Regulation of Alternative Splicing of CD45 by
Antagonistic Effects of SR Protein Splicing
Factors
This information is current as
of June 15, 2017.
Gerdy B. ten Dam, Christian F. Zilch, Diana Wallace, Bé
Wieringa, Peter C. L. Beverley, Lambert G. Poels and Gavin
R. Screaton
J Immunol 2000; 164:5287-5295; ;
doi: 10.4049/jimmunol.164.10.5287
http://www.jimmunol.org/content/164/10/5287
Subscription
Permissions
Email Alerts
This article cites 67 articles, 43 of which you can access for free at:
http://www.jimmunol.org/content/164/10/5287.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2000 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
References
Regulation of Alternative Splicing of CD45 by Antagonistic
Effects of SR Protein Splicing Factors1
Gerdy B. ten Dam,2* Christian F. Zilch,‡ Diana Wallace,§ Bé Wieringa,* Peter C. L. Beverley,§
Lambert G. Poels,† and Gavin R. Screaton¶
I
ntron removal is an essential step during eukaryotic gene
expression. This process requires recognition of the 5⬘ and 3⬘
splice sites by the spliceosome: a multicomponent ribonucleoprotein complex. The major components of the spliceosome are
the small ribonucleoprotein particles (snRNPs)3 U1, U2, and
U4/U6 and the non-snRNP proteins including the family of SR
proteins (reviewed by Green (3) and Kramer (4)). This family of
closely related, highly conserved proteins is characterized by the
presence of one or two N-terminal RNA recognition motifs
(RRMs) and a C-terminal domain rich in arginines and serines (RS
domain). SR proteins are essential for constitutive splicing (5–10)
and alternative splicing (10 –17). The family is highly conserved
between diverse species and have been shown to be essential for
cell survival (18, 19). SR proteins function at multiple steps during
the splicing reaction. In early steps, they facilitate U1 snRNP binding to the 5⬘ splice site (20, 21). Furthermore, they stabilize complex assembly at the 3⬘ splice site by assisting the binding of U2AF
(22) and by forming bridges to connect 5⬘ and 3⬘ splice sites
(23–25).
Substrate-specific effects of individual SR proteins have been
demonstrated in constitutive as well as alternative splicing (7, 11,
Departments of *Cell Biology and †Anatomy, Faculty of Medical Sciences, University of Nijmegen, Nijmegen, The Netherlands; ‡Imperial Cancer Research Fund Tumour Immunology Unit, University College London Medical School, London, United
Kingdom; §The Edward Jenner Institute for Vaccine Research, Compton, Newbury,
United Kingdom; and ¶Institute of Molecular Medicine, John Radcliffe Hospital,
Headington, Oxford, United Kingdom
Received for publication November 1, 1999. Accepted for publication March 1, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
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.),
the Medical Research Council and the Wellcome Trust (to G.R.S.), and the Imperial
Cancer Research Fund.
2
Address correspondence and reprint requests to Dr. Gerdy B. ten Dam at her current
address: Department of Biochemistry, Trigon Building, Faculty of Medical Sciences,
University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
E-mail address:[email protected]
3
Abbreviations used in this paper: SR, serine(S)-arginine(R)-rich; RRM, RNA recognition motif; DAF, decay accelerating factor.
Copyright © 2000 by The American Association of Immunologists
12, 14, 26, 27). SR proteins are expressed in tissue-specific patterns (8, 10, 14, 26) and can be regulated by phosphorylation (28).
The effects of the SR proteins SF2/ASF can be antagonized by the
heterogeneous ribonucleoproteins A1 and A2B1 (29, 30). In addition, a further level of control of SR protein function may be subserved by nucleocytoplasmic shuttling (31, 32).
CD45 is a transmembrane glycoprotein expressed on leukocytes
(reviewed by Thomas (1) and Trowbridge and Thomas (2)). Alternative splicing of three variable exons (4, 5, and 6 or A, B, and
C) allows the production of eight possible isoforms. In rodent
cells, all of these isoforms have been isolated (33–35), whereas in
humans only five have been identified (36, 37). The splicing is
cell-type specific and activation dependent. B cells express mainly
the high m.w. isoform (ABC), whereas T cells express a panel of
different isoform ranging form the smallest isoform (null), found
also on thymocytes, to the largest isoform (ABC). T cell activation
leads to a programmed shift from high to the low m.w. isoforms,
i.e., down-regulation of CD45RA expression and concomitantly
up-regulation of CD45RO expression. It is this change in cell surface phenotype that is used to differentiate naive from memory T
cells (38). During this activation, there is also a decrease in
expression of CD45RC which is higher than the small decrease
observed for CD45RB but less prominent than the CD45RA
decrease (39).
In this study, we sought to identify factors involved in the regulation of alternative splicing of CD45. Members of the SR protein
family were analyzed for their influence on splice site selection by
cotransfection experiments with a CD45 minigene in COS-1 cells
in vitro (40). Furthermore, we analyzed the SR protein profile in T
cells before and after stimulation.
Materials and Methods
DNA cloning
The human CD45 minigene pSEC-S-LCA1-7 was constructed in the pSEC
expression plasmid (41) and contains 1) the 5⬘ CD45 structural gene region
(exons 1 and 2) comprising the ATG codon and the signal peptide; 2) the
genomic CD45 sequence of exon 3 to exon 7 with the alternative exons 4,
5, and 6; and 3) the decay accelerating factor (DAF) GPI anchor attachment
signal to ensure membrane expression of the expressed proteins (Fig. 1A).
0022-1767/00/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
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 exons
(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 exon 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.
5288
REGULATION OF CD45 SPLICING
SR protein cDNAs were cloned in the vector pCGT7 which carries an
N-terminal tag from the bacteriophage T7 gene (31, 42). SR protein products can therefore be recognized by the T7 tag mAb (Novagen, Madison,
WI). SRp75 was cloned into the parental vector pCG, which lacks the
T7 tag.
The SR protein chimeras and deletion mutants are listed in Fig. 5. RRM
or RS domain swaps or deletions were all constructed by PFU-PCR amplification of the desired fragments with specifically designed primers and
subcloning of the fragments in the pCGT7 expression vector. (Nomenclature of the constructs is illustrated for SF2/ASF (30aRRM1–30aRRM2–
30aRS) and SC35 (30bRRM1–30bRS), e.g., substitution of the RS domain
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 ⌬
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
(aa 1–115), and SC35-RS (aa 116 –221) were subcloned in pCGT7 in the
indicated combinations. The SF2-ASF-SRp40 chimeras and SF2/ASF deletion mutants were described by Caceres et al. (31, 32).
In vivo analysis of alternative CD45 splicing
COS-1 cells were cultured in DMEM (Life Technologies, Rockville, MD)
supplemented with 10% FCS (Life Technologies) in 6-well plates. At a cell
density of 30 – 40%, cells were (co)transfected with 1 ␮g of minigene DNA
and the indicated amount of SR protein construct using Lipofectin reagents
according to the manufacturer (Life Technologies).
Transfected cells were harvested 48 –72 h after transfection and RNA
was isolated with the RNAzol B (Cinna/Biotecx Laboratories, Friendswood, TX) method and analyzed by RT-PCR. First-strand specifically
primed (DAF reverse primer, 5⬘-CCTAAATGAAGAGCACAATTGCA3⬘) cDNA synthesized with superscript reverse Transcriptase (Life Technologies) from 200 ng of RNA was amplified for 22 cycles using a 5⬘
end-labeled forward primer (LCA2, 5⬘-ATTGGATCCGCTGACTTCCA
GATATGACC-3⬘) and a nonlabeled reverse primer (LCA7, 5⬘-CCGAGATC
TTCAGAGGCATTAAGGTAGGC-3⬘). PCR products were run on a 6%
denaturing polyacrylamide gel and detection and quantitation were conducted by autoradiography and PhosphoImage analysis (Kodak X-OMAT
S1, Molecular Analyst Bio-Rad, Hercules, CA), respectively.
RNA from PHA-stimulated RO⫹ and RA⫹ T cells was reversed transcribed with the LCA9 primer (5⬘-GTAATCCACAGTGATGTTTGC-3⬘)
and amplified using LCA2 and LCA7 primers. PCR products were run on
a 2% agarose gel.
For immunofluorescence analysis, COS-1 cells were seeded on coverslips in 6-well plates and transfected with the CD45 minigene. Cells were
stained 48 h after transfection at 4°C with the CD45 exon B-specific Ab
PD7/26 (Dakopatts, Copenhagen, Denmark).
SR proteins and immunoblotting
To analyze SR protein products (SR proteins, chimeric constructs, and
deletion mutants) expressed by the pCGT7 vector, transfected COS-1 cells
were lysed in 5% SDS in 50 mM Tris-HCl (pH 6.8) and 20 mM EDTA,
sonicated, and resolved by 12% SDS-polyacrylamide gels. Proteins were
electroblotted and probed with the T7 tag Ab (Novagen) or the 104 mAb (43).
For SR protein detection in lymphocytes, human T cells were isolated
from fresh buffy coats and separated into CD45RA⫹ and CD45RO⫹ populations by selection with the UCHL1 (CD45RO) and the SN130
(CD45RA) Abs. Both populations were stimulated with PHA-P for 0, 12,
36, 72, and 144 h and analyzed at indicated time points for CD45RA/RO
expression by flow cytometry to observe changes in RA/RO expression.
Subsequently, equal amounts of cells were lysed in 5% SDS lysis buffer
and sonicated twice for 20 s. Proteins were resolved on 12% SDS-polyacrylamide gels, electroblotted, and probed with the 104 mAb.
Cell separation and stimulation of CD45RA⫹ and CD45RO⫹ T
cells
PBMC were isolated from buffy coats (North East London Blood Transfusion Service, London, U.K.) by Ficoll-Paque density gradient centrifugation (Pharmacia, St Albans, U.K.). Monocytes and macrophages were
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 1. CD45 alternative splicing. A, Diagrammatic representation of the CD45 minigene. Transcription is driven by the SV40 promoter, introns are
shown as lines, numbered boxes are CD45 exons.
DAF GPI attachment site. The position of the oligonucleotide 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).
The Journal of Immunology
5289
FIGURE 2. Alternative splicing of the CD45 minigene in COS-1 cells.
A, Immunofluorescence analysis of nonfixed transfected COS-1 cells
stained with the CD45RB Ab (PD7/26). B, Isoform expression pattern of
the CD45 minigene after transient transfection in COS-1 cells. The left
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.
M, CD45 marker (637, 295, and 154 bp). Phosphor image quantitation of
the CD45 isoform expression (n ⫽ 3).
removed by adherence to plastic for 1 h at 37°C and these were saved and
used as APC after mitomycin C treatment where required. CD45RA⫹ and
CD45RO⫹ T cells were then negatively selected. Nonadherent cells were
incubated at 4°C for 30 min with a mixture of mAbs, BU12 (anti-CD19, a
gift from N. Ling, University of Birmingham Medical School, Birmingham, U.K.), OKM1 (anti-CD11b; American Type Culture Collection), and
R10 (antiglycophorin, a gift from P. Edwards, University of Cambridge,
Cambridge, U.K.) along with SN130 (anti-CD45RA; Imperial Cancer Research Fund, London, U.K.) for removal of CD45RA⫹ T cells or UCHL1
(anti-CD45RO; Imperial Cancer Research Fund) for removal of CD45RO⫹
T cells. After washing, the cells were incubated with sheep anti mouse
IgG-coated magnetic beads at 4°C for 20 min (Dynal, Bromborough,
U.K.); labeled cells were then removed with a Dynal magnet. After five
rounds of magnetic bead separation, purity was ⬎98% by FACS analysis.
For stimulation, 2 ⫻ 106 CD45RA⫹ or CD45RO⫹ T cells were incubated in 24-well flat-bottom tissue culture plates (Becton Dickinson,
Mountain View, CA) along with 5% mitomycin C-treated adherent cells
and 1 ␮g/ml PHA-P (Sigma, St. Louis, MO) for 0, 12, 36, 72, and 144 h.
Cells were analyzed by flow cytometry at the time points indicated for
alterations in CD45RA and CD45RO expression.
Results
Alternative splicing of CD45 occurs by inclusion or skipping of
three alternative exons (4, 5, and 6 or A, B, and C) and results in
a maximum of eight different isoforms, of which five are expressed
in human hematopoietic cells (Fig. 1B). We studied alternative
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.
COS-1 cells transfected with the CD45 minigene and stained with
a CD45 exon-specific Ab showed a granular membrane staining
pattern (Fig. 2A). Western blot analysis showed expression of the
CD45 isoforms ABC, BC, AB, B, and null with high expression
levels of the low m.w. isoforms (data not shown; Ref. 40). RTPCR analysis of transfected COS-1 cells demonstrated that the
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).
SR proteins display antagonistic effects on the alternative
splicing of CD45
The focus of this paper was to examine the role of SR proteins in
CD45 alternative splicing. SR proteins are essential for constitutive splicing (5–10) and alternative splicing (10 –16) and have
demonstrated substrate-specific effects on splicing (7, 11, 12, 14,
26, 27). The SR proteins are thus candidates to control CD45
splicing.
In COS-1 cells, the CD45 minigene is processed to give a mixture of isoforms (Fig. 2B), of which the smallest is the most abundant (CD45 null, which skips the alternative exons). The ability of
SR proteins to modulate CD45 splicing was assessed by cotransfecting individual SR cDNAs with the CD45 minigene (Fig. 3).
Three groups of SR proteins with different specificities were identified. In the first group, SF2/ASF, SC35, SRp30c, SRp40, and
SRp75 overexpression resulted in down-regulation of the high
m.w. isoforms and up-regulation of the CD45 null isoform with the
CD45 B isoform still detectable (Fig. 3, lanes 6 –9 and 11). The
second group, SRp20 and 9G8, showed the opposite (Fig. 3, lanes
4 and 5), with elevated expression of the largest ABC isoform and
a decrease in expression of the smallest CD45 null isoform. SRp20
and 9G8 both have a single (canonical) RRM and the homology
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 3. Role of SR proteins in regulation of CD45 alternative splicing. COS-1 cells were cotransfected with the CD45 minigene and the indicated 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.
5290
REGULATION OF CD45 SPLICING
between the two proteins in this domain is high (79%) when compared with the canonical RRM of the other SR proteins (35– 45%)
(9). Finally, SRp55 showed no effect on the alternative splicing of
CD45 (Fig. 3, lane 10).
The effects of the antagonistic SR proteins SRp30c and SRp40
(exon skipping) vs SRp20 and 9G8 (exon inclusion) were examined in more detail using incremental amounts of the SR construct
cotransfected with 1 ␮g of the CD45 target (Fig. 4). The shift to
exon inclusion induced by SRp20 was almost complete with only
small amounts of isoforms BC and B still detectable. The effect of
9G8 was less dramatic, the ABC band increased to almost 20% of
the total sum, but the smallest isoform did not decrease and kept
steady at 50%. Interestingly, 9G8 promoted the appearance of the
AB isoform, which is hardly detectable in the other transfections.
Overexpression of SRp30c and SRp40 resulted in a dramatic increase of the smallest isoform skipping exons A, B, and C. The
CD45 ABC and BC isoforms were hardly detectable anymore,
whereas the expression level of the B isoform only decreased marginally or not at all.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
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, Isoforms 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.
These experiments were repeated several times in both COS-1
and HeLa cells and small differences in the levels of CD45 isoform
expression in the control lanes were seen, but changes induced by
SR cotransfection were reproducible in all cases. SR protein expression (except SRp75) was analyzed and confirmed by Western
blotting using the T7 epitope tag. Localization to nuclear speckles
was also verified by immunofluorescence analysis (data not
shown).
Role of structural domains of SR proteins in splice site selection
of the CD45 pre-mRNA
As detailed in the introduction, SR proteins consist of two or three
modular domains, i.e., the RS domain and either one or two Nterminal RRMs. All SR proteins share the canonical RRM characterized by the conserved RNP-1 and RNP-2 submotifs. In addition, a subset of SR proteins also has a central atypical RRM which
lacks the conserved residues in the RNP submotifs (17, 44). To
determine which of these domains is responsible for the switch in
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 chimera 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, promoting 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
of domain deletion and domain swap constructs containing fragments of SRp20, SF2/ASF, SC35, and SRp40. Schematic drawings
of all SR proteins, domain deletion, and domain swap constructs
are listed in Fig. 5. In each case, the expression and integrity of
these constructs was tested by Western blotting using the T7 tag
Ab (data not shown).
To evaluate whether the RRM or RS domain of SRp20 was
responsible for the change in CD45 alternative splicing, we constructed an RS domain deletion mutant (20 ␦ RS) and substituted
the RRM of SRp20 with the corresponding domain of SC35 (30b120RS, Fig. 6, A and C). Deletion of the RS domain of SRp20
promoted skipping of all alternative exons (Fig. 6A, lane 5). When
the RRM of SRp20 was exchanged for the RRM of SC35 (30b120RS), exon skipping was also seen giving a similar pattern to
wild-type SC35 (Fig. 6A, lanes 4 and 7), implying a role of the
RRM of SRp20 in the formation of the ABC isoform.
Domain swap chimeras between SF2/ASF and SRp40 are
shown in Fig. 7. Both the wild-type proteins promote exon skipping, as do many of the mutants except those in which RRM2 of
SRp40 is fused to RRM1 of SF2/ASF (Fig. 7, lanes 6 and 7).
RRM2 of SF2/ASF seems to play a specific role in splice site
selection; when it is deleted, the splicing of SF2/ASF resembles
Discussion
Alternative splicing of CD45 occurs by inclusion or exclusion of
three alternative exons (4, 5, and 6 or A, B, and C) in the Nterminal part of the molecule, resulting in a maximum of eight
different isoforms. cis-Acting sequences and trans-acting factors
have been postulated to influence the alternative splicing pattern of
CD45. Linker scanning analysis revealed that in exon A three segments (positions 8 –10, 40 –91, and 127–137, total length 198 bp)
and in exon C one large segment (positions 16 –137, total length
144 bp) were essential for tissue-specific alternative splicing (46,
47). No such segments were found in exon 5, suggesting that splicing of exon 5 is not regulated in a tissue-specific manner (46, 47).
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
FIGURE 5. Summary of the role of the SR proteins and their modular
domains on the alternative splicing of CD45 in vivo. The SR protein constructs, deletion mutants, and domain swap constructs used in this work are
shown schematically. Promotion of CD45 ABC or null splicing after overexpression of each construct is indicated by ⫹⫹⫹ or ⫹⫹. No effect is
indicated by ⫺ and a single ⫹ indicates no promotion of ABC or null
splicing but formation of intermediates. Overexpression of 9G8 promotes
AB splicing in addition to ABC.
Upon stimulation, naive T cells switch from CD45RA⫹ (exon inclusion, expressing the CD45 isoforms ABC, AB, BC, and B) to
CD45RO⫹ (exon skipping, expressing the isoforms B and null),
whereas mature memory CD45RO⫹ cells remain RO⫹. Resting T
cells were separated into RO⫹ and RA⫹ populations and activated
by culture in the presence of PHA. After 6 days, 99% of the RA⫹
T cells had lost expression of the exon A epitope and become
RO⫹. RT-PCR revealed that at the RNA level in the CD45RA⫹
population, the CD45 ABC, AB, and BC isoforms were downregulated between 36 and 72 h and by 72 h there was a predominant expression of the CD45 null and B isoforms. The CD45RO⫹
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
RA⫹ and RO⫹ 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 increased. Besides this total increase of SR protein expression, several changes occur specifically in CD45RA⫹-stimulated T cells.
SRp75 induction is greater and occurs later in RA⫹- vs RO⫹stimulated cells. Furthermore, in RA⫹-stimulated T cells, a band
appears in the 30-kDa cluster of SR proteins. This 30-kDa SR
protein increased markedly after 72 h of stimulation and judging
by its size it may represent SRp30c; the smallest SR protein in the
30-kDa group, however, direct identification is not possible, as a
specific mAb is not available. We have previously shown induction of SRp30c at the RNA level following T cell activation. Also
apparent from these blots are differences in the ratios of some of
the 30-kDa SR proteins. For instance, the third band possibly representing SC35 is induced more in RA-stimulated cells whereas
the fourth band, possibly 9G8, is not up-regulated in the RA-stimulated cells. SRp55 and SRp20 were hardly detectable in these T
cell populations. T cells from two individual were analyzed which
demonstrated comparable results after Western blot analysis with
the mAb 104.
5292
REGULATION OF CD45 SPLICING
Fusions between T and B cells retain the T cell phenotype producing the null isoform which led to the suggestion that exon inclusion is the default pattern, whereas skipping is regulated by
dominant trans-acting factors in T cells (48). We have previously
demonstrated that regulation of alternative splicing of this CD45
minigene is restricted to lymphoid cells. All nonlymphoid transgenic cells and transfected cell lines such as COS-1, HeLa, and
3T3 showed a specific and stable pattern of splicing to form the
low m.w. isoforms of CD45.
We have shown that SR proteins show antagonistic effects on
alternative splicing of CD45. SRp20 promoted exon inclusion,
leading to elevated levels of the CD45 ABC isoform and to a
decrease in expression levels of the CD45 null, B, and BC isoforms. 9G8 promoted splicing of the ABC and also the AB isoform; however, only splicing of the BC isoform was decreased.
Exon exclusion was promoted by SC35, SF2ASF, SRp30c, SRp40,
and SRp75. All of these SR proteins promoted splicing of the
CD45 null isoform with decreased levels of splicing of the ABC
and BC isoforms. Generally, splicing of the CD45 B isoform was
not affected. This could be explained by the fact that splicing of
exon 5 is not regulated in a tissue-specific fashion (46, 47). Also,
during the switch from CD45RA to CD45RO in activated T cells,
the T cells do not lose expression of the CD45 B isoform (38, 49,
50). These results show that individual SR proteins are able to
switch CD45 splicing; however, to induce a complete shift in
CD45 splicing, additional factors like other SR proteins or non-SR
factors might be involved.
Regulation of CD45 splicing by SRp20 and 9G8 causing exon
inclusion could be explained by the presence of exonic (or intronic) enhancer sequences in the CD45 pre-mRNA. Several in-
dependent studies identified a SRp20-binding sequence with enhancer activity, all sharing the degenerate sequence CUC(U/
G)UC(C/T) (51–54). In addition, application of the so-called
SELEX strategy yielded SRp20-specific sequences with the consensus CA/UA/UC (55). The CUC(U/G)UC(C/T) sequence is also
found in exon 4 of the SRp20 gene, the splicing of which is autoregulated by SRp20 itself (56). In this study, it was suggested
that recognition and splicing of exons with weak splice acceptor
sites is a general function of SRp20. A perfect match to the SRp20
consensus sequence is not found in the exonic sequences of CD45;
however, the sequence CACCACUGCAUUCUCACCC (nt 59 –77
in exon 4) is reminiscent of all of the identified SRp20 enhancer
sequences.
Overexpression of 9G8 promotes CD45 AB and ABC splicing.
This shift is not as complete as the switch induced by SRp20, as
the CD45 null, B, and BC splicing are still detectable. Appearance
of the AB isoform, in combination with the ABC isoform, indicates that 9G8 is specifically involved in exon A splicing. No other
SR protein was able to promote CD45 AB splicing.
Recently, two very divergent 9G8 splicing enhancers have been
described (54). One shows strong homology with the 9G8 consensus sequence AGAC(G/U)ACGAY isolated by the SELEX approach (55), whereas the other is pyrimidine rich and shows some
sequence homology with the Drosophila double sex splicing enhancer (UCUUCAAUCAAACA) which can bind 9G8 specifically
(52). SELEX, using a mutated form of 9G8 lacking the zincknuckle region, yields a pyrimidine-rich sequence (C(A/U)(A/
U)C) that resembles the SRp20 SELEX winner sequence (54, 55).
Sequences closely resembling the 9G8 consensus are found in
CD45 exon 4 but not in exons 5 and 6 (e.g., CD45 exon 4:
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
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 isoform expression patterns shown in A and B, respectively, were quantitated using phosphor imager (BioRad 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 indicated below the figure, and isoform expression level (%)
is indicated on the left.
The Journal of Immunology
306GACTGACTACA316, nucleotide numbers are from the
CD45ABC cDNA), which may explain the ability of 9G8 to promote the use of CD45 isoforms containing exon 4.
CD45 exon exclusion is promoted by SF2/ASF, SC35, SRp30c,
SRp40, and SRp75. It has recently been shown using a CD45
minigene containing only alternative exon 4 that overexpression of
hsSWAP, SF2/ASF, SC35, SRp40, and SRp75 all promoted exclusion of exon 4, which is in agreement with our findings (57, 58).
Lemaire et al. (58) also demonstrated that regulation of CD45 exon
4 splicing is dependent on exon 4 itself and not affected by the
presence of the 3⬘ constitutive exon, exon 7. Information for exon
exclusion must therefore be present in the exonic (or intronic)
sequences of the alternative CD45 exons. Elements that repress
splice sites have been identified in other systems (59 – 66). In Drosophila, binding of Sxl1 to the 3⬘ splice site interferes with U2AF
binding (67). In addition, a splicing silencer (IIIA) present in the
adenovirus late region L1 mRNA was demonstrated to bind SR
proteins, which prevented recruitment of U2snRNP to the spliceosome. This silencer binds a number of SR proteins but is most
FIGURE 8. Dynamic protein expression levels of SR proteins in stimulated CD45RA and CD45RO T cells. CD45RO- and RA-selected T cells,
stimulated with PHA for the indicated times, were analyzed by RT-PCR
(A) or Western blot analysis (B) using the SR protein-specific mAb 104.
Position of the SR proteins are indicated on the right of each panel.
efficiently bound by SF2/ASF-SC35 (59). However, enhancer/silencer elements specific for the SR proteins causing CD45 exon
exclusion have not been identified and will be the subject of our
future studies.
The results of the experiments using chimeric SR proteins are
tabulated in Fig. 5. In summary, these data demonstrate that the
atypical RRM of SF2/ASF determines the specificity for CD45
mRNA splicing. All constructs that include this domain promote
exon skipping, whereas all constructs lacking this domain promoted exon inclusion and behaved like SRp20. The results resemble those with adenovirus E1A where wild-type SF2/ASF favors
the selection of the most proximal 13S site whereas the mutant
lacking RRM2 switches splicing to the 12S site which is also promoted by SRp20 (31).
During T cell activation, we demonstrated specific changes in
SR protein expression levels. We show that SRp75 is up-regulated
to a higher level (but later) in stimulated CD45RA⫹ vs CD45RO⫹
T cells. In addition, the smallest band of the 30-kDa SR proteins
which might represent SRp30c is induced only in CD45RA⫹ cells.
This is concomitant with the fact that both SRp30c and SRp75
promote splicing of the CD45 null isoform in transfected COS-1
cells. One band in the 30-kDa panel of SR proteins, possibly 9G8,
is not up-regulated in stimulated CD45RA⫹, which is in agreement
with the fact that 9G8 promotes CD45 ABC and AB splicing and
not CD45 null splicing.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
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
CD45 minigene after cotransfection with the SRp40 deletion mutant and
the SRp40-SF2/ASF swap constructs. RT-PCR analysis of the cotransfection experiments with the indicated constructs is shown. Control transfection with the CD45 minigene only is shown in lane ⫺. CD45 isoforms are
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
are indicated on the right, constructs used for cotransfection are indicated
below the figure, and percentage of isoform expression as a fraction of the
total is indicated on the left.
5293
5294
The expression level, phosphorylation, cellular location, and
specific mix of SR/hnRNP proteins are believed to be one component in the regulation of specific splice site choices (10, 14, 26).
This model presumes that SR protein expression patterns are tissue
specific, developmentally regulated, and responsive to the metabolic state of the cells. We have demonstrated complex changes in
the expression of SR proteins upon T cell activation, which occur
in parallel with changes in the splicing of CD45. Furthermore, we
have also demonstrated that members of the SR protein family can
have dramatic and antagonistic effects on CD45 splicing by transfection in vivo. The SR protein family is thus a strong candidate
for CD45 regulation in vivo. In future experiments, we hope to
strengthen these observations using T cell transgenic mouse technology and also plan to map the SR-specific intronic and exonic
enhancer/silencer sequences within the CD45 gene.
Acknowledgments
References
1. Thomas, M. L. 1989. The leukocyte common antigen family. Annu. Rev. Immunol. 7:339.
2. Trowbridge, I. S., and M. L. Thomas. 1994. CD45: an emerging role as a protein
tyrosine phosphatase required for lymphocyte activation and development. Annu.
Rev. Immunol. 12:85.
3. Green, M. R. 1991. Biochemical mechanisms of constitutive and regulated premRNA splicing. Annu. Rev. Cell Biol. 7:559.
4. Kramer, A. 1996. The structure and function of proteins involved in mammalian
pre-mRNA splicing. Annu. Rev. Biochem. 65:367.
5. Krainer, A. R., G. C. Conway, and D. Kozak. 1990. Purification and characterization of pre-mRNA splicing factor SF2 from HeLa cells. Genes Dev. 4:1158.
6. Fu, X. D., and T. Maniatis. 1992. Isolation of a complementary DNA that encodes the mammalian splicing factor SC35. Science 256:535.
7. Kim, Y. J., P. Zuo, J. L. Manley, and B. S. Baker. 1992. The Drosophila RNAbinding protein RBP1 is localized to transcriptionally active sites of chromosomes and shows a functional similarity to human splicing factor ASF/SF2.
Genes Dev. 6:2569.
8. Zahler, A. M., W. S. Lane, J. A. Stolk, and M. B. Roth. 1992. SR proteins: a
conserved family of pre-mRNA splicing factors. Genes Dev. 6:837.
9. Cavaloc, Y., M. Popielarz, J. P. Fuchs, R. Gattoni, and J. Stevenin. 1994. Characterization and cloning of the human splicing factor 9G8: a novel 35 kDa factor
of the serine/arginine protein family. EMBO J. 13:2639.
10. Screaton, G. R., J. F. Caceres, A. Mayeda, M. V. Bell, M. Plebanski,
D. G. Jackson, J. I. Bell, and A. R. Krainer. 1995. Identification and characterization of three members of the human SR family of pre-mRNA splicing factors.
EMBO J. 14:4336.
11. Krainer, A. R., G. C. Conway, and D. Kozak. 1990. The essential pre-mRNA
splicing factor SF2 influences 5⬘ splice site selection by activating proximal sites.
Cell 62:35.
12. Ge, H., and J. L. Manley. 1990. A protein factor, ASF, controls cell-specific
alternative splicing of SV40 early pre-mRNA in vitro. Cell 62:25.
13. Fu, X. D., A. Mayeda, T. Maniatis, and A. R. Krainer. 1992. General splicing
factors SF2 and SC35 have equivalent activities in vitro, and both affect alternative 5⬘ and 3⬘ splice site selection. Proc. Natl. Acad. Sci. USA 89:11224.
14. Zahler, A. M., K. M. Neugebauer, W. S. Lane, and M. B. Roth. 1993. Distinct
functions of SR proteins in alternative pre-mRNA splicing. Science 260:219.
15. Caceres, J. F., S. Stamm, D. M. Helfman, and A. R. Krainer. 1994. Regulation of
alternative splicing in vivo by overexpression of antagonistic splicing factors.
Science 265:1706.
16. Wang, J., and J. L. Manley. 1995. Overexpression of the SR proteins ASF/SF2
and SC35 influences alternative splicing in vivo in diverse ways. RNA 1:335.
17. Manley, J. L., and R. Tacke. 1996. SR proteins and splicing control. Genes Dev.
10:1569.
18. Wang, J., Y. Takagaki, and J. L. Manley. 1996. Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability. Genes Dev. 10:2588.
19. Wang, J., S. H. Xiao, and J. L. Manley. 1998. Genetic analysis of the SR protein
ASF/SF2: interchangeability of RS domains and negative control of splicing.
Genes Dev. 12:2222.
20. Eperon, I. C., D. C. Ireland, R. A. Smith, A. Mayeda, and A. R. Krainer. 1993.
Pathways for selection of 5⬘ splice sites by U1 snRNPs and SF2/ASF. EMBO J.
12:3607.
21. Kohtz, J. D., S. F. Jamison, C. L. Will, P. Zuo, R. Luhrmann, M. Garcia Blanco,
and J. L. Manley. 1994. Protein-protein interactions and 5⬘-splice-site recognition
in mammalian mRNA precursors. Nature 368:119.
22. Tarn, W. Y., and J. A. Steitz. 1995. Modulation of 5⬘ splice site choice in premessenger 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
mediates specific interactions between U1 and U2 small nuclear ribonucleoprotein particles at the 3⬘ splice site. Proc. Natl. Acad. Sci. USA 89:1725.
24. Wu, J. Y., and T. Maniatis. 1993. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell 75:1061.
25. Staknis, D., and R. Reed. 1994. SR proteins promote the first specific recognition
of pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein 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
single SR proteins. Nature 365:82.
27. Mayeda, A., G. R. Screaton, S. D. Chandler, X. D. Fu, and A. R. Krainer. 1999.
Substrate specificities of SR proteins in constitutive splicing are determined by
their RNA recognition motifs and composite pre-mRNA exonic elements. Mol.
Cell. Biol. 19:1853.
28. Xiao, S. H., and J. L. Manley. 1998. Phosphorylation-dephosphorylation differentially affects activities of splicing factor ASF/SF2. EMBO J. 17:6359.
29. Mayeda, A., and A. R. Krainer. 1992. Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2. Cell 68:365.
30. Mayeda, A., S. H. Munroe, J. F. Caceres, and A. R. Krainer. 1994. Function of
conserved domains of hnRNP A1 and other hnRNP A/B proteins. EMBO J.
13:5483.
31. Caceres, J. F., T. Misteli, G. R. Screaton, D. L. Spector, and A. R. Krainer. 1997.
Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity. J. Cell Biol. 138:225.
32. Caceres, J. F., G. R. Screaton, and A. R. Krainer. 1998. A specific subset of SR
proteins shuttles continuously between the nucleus and the cytoplasm. Genes
Dev. 12:55.
33. Barclay, A. N., D. I. Jackson, A. C. Willis, and A. F. Williams. 1987. Lymphocyte specific heterogeneity in the rat leucocyte common antigen (T200) is due to
differences in polypeptide sequences near the NH2-terminus. EMBO J. 6:1259.
34. Saga, Y., J. S. Tung, F. W. Shen, and E. A. Boyse. 1987. Alternative use of 5⬘
exons in the specification of Ly-5 isoforms distinguishing hematopoietic cell
lineages. Proc. Natl. Acad. Sci. USA 84:5364.
35. Saga, Y., J. S. Lee, C. Saraiya, and E. A. Boyse. 1990. Regulation of alternative
splicing in the generation of isoforms of the mouse Ly-5 (CD45) glycoprotein.
Proc. Natl. Acad. Sci. USA 87:3728.
36. Ralph, S. J., M. L. Thomas, C. C. Morton, and I. S. Trowbridge. 1987. Structural
variants of human T200 glycoprotein (leukocyte-common antigen). EMBO J.
6:1251.
37. Streuli, M., L. R. Hall, Y. Saga, S. F. Schlossman, and H. Saito. 1987. Differential
usage of three exons generates at least five different mRNAs encoding human
leukocyte common antigens. J. Exp. Med. 166:1548.
38. Akbar, A. N., L. Terry, A. Timms, P. C. Beverley, and G. Janossy. 1988. Loss of
CD45R and gain of UCHL1 reactivity is a feature of primed T cells. J. Immunol.
140:2171.
39. Zapata, J. M., R. Pulido, A. Acevedo, F. Sanchez Madrid, and
M. O. de Landazuri. 1994. Human CD45RC specificity: a novel marker for T
cells at different maturation and activation stages. J. Immunol. 152:3852.
40. ten Dam, G. B., Wieringa, B., and Poels, L. G. 1999. Alternative splicing of
CD45 pre-mRNA is uniquely obedient to conditions in lymphoid cells. Biochim.
Biophys. Acta 1446:317.
41. ten Dam, G. B., Poels, L. G., and Wieringa, B. 1998. Cell surface GPI-anchoring
of CD45 Isoforms. Mol. Biol. Rep. 25:197.
42. Wilson, A. C., M. G. Peterson, and W. Herr. 1995. The HCF repeat is an unusual
proteolytic cleavage signal. Genes Dev. 9:2445.
43. Roth, M. B., C. Murphy, and J. G. Gall. 1990. A monoclonal antibody that
recognizes a phosphorylated epitope stains lampbrush chromosome loops and
small granules in the amphibian germinal vesicle. J. Cell Biol. 111:2217.
44. Fu, X. D. 1995. The superfamily of arginine/serine-rich splicing factors. RNA
1:663.
45. Roth, M. B., A. M. Zahler, and J. A. Stolk. 1991. A conserved family of nuclear
phosphoproteins localized to sites of polymerase II transcription. J. Cell Biol.
115:587.
46. Streuli, M., and H. Saito. 1989. Regulation of tissue-specific alternative splicing:
exon- specific cis-elements govern the splicing of leukocyte common antigen
pre-mRNA. EMBO J. 8:787.
47. Tsai, A. Y., M. Streuli, and H. Saito. 1989. Integrity of the exon 6 sequence is
essential for tissue-specific alternative splicing of human leukocyte common antigen pre-mRNA. Mol. Cell. Biol. 9:4550.
48. Rothstein, D. M., H. Saito, M. Streuli, S. F. Schlossman, and C. Morimoto. 1992.
The alternative splicing of the CD45 tyrosine phosphatase is controlled by negative regulatory trans-acting splicing factors. J. Biol. Chem. 267:7139.
49. Deans, J. P., H. M. Serra, J. Shaw, Y. J. Shen, R. M. Torres, and L. M. Pilarski.
1992. Transient accumulation and subsequent rapid loss of messenger RNA encoding high molecular mass CD45 isoforms after T cell activation. J. Immunol.
148:1898.
50. Smith, S. H., M. H. Brown, D. Rowe, R. E. Callard, and P. C. Beverley. 1986.
Functional subsets of human helper-inducer cells defined by a new monoclonal
antibody, UCHL1. Immunology 58:63.
51. Chiara, M. D., O. Gozani, M. Bennett, P. Champion Arnaud, L. Palandjian, and
R. Reed. 1996. Identification of proteins that interact with exon sequences, splice
sites, and the branchpoint sequence during each stage of spliceosome assembly.
Mol. Cell. Biol. 16:3317.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
We thank Alison Cowper for her excellent technical assistance and Javier
Caceres for the provision of SF2 mutant expression vectors.
REGULATION OF CD45 SPLICING
The Journal of Immunology
52. Lynch, K. W., and T. Maniatis. 1996. Assembly of specific SR protein complexes
on distinct regulatory elements of the Drosophila doublesex splicing enhancer.
Genes Dev. 10:2089.
53. Lou, H., K. M. Neugebauer, R. F. Gagel, and S. M. Berget. 1998. Regulation of
alternative polyadenylation by U1 snRNPs and SRp20. Mol. Cell. Biol. 18:4977.
54. Schaal, T. D., and T. Maniatis. 1999. Selection and characterization of premRNA splicing enhancers: identification of novel SR protein-specific enhancer
sequences. Mol. Cell. Biol. 19:1705.
55. Cavaloc, Y., C. F. Bourgeois, L. Kister, and J. Stevenin. 1999. The splicing
factors 9G8 and SRp20 transactivate splicing through different and specific enhancers. RNA 5:468.
56. Jumaa, H., and P. J. Nielsen. 1997. The splicing factor SRp20 modifies splicing
of its own mRNA and ASF/SF2 antagonizes this regulation. EMBO J. 16:5077.
57. Sarkissian, M., A. Winne, and R. Lafyatis. 1996. Mammalian SWAP regulation
of alternative mRNA splicing of CD45 exon 4 and fibronectin IIICS. J. Biol.
Chem. 271:31106.
58. Lemaire, R., A. Winne, M. Sarkissian, and R. Lafyatis. 1999. SF2 and SRp55 regulation of CD45 exon 4 skipping during T cell activation. Eur. J. Immunol. 29:823.
59. kanopka, A., O. Muhlemann, and G. akusjarvi. 1996. Inhibition by SR proteins
of splicing of a regulated adenovirus pre-mRNA. Nature 381:535.
60. Blanchette, M., and B. Chabot. 1997. A highly stable duplex structure sequesters
the 5⬘ splice site region of hnRNP A1 alternative exon 7B. RNA 3:405.
5295
61. Blanchette, M., and B. Chabot. 1999. Modulation of exon skipping by highaffinity hnRNP A1-binding sites and by intron elements that repress splice site
utilization. EMBO J. 18:1939.
62. Si, Z. H., D. Rauch, and C. M. Stoltzfus. 1998. The exon splicing silencer in
human immunodeficiency virus type 1 Tat exon 3 is bipartite and acts early in
spliceosome assembly. Mol. Cell. Biol. 18:5404.
63. Si, Z., B. A. Amendt, and C. M. Stoltzfus. 1997. Splicing efficiency of human
immunodeficiency virus type 1 tat RNA is determined by both a suboptimal 3⬘
splice site and a 10 nucleotide exon splicing silencer element located within tat
exon. Nucleic Acids Res. 25:861.
64. Chan, R. C., and D. L. Black. 1997. The polypyrimidine tract binding protein
binds upstream of neural cell-specific c-src exon N1 to repress the splicing of the
intron downstream. Mol. Cell. Biol. 17:4667.
65. Chan, R. C., and D. L. Black. 1997. Conserved intron elements repress splicing
of a neuron-specific c-src exon in vitro. Mol. Cell. Biol. 17:2970.
66. Muro, A. F., M. Caputi, R. Pariyarath, F. Pagani, E. Buratti, and F. E. Baralle.
1999. Regulation of fibronectin EDA exon alternative splicing: possible role of
RNA secondary structure for enhancer display. Mol. Cell. Biol. 19:2657.
67. Valcarcel, J., R. Singh, P. D. Zamore, and M. R. Green. 1993. The protein Sexlethal antagonizes the splicing factor U2AF to regulate alternative splicing of
transformer pre-mRNA. Nature 362:171.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017