|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
RED CELLS
From the Centre de Génétique
Moléculaire et Cellulaire, CNRS UMR 5534, Université Lyon
1, Villeurbanne, France; Dana-Farber/Harvard Cancer Center and
Department of Medicine, Brigham and Women's Hospital, Department of
Pediatrics, Children's Hospital of Boston, and Harvard Medical School,
Boston, MA.
The inclusion of exon 16 in the mature protein 4.1R messenger RNA
(mRNA) is a critical event in red blood cell membrane biogenesis. It
occurs during late erythroid development and results in inclusion of
the 10-kd domain needed for stabilization of the spectrin/actin lattice. In this study, an experimental model was established in murine
erythroleukemia cells that reproduces the endogenous exon 16 splicing
patterns from a transfected minigene. Exon 16 was excluded in
predifferentiated and predominantly included after induction. This
suggests that the minigene contained exon and abutting intronic
sequences sufficient for splicing regulation. A systematic analysis of
the cis-acting regulatory sequences that reside within the
exon and flanking introns was performed. Results showed that (1) the
upstream intron of 4.1R pre-mRNA is required for exon recognition and
it displays 2 enhancer elements, a distal element acting in
differentiating cells and a proximal constitutive enhancer that resides
within the 25 nucleotides preceding the acceptor site; (2) the exon
itself contains a strong constitutive splicing silencer; (3) the exon
has a weak 5' splice site; and (4) the downstream intron
contains at least 2 splicing enhancer elements acting in
differentiating cells, a proximal element at the vicinity of the 5'
splice site, and a distal element containing 3 copies of the UGCAUG
motif. These results suggest that the interplay between negative and
positive elements may determine the inclusion or exclusion of exon 16. The activation of the enhancer elements in late erythroid
differentiation may play an important role in the retention of exon 16.
(Blood. 2001;98:3809-3816) Protein 4.1R is a critical 80-kd cytoskeletal
protein found in circulating red blood cells (RBCs). It mediates the
formation and maintenance of spectrin/actin complex and anchors the
cytoskeleton to the overlying lipid bilayer.1 Human 4.1R
is encoded by a single genomic locus over 200 kb in
length,2 and is expressed as multiple isoforms resulting
from complex alternative pre-messenger RNA (pre-mRNA) splicing
pathways. Previous studies have shown that inclusion of a 21-amino acid
sequence motif at the N-terminus of the 10-kd spectrin/actin-binding
(SAB) domain is required to promote cytoskeletal junctional complex
stability.3,4 Genomic cloning of both the mouse and human
genes confirmed that the 63-nucleotide (nt) motif is encoded by
an individual exon, exon 16.2,5 The splicing of this exon
is therefore regulated in a cassette fashion. Exon 16 is omitted from
much of the 4.1R mRNA of pre-erythroid cells but is included in most of
the mRNA produced in late erythroid cells (Figure
1).6,7 Splicing of exon 16 is thus highly regulated in a differentiation stage-specific manner,
and this regulated event is critical for production of 4.1R isoforms
that sustain the function of 4.1R in circulating RBCs.
Alternative splicing of pre-mRNA is a fundamental mechanism for
regulating eukaryotic gene expression.8 In many cases, alternative RNA splicing contributes to developmentally regulated and
cell type-specific patterns of gene expression. Although a great deal
of information is available concerning the general constitutive
splicing reactions of simple splicing units, much less is known about
the molecular mechanisms and cellular factors involved in regulated
alternative splicing.8,9 Studies of vertebrate genes aimed
at understanding the regulation of alternative splicing have led to
identification of a number of pre-mRNA features that influence
alternative splice selection. These include the sequence of the 5' and
3' splice sites, the branch point sequence, the upstream polypyrimidine
(poly[Y]) tract, and specific RNA sequences (enhancers and silencers)
located in exons or introns.10 Elements that stimulate
splicing to an adjacent splice site are known collectively as splicing
enhancers, whereas elements that repress the use of splice site are
known as splicing silencers.
In addition to cis-acting elements present in pre-mRNA, the
general pre-mRNA splicing mechanism is governed by small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing
factors.9,10 Multiple factors are believed to be involved
in the regulation of alternative splicing through a complex network of
interactions between the factors and pre-mRNA elements. Factors exert
both positive and negative effects on the exon recognition. Moreover, several studies have shown that tissue-specific distribution of "constitutive" factors (SR proteins) varies to a certain degree and
that overexpression of SR proteins in vivo alters the splicing pattern.11 Hence, differences in the activities or amounts
of general non-snRNP factors, as well as the presence of "specific" factors, could modulate alternative splicing pathways.
We have shown that exon 16 is preferentially included in mature 4.1R
mRNA in induced mouse erythroleukemia (MEL) cells, and that the protein
isoform bearing exon 16-encoding peptide is preferentially and rapidly
recruited in the membrane.7 We present in this report the
identification of sequences in the 4.1R gene that
regulate the extent of exon 16 inclusion during differentiation
stage-specific splicing events occurring in maturing erythroid cells.
We developed a stable transfection system that reproduces endogenous
4.1R splicing patterns, using inducible MEL cells and transfected
minigene constructs bearing exon 16. Furthermore, we identified
multiple splicing enhancers and silencers that lie within exon 16 and
close-vicinity intronic sequences. Small changes in the wild-type (WT)
sequences altered exon 16 splicing patterns, but only certain of these
elements seem to modulate exon recognition in a stage-specific manner. Our results suggest that exon sequences and abutting intronic sequences
contain both positive and negative elements that mediate control of
alternative splicing of exon 16. These elements must serve coordinately
as primary targets to promote exon skipping in predifferentiated MEL
cells and to allow exon inclusion as the cells differentiate.
Genomic cloning and DNA sequencing
Minigene constructs
Mutant constructs were also prepared as BstEII/NheI fragments and subcloned in p(13,17)/CMV. Large sequence substitutions were generated by the same 2-step PCR method.13 The first PCR step was performed using chimeric primers overlapping exon 16 and exon 7 or exon 16 and globin exon 2, to generate precise insertions of predefined fragments. To construct point-mutated minigenes, primers were designed to contain the sequence changes. Each mutant was then generated by the 2-step PCR method.13 Alternatively, the mutation was incorporated by a PCR site-directed mutagenesis procedure using complementary mutant primers and the WT construct as template (QuickChange Site-Directed Mutagenesis Kit, Stratagene). The BstEII/NheI mutated fragments were further subcloned in p(13,17)/CMV cassette. In all constructs with base substitutions, except construct "AA,"
the nucleotide changes created or abolished new restriction sites,
which allowed rapid miniprep selection of the mutant clones. These
mutant constructs were named after the corresponding restriction endonuclease: B for BglII; L for SalI; UI-Xm for
upstream intron, XmaI; DI-Pm for downstream intron,
PmlI; and so forth. The sequence substitutions are
indicated in Figure 2 and listed in Table
1. The cassette, as well as the WT and
the mutant inserts, were fully sequenced to ascertain the absence of
any additional mismatch, resulting from PCR or cloning errors, which
would interfere with the splicing process or the RNase protection
assays (RPAs).
Cell culture, induction, and transfection Several MEL cell subclones were used. In most of the transfection experiments described here, we used subclone 745-A. Cells were cultured and induced to erythroid differentiation as previously described,7 on fibronectin substratum or in suspension in the absence of fibronectin. For stable expression, proliferative cells (2 × 105 cells) were transfected prior to dimethyl sulfoxide (DMSO) induction with 0.5 to 1 µg plasmid DNA using LipofectAmine (Life Technologies, Gaithersburg, MD) or Escort (Sigma, St Louis, MO) transfection reagents in 35-mm tissue culture plates, according to the manufacturer's procedures. Transfected cells were cultured in complete Dulbecco modified Eagle medium containing 800 µg G-418/mL. Selection of stably transfected clones was performed by incubation of individual cell colonies in 96-well microtiter plates.Semiquantitative reverse transcription-PCR Total RNA was obtained by ultracentrifugation of guanidinium isothiocyanate cell lysate on a CsCl cushion, as previously described,7 or using RNeasy Total RNA Kit (Qiagen, Hilden, Germany). The reverse transcription (RT) step was performed on 1µg total RNA for 20 minutes at 42°C, using random hexamers and 25 U Moloney murine leukemia virus (M-MLV) reverse transcriptase (Perkin Elmer-Cetus, Norwalk, CT). After heat inactivation of the reverse transcriptase, the PCR reagents were added. The resulting master PCR mixture contained in final concentrations: 1 × PCR buffer, 2 mmol/L MgCl2, 0.2 mmol/L of each dNTP, 0.2 µmol/L of each primer, and 1.2 U Taq polymerase. Two sets of primers were used to amplify specifically the transcripts deriving from either the endogenous murine or the transfected human genes. The semiquantitative PCR amplification was carried out for the cycle numbers discussed later, and the PCR products quantified as previously described.14 Quantification measurements were obtained using ImageQuantMac v1.2 software.RPA Total RNA (5 µg) from transfected MEL cells was analyzed by RPA as previously detailed,7 using H(13,16) a human-specific probe. H(13,16) was generated by RT-PCR of reticulocyte RNA, using the forward primer of the cassette (5'-AAGGTGGCGTCCTAGATGCCTCTGCT-3') and a reverse primer within exon 16 (5'-ACATTAAATTGCTATGTCTG-3'). The resulting PCR fragment was subcloned into the pCR II vector (Invitrogen). It is noteworthy that the forward primer of the cassette (which is also used as a sense primer in the PCR amplification of the transfection products [see above] and as a forward primer to design the RPA probe), contains a 12-nt human-specific sequence at its 3' end. This sequence is absent from the mouse 4.1 cDNA.5 This feature allowed us to distinguish (RPA) or select (RT-PCR) the RNA products derived from the transfected minigene from those arising from the endogenous mouse 4.1R genes.
Potential cis-elements within and surrounding exon 16 We isolated a human genomic clone containing exon 16 and adjacent introns.2 The corresponding genomic fragment in the mouse has also been isolated.5 Intron sequences were determined (Figure 2). In addition to a highly conserved exon sequence, several characteristics, remarkably conserved in both human and mouse, have been found within and surrounding the exon (underlined sequences in Figure 2): (1) an exceptionally long poly(Y)-rich tract of 105 and 121 nt in human and mouse, respectively, upstream of the 3' splice site; (2) an exclusive purine sequence containing GAR repeats at the 5' third of the exon similar GAR repeat elements have been described as
splicing enhancers15; (3) 2 UAG exonic tripletsthis motif
is known to act as a splicing silencer in other systems through its
binding to hnRNPA116; (4) a weak 5' splice sitealthough it shows the highly invariable dinucleotide/GT, this splice site diverges from the consensus 5' splice site at positions +3 and +4 where
pyrimidines (TT) are present instead of the usually encountered purines
(A[or G] A)17; and (5) a TGCATG motif, which has been found in other pre-mRNA alternative splicing,18-20
present in 3 copies in the downstream intron of both human and mouse
sequences. Each of these particular sequences has been targeted to
determine their potential role in exon 16 alternative splicing.
Alternative splicing of minigene pre-mRNAs is regulated in induced MEL cells Exon 16 with about 300 nt flanking intron sequences on each side was inserted in a splicing cassette as detailed in "Materials and methods" (Figure 3). We postulated that the resulting minigene would contain the minimum sequence for a proper and regulated splicing and would therefore mimic the behavior of the endogenous gene. Indeed, evidence suggests that the sequence elements required for splicing of most pre-mRNA species are located within ~300-nt region containing the branch point, the exon itself, and the 5' and 3' splice sites on both sides of the exon.17
The MEL cells stably transfected with construct WT were induced to erythroid differentiation and mRNA collected after 5 days of induction. As shown in Figure 3, RPA and semiquantitative RT-PCR revealed a switch from a predominant exon skipping to a predominant exon retention after cell induction. The switch remains partial but very similar to the pattern of endogenous pre-mRNA arising from the mouse genes. Reticulocytes isolated from peripheral blood produce almost exclusively an mRNA isoform that includes exon 16. As previously discussed,7 the presence of some mRNA lacking exon 16 in induced cells is likely due to the heterogeneity of the cell population, within which only a fraction of the cells attain terminal differentiation. These data suggest that the minigene contains sequences that are sufficient for splicing regulation of that particular exon in an erythroid context. In these experiments on WT sequences, as well as in experiments carried out on most mutant constructs (see below), we tested at least 2 different stable clones per construct. We noted differences in the level of total pre-mRNA transcription but not in the qualitative pattern (splicing) expression. Exon inclusion was also measured in different transfection experiments using either a pool of G-418-resistant cells, or individual stably transfected clones. No significant difference in the pattern was observed (not shown). All of these data argued against any artifacts arising from the integration site or transcriptional efficiency of the minigene.21 We also conducted a comparative analysis of RPA and RT-PCR as methods of tracking the changes in splicing patterns. We found that the changes in exon inclusion were basically of the same magnitude in both cases (Figure 3). Upstream intron enhancer elements are required for exon recognition and its splicing regulation The sequence upstream of the 3' splicing site displays an unusually long stretch of poly(Y). Substitution of this poly(Y)-rich region by heterologous intron sequences, which are efficient for constitutive splicing of -globin exon 2 (construct GI, Figure 4), led to complete skipping of the exon
in both uninduced and induced cells. We asked whether this constitutive
skipping results only from intronic sequence changes or a cooperative
interaction with the weak 5' splice site by testing 2 mutant constructs
where either one or both elements were altered (Figure 4). Substitution of the WT pyrimidines at positions +3 and +4 by purines (AA construct) strengthened the 5' splice site and led to constitutive exon inclusion in both uninduced and induced cells (Figure 4). Surprisingly, a
minigene incorporating both changes (eg, globin intron and AA changes;
GI/AA construct in Figure 4), behaved like the GI construct; exon
recognition during induction was not rescued by the strengthening of
the 5' splice site. In keeping with our preliminary
observations,22 these data suggest that exon recognition
requires the upstream intronic sequences, both in WT context as well as
in the presence of a strong 5' splice site.
To locate more precisely the upstream intronic region that exerts a
stimulatory and dominant effect on exon inclusion, we designed 4 constructs by substituting 4.1R intronic sequences for their homologues
on globin intron 1. Three regions have been targeted (Figure 4): (1)
the proximal region, "a," from Thus, the upstream intronic sequences seem to act positively on exon 16 splicing. To know whether this stimulatory effect abolished in constructs GI is due to suppression of a 4.1R splicing enhancer(s) or insertion of globin silencer, we tested 5 additional constructs where the poly(Y)-rich sequence was altered by single nucleotide substitutions (constructs UI, Figure 4). As was true with the constructs containing large fragment substitutions, exon inclusion was virtually abolished in construct UI-Xm (region a) in both predifferentiated and differentiated cells, whereas it was dramatically reduced in constructs UI-Bs (region b), and UI-Ec, UI-Mf, and UI-Ns (region c). In the 2 latter constructs, the reduction occurred most specifically in induced cells (Figure 4). These data suggest that upstream poly(Y)-rich intron sequences contain 2 enhancer elements: a distal element within region c, which stimulates exon inclusion after induction, and a proximal element that resides within the first 25 nt upstream of the exon (region a) and that activates exon inclusion in a constitutive manner. A silencer element within the exon acts in a constitutive manner We next searched for a possible role for exon sequences. Exon 16 is expected to be an intrinsically poor splicing substrate because of the weak 5' splice site (see above). To address the contribution of putative exonic regulatory elements that would enhance exon inclusion after cell induction, we first tested 2 different constitutive exon sequences. To do so, exon 16 was precisely substituted by human-globin exon 2 (construct GE, Figure
5) or 4.1R exon 7 (construct E7). In the
E7 minigene, the whole 5' consensus splice site including the last
codon at the 3' end of the exon was derived from exon 16; however, the
donor 5' splice site in construct GE displayed a consensus (AGG/guuugu)
that is potentially weaker than the WT consensus (GAG/guuugu).
Surprisingly, the internal exon 7 was excluded in both uninduced and
induced cells, whereas globin exon 2 was constitutively and fully
included (Figure 5). These results suggest that exon sequences are
essential for splicing regulation, at least in this particular intronic nucleotide context.
Exon 16 contains a purine-rich sequence in its 5' half (Figure 2). Such
purine-rich sequences within exons have been described as splicing
enhancers in both constitutive and alternative exons (see
"Discussion"). Also, the exon sequence contains several AAA trinucleotide stretches, which are found at a relative high frequency in alternatively spliced exons.23 In construct 15GE, the
whole purine-rich tract was substituted by the 5' 15 nt of To distinguish between these 2 possibilities, 5 mutant constructs were made (Figure 5); in R and C constructs, A triplets were mutated to disrupt the putative purine-rich enhancer element at the 5' end of the exon. Similarly, the downstream part of the exon was also targeted by triplet substitutions; in constructs L and B, an A triplet and a UAG element were targeted, respectively. Construct Ps derived from a single base substitution between these upstream and downstream parts of the exon. Again, care was taken not to generate sequences known to affect splicing, such as cryptic splice sites and stop codons. Exon substitution in construct R seemed to have a slight stimulatory effect in uninduced cells and a slight inhibition effect on the regulated exon inclusion after cell induction. In construct Ps, only a little inhibitory effect, if any, was observed after cell induction. In contrast, in the C, L, and B pre-mRNA substrates, the internal exon was preferentially incorporated into the mature mRNAs in predifferentiated cells. Interestingly, a regulatory effect of induction is partly preserved in the C, L, and B mRNAs; indeed, the isoform bearing exon 16 accumulates at a higher amount in differentiating cells than in proliferating cells. On the other hand, it is noteworthy that construct 15GE appeared to combine the effects observed in R and C constructs; exon selection was increased in uninduced cells (C effect) at a level that was not further enhanced following induction (R effect). Finally, the CLB region of exon 16 was inserted in the Altogether, these results suggest the presence of an inhibitor element encompassing the region mutated in constructs C, L, and B and containing the 2 UAG elements. This latter sequence inhibits the exon selection in uninduced cells, acting therefore as a constitutive exonic splicing silencer. Downstream intron sequences enhance exon inclusion in induced cells To determine the contribution of the downstream intron sequences in the regulated splicing event, we tested a series of single mutants (DI constructs, Figure 6), 4 of which were designed to alter the UGCAUG elements. All these mutations led to a significant decrease of exon inclusion in induced cells. In construct DI-Pm/Nr, 2 of the 3 UGCAUG elements were targeted by mean of cumulating DI-Pm and DI-Nr mutations. This led to a more pronounced inhibiting effect. In agreement with previous data, these results suggest a dose-enhancing effects of the UGCAUG elements.19
Moreover, DI-Bs and most remarkably DI-Bc and DI-Bb showed a strong splicing inhibition of the exon at both predifferentiated and differentiated stages. Interestingly, Bc mutation alters a UCUU sequence. Such a pyrimidine sequence functions in most cases through a competitive binding to PTB over U2AF upstream of the 3' splice site and prevents selection of the downstream exon.24,25 In any case, our data suggest the presence of enhancer elements spanning a region from the 5' consensus splice site to the last UGCAUG repeat element downstream. Among these elements, the sequence encompassed by DI-Bc and DI-Bb seems to have a strong impact at the constitutive exon recognition, whereas the regulated splicing appears to be under the control of sequences present on both sides of this region.
Several studies have shown that the expression of exon 16 of protein 4.1R during late erythroid differentiation is required for its interaction with spectrin and actin.3,4,6,7 However, little is known about the developmental regulated alternative splicing mechanism that governs the selection of exon 16 during erythroid differentiation. In this study, we established an exon 16-minigene system in MEL cells that mimics endogenous exon 16 splicing patterns during DMSO-induced erythroid differentiation. Moreover, we used mutagenesis analysis and demonstrated that a complex interplay of positive and negative regulatory signals present in the RNA controls the developmental stage-specific splicing of exon 16. Protein 4.1R exon 16 splicing has been examined previously in
Xenopus oocytes and the HeLa system.26
Transcripts from an exon 16 minigene resulted in exon 16 inclusion when
microinjected into Xenopus oocytes and exclusion in HeLa
nuclear extracts. This difference suggests that specific alternative
splicing factors are present differently in oocytes and HeLa. These 2 splicing systems may or may not reflect the same mechanism responsible for alternative splicing of exon 16 during erythroid differentiation. MEL cells have been shown to successfully reproduce the erythroid pattern of regulated pre-mRNA splicing of The analysis of cis-elements required for exon 16 splicing revealed several important features within the exon and its surrounding intron sequences. In exon 16, the 3' splice site is in a sequence context similar to the consensus.17 However, the 5' splice site is a weak splice site; the critical positions +3 and +4 are pyrimidine residues that deviate from the consensus purines.17 Strengthening of this 5' splice site to consensus sequence as in AA construct led to the selection of the upstream exon 16 in a constitutive manner (Figure 4 and Baklouti and colleagues22). Both the 5' and 3' splice sites are required for spliceosome complex formation in which the 5' splice site is recognized by U1 snRNA base pairing and associated with SR splicing factors, and the 3' splice site is recognized by U2 auxiliary factor (U2AF). Mutation of a weak 5' splice site toward U1 snRNA complementarity has been shown to enhance exon selection.29 The ability of SR proteins to direct the use of alternative 5' splice site by promoting the binding of U1 snRNP to 5' splice site has also been documented.30,31 It seems that the weak 5' splice site contributes to the omission of exon 16 prior to induction. In the induced cell, conditions must be created that permit the 5' splice site to be used efficiently. The cis-acting enhancing elements that direct the splicing
machinery to proximal, usually suboptimal, splice sites fall into 2 general groups, intronic and exonic. A number of intronic
splicing enhancers from both vertebrates and invertebrates that
influence alternative splicing in a cell-specific manner have been
described.18-20,32-36 In the majority of these, 2 recurring types of sequence elements mediate alternative splice usage:
poly(Y) tracts32,36 and short sequence
repeats.18-20 The upstream intron sequences of exon 16 contain an unusually long poly(Y) stretch (Figure 2) in both mouse and
human 4.1R. Similar features have been found in the Another recurring downstream intronic enhancing element is the hexamer TGCATG, which are found near exon 16. The sequence has been demonstrated to function as an enhancer in the alternative splicing of exon IIIB of the fibronectin gene,18,19 and the neuron-specific 18-base N1 exon of the mouse c-src gene.20 These elements are well conserved near exon 16 between mouse and human (Figure 2). There are 3 UGCAUG elements; the alteration of these inhibited exon inclusion in induced cells. These UGCAUG motifs seem also to function in tissue- and developmental-specific manners.18,20 Exon size and sequence play critical roles in the selection of splice sites.37,38 The different splicing patterns observed with GE and E7 constructs (Figure 5), that is, with 2 different constitutive exons positioned within the same flanking intron sequences, further support this view. Most exonic enhancers are composed of purine-rich sequences, which are
thought to mediate their activity mainly through the binding of
constitutive SR proteins.11 Purine-rich sequences at the
5' end of exon 16 match most of the consensus octamer RGAAGAAC proposed
as an ASF/SF2 enhancer core element.39 Strikingly, disruption of this element in constructs C and 15GE, led to a dramatic
increase in exon recognition rather than exon skipping (Figure 5). In
addition to this 5' purine-rich sequence, exon 16 contains 2 copies of
UAG triplet, which is known as a core element for hnRNPA1-binding exon
splicing silencer.16,40 The 42 base pairs at the 3' of
exon 16 is conserved across many species, including human, mouse, rat,
and Xenopus suggesting their importance in regulated
splicing. The substitution of this region with corresponding sequences
from The MEL cell model used in this work suggests that exon 16 skipping is the default pathway, occurring in predifferentiated erythroid cells. It would result from a predominant, and possibly cooperative, effect of the exon splicing silencer and the weak downstream 5' splice site. The regulatory pathway providing for splicing in differentiating cells could result from a stimulatory effect that acts through interactions between the upstream and the downstream intron enhancers and possibly the exonic purine-rich element. These positive interactions would overcome constitutive inhibition following cell commitment. Exon selection resulting from the coordinate activation of both
flanking introns has been described in the neuron-specific and
developmental splicing regulation of the 24-nt exon of the GABA(A)
receptor Several erythroid differentiation stage-specific alternative splicing phenomena have been reported. The third enzyme of the heme synthesis pathway, porphobilinogen deaminase, is expressed both as a "housekeeping" form present in all cell types, and a second isoform expressed only in erythroid cells.45 Erythroid-specific splice forms involving the first 3 exons of the second (5-aminolevulinate dehydratase),46 and the fourth (uroporphyringen III synthase)47 heme biosynthesis enzymes have also been described. These data suggest that erythroid-specific cell development depends in part on regulated splicing. Many mutations result in splicing defects by destroying splice sites, creating cryptic splice sites, or altering splicing enhancer or silencer elements. Erythroid genes whose expression is affected by such mutations include protein 4.1R.14 The elucidation of the mechanisms controlling alternative splicing of 4.1R pre-mRNA will enhance our understanding of the role of regulated splicing in RBC membrane biogenesis, and possibly in the expression of other key erythroid protein forms during late erythropoiesis.
We thank Dr V. T. Marchesi and Dr J. Delaunay for their constant support, and H. Cheng and P. Graham for technical assistance.
Submitted March 8, 2001; accepted August 2, 2001.
Supported by grants from the National Institutes of Health (HL 24385), the Association pour la Recherche sur le Cancer, the Ligue Contre le Cancer, Comité de la Loire, the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Région Rhône-Alpes, the Association Française contre les Myopathies, and the Fondation de France.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Faouzi Baklouti, CNRS UMR 5534, Centre de Génétique Moléculaire et Cellulaire, Université Lyon I, 43, Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France; e-mail: baklouti{at}univ-lyon1.fr.
1. Bennett V, Gilligan DM. The spectrin-based membrane skeleton and micron-scale organization of the plasma membrane. Annu Rev Cell Biol. 1993;9:27-66[CrossRef]. 2. Baklouti F, Huang SC, Vulliamy TJ, Delaunay J, Benz EJ Jr. Organization of the human protein 4.1 genomic locus: new insights into the tissue-specific alternative splicing of the pre-mRNA. Genomics. 1997;39:289-302[CrossRef][Medline] [Order article via Infotrieve].
3.
Horne WC, Huang SC, Becker PS, Tang TK, Benz EJ Jr.
Tissue-specific alternative splicing of protein 4.1 inserts an exon necessary for formation of the ternary complex with erythrocyte spectrin and F-actin.
Blood.
1993;82:2558-2563
4.
Discher DE, Winardi R, Schischmanoff PO, Parra M, Conboy JG, Mohandas N.
Mechanochemistry of protein 4.1's spectrin-actin-binding domain: ternary complex interactions, membrane binding, network integration, structural strengthening.
J Cell Biol.
1995;130:897-907
5.
Huang JP, Tang CJ, Kou GH, Marchesi VT, Benz EJ Jr, Tang TK.
Genomic structure of the locus encoding protein 4.1. Structural basis for complex combinational patterns of tissue-specific alternative RNA splicing.
J Biol Chem.
1993;268:3758-3766 6. Chasis JA, Coulombel L, Conboy J, et al. Differentiation-associated switches in protein 4.1 expression. Synthesis of multiple structural isoforms during normal human erythropoiesis. J Clin Invest. 1993;91:329-338.
7.
Baklouti F, Huang SC, Tang TK, Delaunay J, Marchesi VT, Benz EJ Jr.
Asynchronous regulation of splicing events within protein 4.1 pre-mRNA during erythroid differentiation.
Blood.
1996;87:3934-3941 8. Lopez AJ. Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation. Annu Rev Genet. 1998;32:279-305[CrossRef][Medline] [Order article via Infotrieve]. 9. Smith CW, Valcarcel J. Alternative pre-mRNA splicing: the logic of combinatorial control. Trends Biochem Sci. 2000;25:381-388[CrossRef][Medline] [Order article via Infotrieve]. 10. Hastings ML, Krainer AR. Pre-mRNA splicing in the new millennium. Curr Opin Cell Biol. 2001;13:302-309[CrossRef][Medline] [Order article via Infotrieve]. 11. Graveley BR. Sorting out the complexity of SR protein functions. RNA. 2000;6:1197-1211[CrossRef][Medline] [Order article via Infotrieve].
12.
Tang TK, Qin Z, Leto T, Marchesi VT, Benz EJ Jr.
Heterogeneity of mRNA and protein products arising from the protein 4.1 gene in erythroid and nonerythroid tissues.
J Cell Biol.
1990;110:617-624 13. Maillet P, Delaunay J, Baklouti F. Chimeric probe-mediated ribonuclease protection assay for molecular diagnosis of mRNA deficiencies. Hum Mutat. 1996;7:61-64[CrossRef][Medline] [Order article via Infotrieve].
14.
Morinière M, Ribeiro L, Dalla Venezia N, et al.
Elliptocytosis in patients with C-terminal domain mutations of protein 4.1 correlates with encoded messenger RNA levels rather than with alterations in primary protein structure.
Blood.
2000;95:1834-1841
15.
Tanaka K, Watakabe A, Shimura Y.
Polypurine sequences within a downstream exon function as a splicing enhancer.
Mol Cell Biol.
1994;14:1347-1354
16.
Del Gatto-Konczak F, Olive M, Gesnel MC, Breathnach R.
hnRNP A1 recruited to an exon in vivo can function as an exon splicing silencer.
Mol Cell Biol.
1999;19:251-260 17. Reed R. Initial splice-site recognition and pairing during pre-mRNA splicing. Curr Opin Genet Dev. 1996;6:215-220[CrossRef][Medline] [Order article via Infotrieve].
18.
Huh GS, Hynes RO.
Regulation of alternative pre-mRNA splicing by a novel repeated hexa-nucleotide element.
Genes Dev.
1994;8:1561-1574
19.
Lim LP, Sharp PA.
Alternative splicing of the fibronectin EIIIB exon depends on specific TGCATG repeats.
Mol Cell Biol.
1998;18:3900-3906 20. Modafferi EF, Black DL. A complex intronic splicing enhancer from the c-src pre-mRNA activates inclusion of a heterologous exon. Mol Cell Biol. 1997;17:6537-6545[Abstract]. 21. Cramer P, Caceres JF, Cazalla D, et al. Coupling of transcription with alternative splicing: RNA pol II promoters modulate SF2/ASF and 9G8 effects on an exonic splicing enhancer. Mol Cell. 1999;4:251-258[CrossRef][Medline] [Order article via Infotrieve]. 22. Baklouti F, Zhou J, Delaunay J, Huang SC, Benz EJ Jr. Characterization of cis-elements modulating alternative splicing of exons encoding the spectrin/actin binding domain in protein 4.1 pre-mRNA [abstract]. Blood. 1994;84(suppl 1):361a.
23.
Stamm S, Zhang MQ, Marr TG, Helfman DM.
A sequence compilation and comparison of exons that are alternatively spliced in neurons.
Nucleic Acids Res.
1994;22:1515-1526 24. Chan RC, Black DL. Conserved intron elements repress splicing of a neuron-specific c-src exon in vitro [published erratum appears in Mol Cell Biol 1997 May;17(5):2970]. Mol Cell Biol. 1995;15:6377-6385[Abstract]. 25. Gooding C, Roberts GC, Smith CW. Role of an inhibitory pyrimidine element and polypyrimidine tract binding protein in repression of a regulated alpha-tropomyosin exon. RNA. 1998;4:85-100[Abstract].
26.
Gee SL, Aoyagi K, Lersch R, Hou V, Wu M, Conboy JG.
Alternative splicing of protein 4.1R exon 16: ordered excision of flanking introns ensures proper splice site choice.
Blood.
2000;95:692-699
27.
Chu ZL, Wickrema A, Krantz SB, Winkelmann JC.
Erythroid-specific processing of human beta spectrin I pre-mRNA.
Blood.
1994;84:1992-1999
28.
Orkin SH, Harosi FI, Leder P.
Differentiation in erythroleukemic cells and their somatic hybrids.
Proc Natl Acad Sci U S A.
1975;72:98-102
29.
Dirksen WP, Hampson RK, Sun Q, Rottman FM.
A purine-rich exon sequence enhances alternative splicing of bovine growth hormone pre-mRNA.
J Biol Chem.
1994;269:6431-6436 30. Eperon IC, Ireland DC, Smith RA, Mayeda A, Krainer AR. Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF. EMBO J. 1993;12:3607-3617[Medline] [Order article via Infotrieve].
31.
Zahler AM, Neugebauer KM, Lane WS, Roth MB.
Distinct functions of SR proteins in alternative pre-mRNA splicing.
Science.
1993;260:219-222 32. Gallego ME, Gattoni R, Stevenin J, Marie J, Expert-Bezancon A. The SR splicing factors ASF/SF2 and SC35 have antagonistic effects on intronic enhancer-dependent splicing of the beta-tropomyosin alternative exon 6A. EMBO J. 1997;16:1772-1784[CrossRef][Medline] [Order article via Infotrieve].
33.
Carstens RP, McKeehan WL, Garcia-Blanco MA.
An intronic sequence element mediates both activation and repression of rat fibroblast growth factor receptor 2 pre-mRNA splicing.
Mol Cell Biol.
1998;18:2205-2217
34.
Cooper TA.
Muscle-specific splicing of a heterologous exon mediated by a single muscle-specific splicing enhancer from the cardiac troponin T gene.
Mol Cell Biol.
1998;18:4519-4525
35.
Kosaki A, Nelson J, Webster NJ.
Identification of intron and exon sequences involved in alternative splicing of insulin receptor pre-mRNA.
J Biol Chem.
1998;273:10331-10337
36.
Markovtsov V, Nikolic JM, Goldman JA, Turck CW, Chou MY, Black DL.
Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein.
Mol Cell Biol.
2000;20:7463-7479 37. Reed R, Maniatis T. A role for exon sequences and splice-site proximity in splice-site selection. Cell. 1986;46:681-690[CrossRef][Medline] [Order article via Infotrieve].
38.
Watakabe A, Tanaka K, Shimura Y.
The role of exon sequences in splice site selection.
Genes Dev.
1993;7:407-418 39. Tacke R, Manley JL. The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities. EMBO J. 1995;14:3540-3551[Medline] [Order article via Infotrieve].
40.
Si Z, Amendt BA, Stoltzfus CM.
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 2.
Nucleic Acids Res.
1997;25:861-867
41.
Caceres JF, Stamm S, Helfman DM, Krainer AR.
Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors.
Science.
1994;265:1706-1709 42. Hou VC, Gee SL, Wu M, Turck CW, Mayeda A, Conboy JG. Developmentally-regulated expression of hnRNPA1 can regulate alternative splicing of protein 4.1R exon 16 through an interaction with an exonic splicing silencer [abstract]. Blood. 2000;96:441a. 43. Ashiya M, Grabowski PJ. A neuron-specific splicing switch mediated by an array of pre-mRNA repressor sites: evidence of a regulatory role for the polypyrimidine tract binding protein and a brain-specific PTB counterpart. RNA. 1997;3:996-1015[Abstract]. 44. Huang SC, Baklouti F, Cheng H, Benz EJ Jr. Differential expression of the SR protein splicing factors and alternative splicing of protein 4.1R exon 16 during erythroid maturation [abstract]. Blood. 2000;96:592a.
45.
Chretien S, Dubart A, Beaupain D, et al.
Alternative transcription and splicing of the human porphobilinogen deaminase gene result either in tissue-specific or in housekeeping expression.
Proc Natl Acad Sci U S A.
1988;85:6-10 46. Kaya AH, Plewinska M, Wong DM, Desnick RJ, Wetmur JG. Human delta-aminolevulinate dehydratase (ALAD) gene: structure and alternative splicing of the erythroid and housekeeping mRNAs. Genomics. 1994;19:242-248[CrossRef][Medline] [Order article via Infotrieve].
47.
Aizencang GI, Bishop DF, Forrest D, Astrin KH, Desnick RJ.
Uroporphyrinogen III synthase. An alternative promoter controls erythroid-specific expression in the murine gene.
J Biol Chem.
2000;275:2295-2304
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  S.-C. Huang, A. Cho, S. Norton, E. S. Liu, J. Park, A. Zhou, I. D. Munagala, A. C. Ou, G. Yang, A. Wickrema, et al. Coupled transcription-splicing regulation of mutually exclusive splicing events at the 5' exons of protein 4.1R gene Blood, November 5, 2009; 114(19): 4233 - 4242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Marques-Garcia, N. Ferrandiz, R. Fernandez-Alonso, L. Gonzalez-Cano, M. Herreros-Villanueva, M. Rosa-Garrido, B. Fernandez-Garcia, J. P. Vaque, M. M. Marques, M. E. Alonso, et al. p73 Plays a Role in Erythroid Differentiation through GATA1 Induction J. Biol. Chem., August 7, 2009; 284(32): 21139 - 21156. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Yang, S.-C. Huang, J. Y. Wu, and E. J. Benz Jr Regulated Fox-2 isoform expression mediates protein 4.1R splicing during erythroid differentiation Blood, January 1, 2008; 111(1): 392 - 401. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kuroyanagi, G. Ohno, S. Mitani, and M. Hagiwara The Fox-1 Family and SUP-12 Coordinately Regulate Tissue-Specific Alternative Splicing In Vivo Mol. Cell. Biol., December 15, 2007; 27(24): 8612 - 8621. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H.-L. Zhou, A. P. Baraniak, and H. Lou Role for Fox-1/Fox-2 in Mediating the Neuronal Pathway of Calcitonin/Calcitonin Gene-Related Peptide Alternative RNA Processing Mol. Cell. Biol., February 1, 2007; 27(3): 830 - 841. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Ponthier, C. Schluepen, W. Chen, R. A. Lersch, S. L. Gee, V. C. Hou, A. J. Lo, S. A. Short, J. A. Chasis, J. C. Winkelmann, et al. Fox-2 Splicing Factor Binds to a Conserved Intron Motif to Promote Inclusion of Protein 4.1R Alternative Exon 16 J. Biol. Chem., May 5, 2006; 281(18): 12468 - 12474. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. Baraniak, J. R. Chen, and M. A. Garcia-Blanco Fox-2 Mediates Epithelial Cell-Specific Fibroblast Growth Factor Receptor 2 Exon Choice Mol. Cell. Biol., February 15, 2006; 26(4): 1209 - 1222. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. G. Underwood, P. L. Boutz, J. D. Dougherty, P. Stoilov, and D. L. Black Homologues of the Caenorhabditis elegans Fox-1 Protein Are Neuronal Splicing Regulators in Mammals Mol. Cell. Biol., November 15, 2005; 25(22): 10005 - 10016. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Sironi, G. Menozzi, G. P. Comi, R. Cagliani, N. Bresolin, and U. Pozzoli Analysis of intronic conserved elements indicates that functional complexity might represent a major source of negative selection on non-coding sequences Hum. Mol. Genet., September 1, 2005; 14(17): 2533 - 2546. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Lei, I. N. M. Day, and I. Vorechovsky Exonization of AluYa5 in the human ACE gene requires mutations in both 3' and 5' splice sites and is facilitated by a conserved splicing enhancer Nucleic Acids Res., July 14, 2005; 33(12): 3897 - 3906. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nakahata and S. Kawamoto Tissue-dependent isoforms of mammalian Fox-1 homologs are associated with tissue-specific splicing activities Nucleic Acids Res., April 11, 2005; 33(7): 2078 - 2089. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Yang, S.-C. Huang, J. Y. Wu, and E. J. Benz Jr An erythroid differentiation-specific splicing switch in protein 4.1R mediated by the interaction of SF2/ASF with an exonic splicing enhancer Blood, March 1, 2005; 105(5): 2146 - 2153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. P. Baraniak, E. L. Lasda, E. J. Wagner, and M. A. Garcia-Blanco A Stem Structure in Fibroblast Growth Factor Receptor 2 Transcripts Mediates Cell-Type-Specific Splicing by Approximating Intronic Control Elements Mol. Cell. Biol., December 15, 2003; 23(24): 9327 - 9337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Sorek and G. Ast Intronic Sequences Flanking Alternatively Spliced Exons Are Conserved Between Human and Mouse Genome Res., July 1, 2003; 13(7): 1631 - 1637. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Miriami, H. Margalit, and R. Sperling Conserved sequence elements associated with exon skipping Nucleic Acids Res., April 1, 2003; 31(7): 1974 - 1983. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Su, J. R. Testaverde, C. N. Davis, W. A. Hayajneh, R. Adair, and A. M. Colberg-Poley Human cytomegalovirus UL37 immediate early target minigene RNAs are accurately spliced and polyadenylated J. Gen. Virol., January 1, 2003; 84(1): 29 - 39. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
Charon human genomic library was screened using a
protein 4.1R complementary DNA (cDNA) probe pLym.
16 indicate the inclusion or exclusion of exon 16, respectively. Neg indicates negative. (C) RPA using probe
H(13,16). Mock uninduced (1) and induced (2) MEL cells;
uninduced (3) and induced (4) MEL cells transfected with WT construct;
uninduced (5) and induced (6) MEL cells transfected with the
p(13,17)/CMV cassette without the 0.7-kb exon 16 fragment. Y, yeast
RNA; M, size markers; P, undigested probe. The lower diffused band
corresponds to the endogenous 4.1R mRNA.
-and 
2 pre-mRNA.