Two exonic elements in the flanking constitutive exons control the alternative splicing of the alpha exon of the ZO-1 pre-mRNA

The 240-bp alpha exon of the tight junction (TJ) protein ZO-1 pre-mRNA is alternatively spliced. Expression of both ZO-1alpha+/ZO-1alpha- isoforms results in hermetic TJs, and these become leaky when ZO-1alpha- expression prevails. The alpha exon inclusion/skipping mechanism was studied by in vivo RT-PCR splicing assays in neural and epithelial cells, utilizing a canine minigene construct containing the alpha exon, and the flanking introns and exons. Inclusion of the alpha exon always occurs in wild-type MDCK cells and it is detectable in transfected HeLa cells. However, the alpha exon is skipped in transfected neural cells. Accordingly, both 5' and 3' splice sites surrounding the alpha exon appear to be suboptimal and no cis-acting splicing control elements were found in this exon. Deletion analysis revealed an 83-bp splicing enhancer in the downstream exon and a 35-bp splicing silencer at the beginning of the upstream exon. In epithelial cells all constructs rendered alpha exon inclusion. We conclude that, in neural cells, skipping of the alpha exon depends on two antagonistic exonic elements located in the flanking constitutive exons.     

Expression of the unique NCAM VASE exon is independently regulated in distinct tissues during development

During development of the rat central nervous system, neural cell adhesion molecule (NCAM) mRNAs containing in the extracellular domain a 30-bp alternative exon, here named VASE, replace RNAs that lack this exon. The presence of this alternative exon between previously described exons 7 and 8 changes the predicted loop structure of the derived polypeptide from one resembling an immunoglobulin constant region domain to one resembling an immunoglobulin variable domain. This change could have significant effects on NCAM polypeptide function and cell-cell interaction. In this report we test multiple rat tissues for the presence of additional alternative exons at this position and also examine the regulation of splicing of the previously described exon. To sensitively examine alternative splicing, polymerase chain reactions (PCRs) with primers flanking the exon 7/exon 8 alternative splicing site were performed. Four categories of RNA samples were tested for new exons: whole brain from embryonic day 11 to adult, specific brain regions dissected from adult brain, clonal lines of neural cells in vitro, and muscle cells and tissues cultured in vitro and obtained by dissection. Within the limits of the PCR methodology, no evidence for any alternative exon other than the previously identified VASE was obtained. The regulation of expression of this exon was found to be complex and tissue specific. Expression of the 30-bp exon in the heart and nervous system was found to be regulated independently; a significant proportion of embryonic day 15 heart NCAM mRNAs contain VASE while only a very small amount of day 15 nervous system mRNAs contain VASE. Some adult central nervous system regions, notably the olfactory bulb and the peripheral nervous system structures adrenal gland and dorsal root ganglia, express NCAM which contains very little VASE. VASE is undetectable in NCAM PCR products from the olfactory epithelium. Other nervous system regions express significant quantities of NCAM both with and without VASE. Clonal cell lines in culture generally expressed very little VASE. These results indicate that a single alternative exon, VASE, is found in NCAM immunoglobulin-like loop 4 and that distinct tissues and nervous system regions regulate expression of VASE independently both during development and in adult animals.     

Elements of the rat tropoelastin gene associated with alternative splicing

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA.     

NCAM polypeptides in heart development: association with Z discs of forms that contain the muscle-specific domain

Previous studies of neural cell adhesion molecule (NCAM) cDNAs have revealed an alternatively spliced set of small exons (12A, 12B, 12C, and 12D) that encode a region in the extracellular portion of the molecule known as the muscle-specific domain (MSD). The entire MSD region can be expressed in skeletal muscle, heart, and skin; only exons 12A and 12D have been found in brain. These studies did not reveal which NCAM polypeptides contain the MSD region or the immunohistochemical distribution of these NCAM molecules. To address these questions, we prepared antibodies against the oligopeptides encoded by exons 12A and 12B and by exons 12C and 12D, and we used these antibodies to study the forms of NCAM containing the MSD region expressed during embryonic chicken heart development. These antibodies recognize certain forms of NCAM found in the heart, but they do not recognize brain NCAM. In the heart, each of the splice variants of NCAM (large cytoplasmic domain, small cytoplasmic domain, and small surface domain) that differ in their mode of attachment to the plasma membrane or in the size of their cytoplasmic domain is expressed in a form that contains and in a form that lacks the MSD region. No microheterogeneity is observed in the size of NCAM molecules containing the MSD region, even at the level of cyanogen bromide fragments, suggesting that exons 12A-D are expressed as a single unit. Depending on the site and the stage of development, the percent of NCAM molecules containing the MSD region can vary from nearly 0 to 100%. In general, this percentage increases during development. In immunohistochemical studies of hearts from stage 18 embryos, forms of NCAM containing the MSD region colocalized with Z discs. No other adhesion molecules were found in this distribution at this early stage of development. Studies on isolated cells in vitro demonstrate that the colocalization with Z discs of NCAM molecules containing the MSD region does not depend on cell-cell contact, and they raise the possibility that this form of NCAM is involved in cell-extracellular matrix interactions. The association of NCAM molecules containing the MSD region with Z discs suggests that this form of NCAM is involved in early myofibrillogenesis.     

Natural base-pairing interactions between 5' splice site and branch site sequences affect mammalian 5' splice site selection.

In the murine gene encoding the neuronal cell adhesion molecule (NCAM), the integrity of the 5' splice site of exon 18 (E18) is essential for regulation of alternative splicing. To further study the contribution of 5' splice site sequences, we used a simple NCAM pre-mRNA containing a portion of E18 fused to E19 and separated by a shortened intron. This RNA is spliced in vitro to produce five sets of lariat intermediates and products, the most abundant set displaying aberrant migration in acrylamide/urea gels. Base pairing interactions between positions +5 and +8 of the intron and positions -3 and -6 from the branch point were responsible for the faster migration of this set of lariat molecules. To test whether the duplex structure forms earlier and contributes to 5' splice site selection, we used NCAM substrates carrying the 5' splice sites of E17 and E18 in competition for the 3' splice site of E19. Mutations upstream of the major branch site improve E18/E19 splicing in NIH3T3 extracts, whereas compensatory mutations at positions +7 and +8 neutralize the effect of branch site mutations and curtail E18/E19 splicing. Our data suggest that duplex formation occurs early and interferes with the assembly of complexes initiated on the 5' splice site of NCAM E18. This novel type of intron interaction may exist in the introns of other mammalian pre-mRNAs.     

An element in the 5' common exon of the NCAM alternative splicing unit interacts with SR proteins and modulates 5' splice site selection.

The neural cell adhesion molecule (NCAM) gene contains an 801 nt exon that is included preferentially in neuronal cells. We have set up an in vitro splicing system that mimics the neuro-specific alternative splicing profile of NCAM exon 18. Splicing regulation is observed using model pre-mRNAs that contain competing 5' or 3' splice sites, suggesting that distinct pathways regulate NCAM 5' and 3' splice site selection. While inclusion of exon 18 is the predom-inant choice in neuronal cells, an element in the 5' common exon 17 improves exon 17/exon 19 splicing in a neuronal cell line. A similar behavior is observed in vitro as the element can stimulate the 5' splice site of exon 17 or a heterologous 5' splice site. The minimal 32 nt sequence of the exon 17 enhancer consists of purine stretches and A/C motifs. Mutations in the purine stretches compromise the binding of SR proteins and decreases splicing stimulation in vitro. Mutations in the A/C motifs do not affect SR protein binding but reduce enhancing activity. Our results suggest that the assembly of an enhancer complex containing SR proteins in a 5' common exon ensures that NCAM mRNAs lacking exon 18 are made in neuronal cells.     

A DNA damage signal activates and derepresses exon inclusion in Drosophila TAF1 alternative splicing

Signal-dependent alternative splicing is important for regulating gene expression in eukaryotes, yet our understanding of how signals impact splicing mechanisms is limited. A model to address this issue is alternative splicing of Drosophila TAF1 pre-mRNA in response to camptothecin (CPT)-induced DNA damage signals. CPT treatment of Drosophila S2 cells causes increased inclusion of TAF1 alternative cassette exons 12a and 13a through an ATR signaling pathway. To evaluate the role of TAF1 pre-mRNA sequences in the alternative splicing mechanism, we developed a TAF1 minigene (miniTAF1) and an S2 cell splicing assay that recapitulated key aspects of CPT-induced alternative splicing of endogenous TAF1. Analysis of miniTAF1 indicated that splice site strength underlies independent and distinct mechanisms that control exon 12a and 13a inclusion. Mutation of the exon 13a weak 5' splice site or weak 3' splice site to a consensus sequence was sufficient for constitutive exon 13a inclusion. In contrast, mutation of the exon 12a strong 5' splice site or moderate 3' splice site to a consensus sequence was only sufficient for constitutive exon 12a inclusion in the presence of CPT-induced signals. Analogous studies of the exon 13 3' splice site suggest that exon 12a inclusion involves signal-dependent pairing between constitutive and alternative splice sites. Finally, intronic elements identified by evolutionary conservation were necessary for full repression of exon 12a inclusion or full activation of exon 13a inclusion and may be targets of CPT-induced signals. In summary, this work defines the role of sequence elements in the regulation of TAF1 alternative splicing in response to a DNA damage signal.     

<Emphasis Type="Italic">HLA-G</Emphasis> allelic variants are associated with differences in the<Emphasis Type="Italic"> HLA-G</Emphasis> mRNA isoform profile and<Emphasis Type="Italic"> HLA-G</Emphasis> mRNA levels

During pregnancy, the human extra-villous trophoblast in the contact zone between maternal and fetal tissue in the placenta does not express the classical MHC class I and II molecules. Instead, HLA-G and -C, and possibly HLA-E, are expressed. HLA-G may modulate the immunological relationship between mother and fetus in several ways. Finally, the expression of membrane-bound HLA-G and soluble HLA-G has been proposed to influence the outcome of pregnancy, and an aberrant HLA-G expression in pre-eclamptic placentas and spontaneous abortions has been reported. Here, an association between certain HLA-G polymorphisms and the mRNA levels of the different alternatively spliced HLA-G isoforms in first trimester trophoblast cell populations is reported. Several alternatively spliced HLA-G mRNA isoforms, including a 14-bp polymorphism in the 3'UTR end (exon 8) of the HLA-G gene, are expressed at a significantly lower level than the corresponding HLA-G mRNA isoforms with the 14-bp sequence deleted. Furthermore, characteristic HLA-G mRNA isoform expression patterns were associated with specific HLA-G genotypes and alleles. In the HLA-G*01012 and - G*01013 alleles that include the 14-bp sequence, an additional alternative splicing was observed, with the first 92-bp of exon 8 spliced out. This was most pronounced in HLA-G genotypes with G*01013. These findings may have functional implications for the recent reports of aberrant HLA-G expression and reproductive success.     

Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle.

A two-site model for the binding of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was tested in order to understand how exon partners are selected in complex pre-mRNAs containing alternative exons. In this model, it is proposed that two U1 snRNPs define a functional unit of splicing by base pairing to the 3' boundary of the downstream exon as well as the 5' boundary of the intron to be spliced. Three-exon substrates contained the alternatively spliced exon 4 (E4) region of the preprotachykinin gene. Combined 5' splice site mutations at neighboring exons demonstrate that weakened binding of U1 snRNP at the downstream site and improved U1 snRNP binding at the upstream site result in the failure to rescue splicing of the intron between the mutations. These results indicate the stringency of the requirement for binding a second U1 snRNP to the downstream 5' splice site for these substrates as opposed to an alternative model in which a certain threshold level of U1 snRNP can be provided at either site. Further support for the two-site model is provided by single-site mutations in the 5' splice site of the third exon, E5, that weaken base complementarity to U1 RNA. These mutations block E5 branchpoint formation and, surprisingly, generate novel branchpoints that are specified chiefly by their proximity to a cryptic 5' splice site located at the 3' terminus of the pre-mRNA. The experiments shown here demonstrate a true stimulation of 3' splice site activity by the downstream binding of U1 snRNP and suggest a possible mechanism by which combinatorial patterns of exon selection are achieved for alternatively spliced pre-mRNAs.