Functionally distinct insulin receptors generated by tissue-specific alternative splicing.

Cloning of the insulin receptor cDNA has earlier revealed the existence of two alternative forms of the receptor differing by the presence or absence of 12 amino acids near the C-terminus of the receptor alpha-subunit. This insert has been shown by others to be encoded by a discrete exon, and alternative splicing of this exon leads to tissue-specific expression of two receptor isoforms. We have studied the functional significance of the receptor isoforms and have confirmed that they are generated by alternative splicing. When cDNAs encoding the two forms of the insulin receptors are expressed in Rat 1 cells, the receptor lacking the insert (HIR-A) has a significantly higher affinity for insulin than the receptor with the insert (HIR-B). This difference in affinity is maintained when insulin binding activity is assayed in solution using detergent solubilized, partially purified receptors. These data, combined with the tissue specificity of HIR-A and HIR-B expression, suggest that alternative splicing may result in the modulation of insulin metabolism or responsiveness by different tissues.     

Use of an alternate splice donor site in the human GAP gene is responsible for synthesis of the p100 isoform

GAP (GTPase-activating protein), a negative regulator of the receptor tyrosine kinase signal transduction pathway, exists as two isoforms: a ubiquitous, p120 form and a primate placenta-specific p100 form lacking the N-terminal hydrophobic domain. The cDNA species encoding p120 and p100 GAP are identical except that p100 GAP cDNA contains a 65-bp insert not present in p120 cDNA. The purpose of this study was to locate the 65-bp insert in the genomic GAP sequence, thereby determining the mechanism by which alternate splicing produces the two mRNA species. It was found that the 65-bp insert is located just 3′ to the sequence encoding the hydrophobic domain, indicating that the p100 form of GAP results from utilization of an alternate splice donor site. In addition, the sequence encoding the hydrophobic domain was found to be contained within a single large exon. The intron separating this exon from the exon encoding the 5′-portion of the SH2-N domain was determined to be at least 40 kb in length. Finally, it was found that the sequence encoding the SH2-N domain contains an intron 1006 bp long, and the sequence of this intron has been deduced. It is anticipated that the data presented in this paper will provide the basis for elucidating RNA processing mechanisms responsible for preferential expression of p100 GAP in the human placenta.     

Alternative splicing of the Drosophila Dscam pre-mRNA is both temporally and spatially regulated

The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene encodes an axon guidance receptor that can express 38,016 different mRNAs by virtue of alternative splicing. The Dscam gene contains 95 alternative exons that are organized into four clusters of 12, 48, 33, and 2 exons each. Although numerous Dscam mRNA isoforms can be synthesized, it remains to be determined whether different Dscam isoforms are synthesized at different times in development or in different tissues. We have investigated the alternative splicing of the Dscam exon 4 cluster, which contains 12 mutually exclusive alternative exons, and found that Dscam exon 4 alternative splicing is developmentally regulated. The most highly regulated exon, 4.2, is infrequently used in early embryos but is the predominant exon 4 variant used in adults. Moreover, the developmental regulation of exon 4.2 alternative splicing is conserved in D. yakuba. In addition, different adult tissues express distinct collections of Dscam mRNA isoforms. Given the role of Dscam in neural development, these results suggest that the regulation of alternative splicing plays an important role in determining the specificity of neuronal wiring. In addition, this work provides a framework to determine the mechanisms by which complex alternative splicing events are regulated.     

Regulatory roles of heterogeneous nuclear ribonucleoprotein M and Nova-1 protein in alternative splicing of dopamine D2 receptor pre-mRNA

The dopamine D2 receptor (D2R) plays a crucial role in the regulation of diverse key physiological functions, including motor control, reward, learning, and memory. This receptor is present in vivo in two isoforms, D2L and D2S, generated from the same gene by alternative pre-mRNA splicing. Each isoform has a specific role in vivo, underlining the importance of a strict control of its synthesis, yet the molecular mechanism modulating alternative D2R pre-mRNA splicing has not been completely elucidated. Here, we identify heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a key molecule controlling D2R splicing. We show that binding of hnRNP M to exon 6 inhibited the inclusion of this exon in the mRNA. Importantly, the splicing factor Nova-1 counteracted hnRNP M effects on D2R pre-mRNA splicing. Indeed, mutations of the putative Nova-1-binding site on exon 6 disrupted Nova-1 RNA assembly and diminished the inhibitory effect of Nova-1 on hnRNP M-dependent exon 6 exclusion. These results identify Nova-1 and hnRNP M as D2R pre-mRNA-binding proteins and show their antagonistic role in the alternative splicing of D2R pre-mRNA.     

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.     

The expression analysis of mouse interleukin-6 splice variants argued against their biological relevance

Alternative splicing generates several interleukin-6 (IL-6) isoforms; for them an antagonistic activity to the wild-type IL-6 has been proposed. In this study we quantified the relative abundance of IL-6 mRNA isoforms in a panel of mouse tissues and in C2C12 cells during myoblast differentiation or after treatment with the Ca(2+) ionophore A23187, the AMP-mimetic AICAR and TNF-α. The two mouse IL-6 isoforms identified, IL-6δ5 (deletion of the first 58 bp of exon 5) and IL-6δ3 (lacking exon 3), were not conserved in rat and human, did not exhibit tissue specific regulation, were expressed at low levels and their abundance closely correlated to that of full-length IL-6. Species-specific features of the IL-6 sequence, such as the presence of competitive 3' acceptor site in exon 5 and insertion of retrotransposable elements in intron 3, could explain the production of IL-6δ5 and IL-6δ3. Our results argued against biological significance for mouse IL-6 isoforms.     

Differential expression of CD45 isoforms is controlled by the combined activity of basal and inducible splicing-regulatory elements in each of the variable exons

The human CD45 gene encodes five isoforms of a transmembrane tyrosine phosphatase that differ in their extracellular domains as a result of alternative splicing of exons 4-6. Expression of the CD45 isoforms is tightly regulated in peripheral T cells such that resting cells predominantly express the larger CD45 isoforms, encoded by mRNAs containing two or three variable exons. In contrast, activated T cells express CD45 isoforms encoded by mRNAs lacking most or all of the variable exons. We have previously identified the sequences within CD45 variable exon 4 that control its level of inclusion into spliced mRNAs. Here we map the splicingregulatory sequences within CD45 variable exons 5 and 6. We show that, like exon 4, exons 5 and 6 each contain an exonic splicing silencer (ESS) and an exonic splicing enhancer (ESE), which together determine the level of exon inclusion in na?ve cells. We further demonstrate that the primary activation-responsive silencing motif in exons 5 and 6 is homologous to that in exon 4 and, as in exon 4, binds specifically to the protein heterogeneous nuclear ribonucleoprotein L. Together these studies reveal common themes in the regulation of the CD45 variable exons and provide a mechanistic explanation for the observed physiological expression of CD45 isoforms.     

Activation of nuclear Ca(2+)/calmodulin-dependent protein kinase II and brain-derived neurotrophic factor gene expression by stimulation of dopamine D2 receptor in transfected NG108-15 cells

We identified the isoforms of Ca(2+) /calmodulin-dependent protein kinase II (CaM kinase II) subunits in rat striatum. All four subunits of CaM kinase II alpha, beta, gamma and delta were detected including the isoforms of alphaB, gammaA, gammaA', gammaA.B, delta3 and delta7 with nuclear localization signal. We established NG108-15 cells with the stably expressed dopamine D2L receptor (D2LR, long form), which is an alternative splicing variant. The cells were termed NGD2L. Immunostaining demonstrated that D2LR was localized in plasma membranes. Calcium imaging with fluo-3 AM revealed that quinpirole, a D2R agonist, increased the intracellular Ca(2+), which was blocked by treatment with sulpiride and pertussis toxin in NGD2L cells, but not in mock cells. Furthermore, stimulation of D2LR with quinpirole in NGD2L cells activated the nuclear isoform of CaM kinase II. Stimulation of D2LR increased the expression of exon III- and IV-BDNF mRNA. Overexpression of CaM kinase II delta3 increased exon IV- but not exon III-BDNF mRNA. These results suggest that D2R is involved in the activation of the nuclear isoform of CaM kinase II and thereby in stimulation of gene expression through Ca(2+) signaling.