular domains of SRp20, SC35, and SF2/ASF in CD45 splicing. A, RS domain deletion mutants and domain swap constructs of SRp20 and SC35 were cotransfected with the CD45 minigene and analyzed by RT-PCR. Lane M, CD45 marker and lane 2, control transfection with the CD45 minigene only. B, The indicated deletion mutants of SF2/ASF

and the construct 30b1030aRS were cotransfected with the CD45 minigene and analyzed by RT-PCR. Single transfection with the CD45 minigene is shown in lane 2. CD45 isoforms are indicated on the right, isoform size (bp) is indicated on the left. C and D, CD45 iso- form expression patterns shown in A and B, respec- tively, were quantitated using phosphor imager (Bio- Rad molecular analyst) analysis and reflected in a graph. Relative amounts of CD45 isoforms are shown as a percentage of the sum. Isoforms are indicated on the right and also in gray, cotransfected constructs are indi- cated below the figure, and isoform expression level (%) is indicated on the left.

by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. Fusions between T and B cells retain the T cell phenotype pro- dependent studies identified a SRp20-binding sequence with en- ducing the null isoform which led to the suggestion that exon in- hancer activity, all sharing the degenerate sequence CUC(U/ clusion is the default pattern, whereas skipping is regulated by G)UC(C/T) (51–54). In addition, application of the so-called dominant trans-acting factors in T cells (48). We have previously SELEX strategy yielded SRp20-specific sequences with the con- demonstrated that regulation of alternative splicing of this CD45 sensus CA/UA/UC (55). The CUC(U/G)UC(C/T) sequence is also minigene is restricted to lymphoid cells. All nonlymphoid trans- found in exon 4 of the SRp20 gene, the splicing of which is au- genic cells and transfected cell lines such as COS-1, HeLa, and toregulated by SRp20 itself (56). In this study, it was suggested 3T3 showed a specific and stable pattern of splicing to form the that recognition and splicing of exons with weak splice acceptor low m.w. isoforms of CD45. sites is a general function of SRp20. A perfect match to the SRp20 http://classic.jimmunol.org We have shown that SR proteins show antagonistic effects on consensus sequence is not found in the exonic sequences of CD45; alternative splicing of CD45. SRp20 promoted exon inclusion, however, the sequence CACCACUGCAUUCUCACCC (nt 59–77 leading to elevated levels of the CD45 ABC isoform and to a in exon 4) is reminiscent of all of the identified SRp20 enhancer decrease in expression levels of the CD45 null, B, and BC iso- sequences. forms. 9G8 promoted splicing of the ABC and also the AB iso- Overexpression of 9G8 promotes CD45 AB and ABC splicing. form; however, only splicing of the BC isoform was decreased. This shift is not as complete as the switch induced by SRp20, as

Downloaded from Exon exclusion was promoted by SC35, SF2ASF, SRp30c, SRp40, the CD45 null, B, and BC splicing are still detectable. Appearance and SRp75. All of these SR proteins promoted splicing of the of the AB isoform, in combination with the ABC isoform, indi- CD45 null isoform with decreased levels of splicing of the ABC cates that 9G8 is specifically involved in exon A splicing. No other and BC isoforms. Generally, splicing of the CD45 B isoform was SR protein was able to promote CD45 AB splicing. not affected. This could be explained by the fact that splicing of Recently, two very divergent 9G8 splicing enhancers have been exon 5 is not regulated in a tissue-specific fashion (46, 47). Also, described (54). One shows strong homology with the 9G8 consen- during the switch from CD45RA to CD45RO in activated T cells, sus sequence AGAC(G/U)ACGAY isolated by the SELEX ap- the T cells do not lose expression of the CD45 B isoform (38, 49, proach (55), whereas the other is pyrimidine rich and shows some 50). These results show that individual SR proteins are able to sequence homology with the Drosophila double sex splicing en- switch CD45 splicing; however, to induce a complete shift in hancer (UCUUCAAUCAAACA) which can bind 9G8 specifically CD45 splicing, additional factors like other SR proteins or non-SR (52). SELEX, using a mutated form of 9G8 lacking the zinc- factors might be involved. knuckle region, yields a pyrimidine-rich sequence (C(A/U)(A/ Regulation of CD45 splicing by SRp20 and 9G8 causing exon U)C) that resembles the SRp20 SELEX winner sequence (54, 55). inclusion could be explained by the presence of exonic (or in- Sequences closely resembling the 9G8 consensus are found in tronic) enhancer sequences in the CD45 pre-mRNA. Several in- CD45 exon 4 but not in exons 5 and 6 (e.g., CD45 exon 4: The Journal of Immunology 5293

FIGURE 7. Role of modular domains of SRp40 and SF2/ASF on the alternative splicing of CD45. A, Alteration in the splicing pattern of the FIGURE 8. Dynamic protein expression levels of SR proteins in stim- CD45 minigene after cotransfection with the SRp40 deletion mutant and ulated CD45RA and CD45RO T cells. CD45RO- and RA-selected T cells, by guest on October 1, 2021. Copyright 2000 Pageant Media Ltd. the SRp40-SF2/ASF swap constructs. RT-PCR analysis of the cotransfec- stimulated with PHA for the indicated times, were analyzed by RT-PCR tion experiments with the indicated constructs is shown. Control transfec- (A) or Western blot analysis (B) using the SR protein-specific mAb 104. tion with the CD45 minigene only is shown in lane Ϫ. CD45 isoforms are Position of the SR proteins are indicated on the right of each panel. indicated on the right, isoform size (bp) is indicated on the left. B, Graph of CD45 isoform expression pattern after cotransfection with the SRp40 deletion mutant and the SRp40/SRp30a swap constructs. CD45 isoforms efficiently bound by SF2/ASF-SC35 (59). However, enhancer/si- are indicated on the right, constructs used for cotransfection are indicated lencer elements specific for the SR proteins causing CD45 exon below the figure, and percentage of isoform expression as a fraction of the exclusion have not been identified and will be the subject of our total is indicated on the left. future studies. http://classic.jimmunol.org The results of the experiments using chimeric SR proteins are tabulated in Fig. 5. In summary, these data demonstrate that the

306GACTGACTACA316, nucleotide numbers are from the atypical RRM of SF2/ASF determines the specificity for CD45 CD45ABC cDNA), which may explain the ability of 9G8 to pro- mRNA splicing. All constructs that include this domain promote mote the use of CD45 isoforms containing exon 4. exon skipping, whereas all constructs lacking this domain pro- CD45 exon exclusion is promoted by SF2/ASF, SC35, SRp30c, moted exon inclusion and behaved like SRp20. The results resem-

Downloaded from SRp40, and SRp75. It has recently been shown using a CD45 ble those with adenovirus E1A where wild-type SF2/ASF favors minigene containing only alternative exon 4 that overexpression of the selection of the most proximal 13S site whereas the mutant hsSWAP, SF2/ASF, SC35, SRp40, and SRp75 all promoted ex- lacking RRM2 switches splicing to the 12S site which is also pro- clusion of exon 4, which is in agreement with our findings (57, 58). moted by SRp20 (31). Lemaire et al. (58) also demonstrated that regulation of CD45 exon During T cell activation, we demonstrated specific changes in 4 splicing is dependent on exon 4 itself and not affected by the SR protein expression levels. We show that SRp75 is up-regulated presence of the 3Ј constitutive exon, exon 7. Information for exon to a higher level (but later) in stimulated CD45RAϩ vs CD45ROϩ exclusion must therefore be present in the exonic (or intronic) T cells. In addition, the smallest band of the 30-kDa SR proteins sequences of the alternative CD45 exons. Elements that repress which might represent SRp30c is induced only in CD45RAϩ cells. splice sites have been identified in other systems (59–66). In Dro- This is concomitant with the fact that both SRp30c and SRp75 sophila, binding of Sxl1 to the 3Ј splice site interferes with U2AF promote splicing of the CD45 null isoform in transfected COS-1 binding (67). In addition, a splicing silencer (IIIA) present in the cells. One band in the 30-kDa panel of SR proteins, possibly 9G8, adenovirus late region L1 mRNA was demonstrated to bind SR is not up-regulated in stimulated CD45RAϩ, which is in agreement proteins, which prevented recruitment of U2snRNP to the spliceo- with the fact that 9G8 promotes CD45 ABC and AB splicing and some. This silencer binds a number of SR proteins but is most not CD45 null splicing. 5294 REGULATION OF CD45 SPLICING

The expression level, phosphorylation, cellular location, and 22. Tarn, W. Y., and J. A. Steitz. 1995. Modulation of 5Ј splice site choice in pre- specific mix of SR/hnRNP proteins are believed to be one com- messenger RNA by two distinct steps. Proc. Natl. Acad. Sci. USA 92:2504. 23. Fu, X. D., and T. Maniatis. 1992. The 35-kDa mammalian splicing factor SC35 ponent in the regulation of specific splice site choices (10, 14, 26). mediates specific interactions between U1 and U2 small nuclear ribonucleopro- This model presumes that SR protein expression patterns are tissue tein particles at the 3Ј splice site. Proc. Natl. Acad. Sci. USA 89:1725. specific, developmentally regulated, and responsive to the meta- 24. Wu, J. Y., and T. Maniatis. 1993. Specific interactions between proteins impli- cated in splice site selection and regulated alternative splicing. Cell 75:1061. bolic state of the cells. We have demonstrated complex changes in 25. Staknis, D., and R. Reed. 1994. SR proteins promote the first specific recognition the expression of SR proteins upon T cell activation, which occur of pre-mRNA and are present together with the U1 small nuclear ribonucleopro- in parallel with changes in the splicing of CD45. Furthermore, we tein particle in a general splicing enhancer complex. Mol. Cell. Biol. 14:7670. 26. Fu, X. D. 1993. Specific commitment of different pre-mRNAs to splicing by have also demonstrated that members of the SR protein family can single SR proteins. Nature 365:82. have dramatic and antagonistic effects on CD45 splicing by trans- 27. Mayeda, A., G. R. Screaton, S. D. Chandler, X. D. Fu, and A. R. Krainer. 1999. fection in vivo. The SR protein family is thus a strong candidate Substrate specificities of SR proteins in constitutive splicing are determined by their RNA recognition motifs and composite pre-mRNA exonic elements. Mol. for CD45 regulation in vivo. In future experiments, we hope to Cell. 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