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PubMed2007

MicroRNAs regulate the expression of the alternative splicing factor nPTB during muscle development.

Alternative pre-mRNA splicing determines many changes in gene expression during development. Two regulators known to control splicing patterns during neuron and muscle differentiation are the polypyrimidine tract-binding protein (PTB) and its neuronal homolog nPTB. These proteins repress certain exons in early myoblasts, but upon differentiation of mature myotubes PTB/nPTB expression is reduced, leading to increased inclusion of their target exons. We show here that the repression of nPTB expression during myoblast differentiation results from its targeting by the muscle-restricted microRNA miR-133. During differentiation of C2C12 myoblasts, nPTB protein but not mRNA expression is strongly reduced, concurrent with the up-regulation of miR-133 and the induction of splicing for several PTB-repressed exons. Introduction of synthetic miR-133 into undifferentiated C2C12 cells leads to a decrease in endogenous nPTB expression. Both the miR-133 and the coexpressed miR-1/206 microRNAs are extremely conserved across animal species, and PTB proteins are predicted targets for these miRNAs in Drosophila, mice, and humans. There are two potential miR-133-responsive elements (MRE) within the nPTB 3' untranslated region (UTR), and a luciferase reporter carrying this 3' UTR is repressed by miR-133 in an MRE-dependent manner. Transfection of locked nucleic acid (LNA) oligonucleotides designed to block the function of miR-133 and miR-1/206 increases expression of nPTB and decreases the inclusion of PTB dependent exons. These results indicate that miR-133 directly down-regulates a key splicing factor during muscle development and establishes a role for microRNAs in the control of a developmentally dynamic splicing program.

3' Untranslated Regions↗
PubMed1989

Structure and expression of a family of Ultrabithorax mRNAs generated by alternative splicing and polyadenylation in Drosophila.

The 77-kb primary transcript of the homeotic Ultrabithorax (Ubx) gene is alternatively spliced to yield at least five different coding regions. Each is restricted to either a 3.2- or a 4.3-kb size class generated by alternative polyadenylation. The pathways for splicing and polyadenylation are therefore coordinately regulated, and because the relative abundance of the respective mRNAs varies throughout development, these pathways also appear to be developmentally regulated. Translation of these mRNAs yields a family of Ubx proteins characterized by constant amino- and carboxy-proximal regions of 247 and 99 amino acid residues, respectively. Members of this family are distinguished by a short variable region that links the constant regions and consists of different combinations of three optional elements of 9, 17, and 17 residues. Only four amino acid residues separate this variable region from the 60-residue homeo domain of the carboxy-terminal constant region. This proximity suggests that functional differences among the Ubx proteins derive from the differential effects of their variable regions on the DNA-binding capacity of the homeo domain. An argument is made that these functional differences are tissue specific.

Amino Acid Sequence↗
PubMed1993

The ability of agrin to cluster AChRs depends on alternative splicing and on cell surface proteoglycans.

Agrin, which induces acetylcholine receptor (AChR) clustering at the developing neuromuscular synapse, occurs in multiple forms generated by alternative splicing. Some of these isoforms are specific to the nervous system; others are expressed in both neural and nonneural tissues, including muscle. We have compared the AChR clustering activity of agrin forms varying at each of the three identified splicing sites, denoted x, y, and z. Agrin isoforms were assayed by applying either transfected COS cells, with agrin bound to their surfaces, or soluble agrin to myotubes of the C2 muscle line, or of two variant lines having defective proteoglycans. Dramatic differences in activity were seen between z site isoforms and lesser differences between y site isoforms. The most active agrin forms contained splicing inserts of 4 amino acids at the y site and 8 amino acids at the z site. These forms are found exclusively in neural tissue. All forms were active on C2 myotubes in cell-attached assays, but muscle forms were less active than neural forms. AChR clustering activity of all agrin forms was decreased when assayed on the proteoglycan-deficient lines, suggesting that proteoglycans may help mediate the action of agrin. As neural agrin forms are more active than muscle forms, they are likely to play a primary role in synaptogenesis.

Agrin↗
PubMed1997

Anatomic localization of alternatively spliced leptin receptors (Ob-R) in mouse brain and other tissues.

Leptin's effects are mediated by interactions with a receptor that is alternatively spliced, resulting in at least five different murine forms: Ob-Ra, Ob-Rb, Ob-Rc, Ob-Rd, and Ob-Re. A mutation in one splice form, Ob-Rb, results in obesity in mice. Northern blots, RNase protection assays, and PCR indicate that Ob-Rb is expressed at a relatively high level in hypothalamus and low level in several other tissues. Ob-Ra is expressed ubiquitously, whereas Ob-Rc, -Rd, and -Re RNAs are only detectable using PCR. In hypothalamus, Ob-Rb is present in the arcuate, ventromedial, dorsomedial, and lateral hypothalamic nuclei but is not detectable in other brain regions. These nuclei are known to regulate food intake and body weight. The level of Ob-Rb in hypothalamus is reduced in mice rendered obese by gold thioglucose (GTG), which causes hypothalamic lesions. The obesity in GTG-treated mice is likely to be caused by ablation of Ob-Rb-expressing neurons, which results in leptin resistance.

Alternative Splicing↗
PubMed2004

Alternative splicing of the human luteal LH receptor during luteolysis and maternal recognition of pregnancy.

Deletion of exon 10 of the human LH receptor impairs LH but not hCG action. Other splice variants of the LH receptor impair both LH and hCG action in other species. We hypothesized that alternatively spliced LH receptors are involved in luteolysis and luteal rescue with hCG in women. mRNA was extracted from human luteinized granulosa cells and from corpora lutea from across the luteal phase and after luteal rescue in vivo with exogenous hCG. Splice variants were detected by RT-PCR using carefully designed primer pairs. Products were visualized on agarose gels, extracted, purified and sequenced. Three splice variants of the human LH receptor were detected and characterized. These demonstrate a region of multiple splicing between exons 8 and 11 of the receptor. A naturally occurring splice variant with exon 10 alone removed was not identified. There was no obvious change in the pattern of splice variants across the luteal phase in the presence or absence of hCG. These data do not support the hypothesis that qualitative changes in LH receptor splicing have a role in luteolysis or that a naturally occurring LH receptor lacking exon 10 has a role in maternal recognition of pregnancy.

Alternative Splicing↗
PubMed1994

Wild-type alternatively spliced p53: binding to DNA and interaction with the major p53 protein in vitro and in cells.

A p53 variant protein (p53as) generated from alternatively spliced p53 RNA is expressed in normal and malignant mouse cells and tissues, and p53as antigen activity is preferentially associated with the G2 phase of the cell cycle, suggesting that p53as and p53 protein may have distinct properties. Using p53as and p53 proteins translated in vitro, we now provide evidence that p53as protein has efficient sequence-specific DNA-binding ability. DNA binding by p53 protein is inefficient in comparison and requires activation. Furthermore, p53as and p53 proteins formed hetero-oligomers when co-translated in vitro, resulting in inactivation of p53as DNA-binding activity. Gel filtration indicated that p53as translated in vitro, like p53, formed tetramers. In support of a functional role of p53as in cells, p53as/p53 hetero-oligomers were coimmunoprecipitated from mouse cells, and both protein forms were detectable in nuclear extracts by electrophoretic mobility shift assays. These results suggest that the biochemical functions of p53 are mediated by interaction between two endogenous protein products of the wild-type p53 gene.

Alternative Splicing↗
PubMed1993

Alternative splicing of a neural-specific Src mRNA (Src+) is a rapid and protein synthesis-independent response to neural induction in Xenopus laevis.

Alternative splicing is used by the src protooncogene to derive two gene products, pp60c-src and pp60+. pp60+ is expressed exclusively in neurons, whereas pp60c-src is expressed in many tissues. The specific role of the src+ gene product in neurons is not yet understood. Expression of src+ mRNA begins early in neural development and is restricted to the neural plate in Xenopus embryos. Using a reverse transcription/polymerase chain reaction assay, we demonstrate that expression of src+ mRNA is dependent on neural induction. Src+ mRNA is expressed in animal cap ectoderm which is cocultured with mesoderm tissue, but not in ectoderm cultured alone. The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA) induces src+ mRNA expression in cultured stage 10+ animal cap ectoderm. This induction is rapid, with src+ mRNA being detected within 1 hr of TPA addition. Only dorsal ectoderm tissue is competent to express src+ mRNA upon TPA treatment, and stage 10+ ectoderm tissue becomes unresponsive to TPA after 4 hr in culture. TPA-dependent induction of src+ mRNA in ectoderm tissue is not blocked by cycloheximide and therefore is not dependent on protein synthesis.

Alternative Splicing↗
PubMed2004

Alternative splicing of Drosophila Dscam generates axon guidance receptors that exhibit isoform-specific homophilic binding.

Dscam is an immunoglobulin (Ig) superfamily protein required for the formation of neuronal connections in Drosophila. Through alternative splicing, Dscam potentially gives rise to 19,008 different extracellular domains linked to one of two alternative transmembrane segments, resulting in 38,016 isoforms. All isoforms share the same domain structure but contain variable amino acid sequences within three Ig domains in the extracellular region. We demonstrate that different isoforms exhibit different binding specificity. Each isoform binds to itself but does not bind or binds poorly to other isoforms. The amino acid sequences of all three variable Ig domains determine binding specificity. Even closely related isoforms sharing nearly identical amino acid sequences exhibit isoform-specific binding. We propose that this preferential homophilic binding specificity regulates interactions between cells and contributes to the formation of complex patterns of neuronal connections.

Alternative Splicing↗
PubMed1999

Tissue-specific alternative splicing of rat brain fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase.

We have reported the occurrence of eight splice variants of rat brain fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase (RB2K). In the present study, we quantified these splice variants in various tissues using a RNAse protection assay and found a tissue-specific pattern of alternative splicing of the RB2K gene. Splice variants containing exon F were specifically expressed in brain. Moreover, exons D and E were spliced in brain, skeletal muscle and heart. Consequently, eight, six, four and two splice variants were expressed in brain, skeletal muscle, heart and liver plus testis, respectively. These results suggest that distinct RB2K isoforms could be involved in regulation of glycolysis in a tissue-specific manner.

Alternative Splicing↗
PubMed1997

Amyloid precursor protein in guinea pigs--complete cDNA sequence and alternative splicing.

We present the cDNA sequence of the guinea pig amyloid precursor protein comprising the complete coding sequence of 770 amino-acid residues. By alternative splicing of three exons transcripts encoding for 695, 714 and 751 amino acids and all forms previously denoted as L-APP are also generated. Guinea pig amyloid precursor protein was shown to exhibit extensive sequence similarity to its human and murine homologues of approx. 97% at the protein level which implies an evolutionary conserved but yet not fully understood physiological function.

Alternative Splicing↗
PubMed1997

Structure-function studies of recombinant murine tripeptidyl-peptidase II: the extra domain which is subject to alternative splicing is involved in complex formation.

Tripeptidyl-peptidase II (TPP II) is an exopeptidase with a remarkably high native Mr (> 10(6)). Recently, an alternatively spliced, murine cDNA variant was identified which contains an additional 39 bp, encoding 13 amino acids in the C-terminal end of the protein. The two enzyme variants were expressed in human kidney 293 cells. Both types of subunit were found to form the active oligomers. In addition, subunits containing the extra 13 amino acids formed an even larger complex eluting in the void volume of a Sepharose CL-4B column. Thus, it appears that this sequence is important for aggregation of subunits.

Alternative Splicing↗
PubMed2006

KRAB-containing zinc finger gene ZNF268 encodes multiple alternatively spliced isoforms that contain transcription regulatory domains.

The ZNF268 gene was originally isolated from an early human embryo cDNA library. Several different transcripts have been isolated for the ZNF268 gene and developmental expression studies suggest that ZNF268 plays a role in the development of human fetal liver and the differentiation of blood cells. In our effort to study the functions of ZNF268 in different organs during development and in pathogenesis, we have now identified 3 novel splicing isoforms, ZNF268e, ZNF268f and ZNF268g, in human fetal tissues and human tumor derived cell lines. The 8 alternatively spliced mRNAs discovered so for are predicted to encode 3 protein isoforms. Expression analysis showed that different mRNA isoforms have different expression profiles. In particular, ZNF268c mRNA was detected only in tumor cells, and ZNF268f appeared to be tissue-specific. By Western blot analysis, all 3 ZNF268 protein isoforms, ZNF268a, ZNF268b1 and ZNF268b2, were expressed in tumor cell lines, while only two protein products, ZNF268b1 and ZNF268b2, were detected in human fetal tissues. Subcellular localization analysis showed that ZNF268a and ZNF268b2 distributed diffusely throughout the cell, while ZNF268b1 mainly localized in the cytoplasm. Moreover, using a CAT reporter system fused to the Gal4 DNA binding domain of the ZNF268 gene, the ZNF268a and b2 activated the CAT reporter gene expression, while the KRAB domain, corresponding to the ZNF268b1 repressed the reporter gene expression. Taken together, our results showed that multiple ZNF268 splicing products encode multiple ZNF268 protein isoforms with different subcellular localization, and that the ZNF268 gene may function as a transcriptional activator in the growth and differentiation of cells in development and/or pathogenesis.

Alternative Splicing↗
PubMed1993

Cloning of alternately spliced mRNA transcripts coding for variants of ovine testicular follitropin receptor lacking the G protein coupling domains.

We report the cloning and characterization of two alternately spliced forms of ovine testicular follitropin receptor mRNA. A smaller receptor cDNA (151 A1) of 727 bp codes for a possible soluble receptor protein of 134 amino acids arising from exons 1-4 of the full length receptor. The 1.1 Kb cDNA clone (HK 18) extending up to the 8th exon codes for a mature protein of 259 amino acids with a single membrane spanning domain predicted by hydropathy analysis. As these structures account for 34% and 61% respectively of the extracellular domain of the full length receptor, we suggest that their putative protein products are likely to possess moderate or high affinity binding sites of physiological significance.

Alternative Splicing↗
PubMed1996

Follitropin signal transduction: alternative splicing of the FSH receptor gene produces a dominant negative form of receptor which inhibits hormone action.

We have studied the functional properties of an alternately spliced form of sheep testicular FSH receptor cDNA that codes for a protein similar to a previously described active receptor but differs in the carboxy terminus in sequence and is also shorter by 25 residues. The receptor expressed in HEK 293 cells fails to activate adenylate cyclase. Cotransfection of stably expressing cells bearing FSH receptor that activates (Gs) adenylate cyclase with the altered receptor cDNA abrogates hormone response. In cells expressing this cDNA. FSH also inhibited cyclic AMP accumulation induced by non hormonal agents such as forskolin and cholera toxin which bypass the receptor. We propose that this altered receptor is a dominant negative receptor which may be coupled to G1 protein(s) or other inhibitory mechanisms.

Adenylyl Cyclases↗
PubMed1996

Characterization of the human AMPD3 gene reveals that 5' exon useage is subject to transcriptional control by three tandem promoters and alternative splicing.

Previous work has identified multiple human AMPD3 transcripts proposed to differ by mutually exclusive alternative splicing of three exons located at, or near, the 5' end of the gene. In this study, we perform a more comprehensive evaluation of human AMPD3 gene expression. Combined Northern blot and RNase protection analyses show that alternative mRNAs are widely expressed in human tissues and cells, but at variable relative abundances. Sequencing of human genomic clones, together with human-mouse somatic cell hybrid analysis, demonstrates that the entire gene is comprised of seventeen exons spanning approx. 60 kilobases on the short arm of chromosome 11 in the region p13-pter. Together, RT-PCR and additional RNase protection analyses establish that exons 1a, 1b, and 1c are 5' terminal sequences in alternative transcripts. Transient transfection experiments show fusion constructs containing proximal flanking and 5' untranslated sequence from each of these exons are able to direct expression of a reporter luciferase gene in mammalian cell lines. These combined results reveal that AMPD3 gene expression is subject to transcriptional control by three tandem promoters. Differential regulation of the exon 1b promoter in skeletal myocytes, as compared to retinal pigment epithelial cells, is proposed to be mediated by skeletal muscle-specific basic helix-loop-helix protein/E-box interactions. Finally, an internal splice acceptor site in exon 1c is shown to be used alternatively to retain the 3' portion of this exon in mature AMPD3 transcripts initiating upstream in exon 1b.

AMP Deaminase↗
PubMed1998

Co-transcriptional commitment to alternative splice site selection.

Production of mRNA in eukaryotic cells involves not only transcription but also various processing reactions such as splicing. Recent experiments have indicated that there are direct physical connections between components of the transcription and processing machinery, supporting previous suggestions that pre-mRNA splicing occurs co-transcriptionally. Here we have used a novel functional approach to demonstrate co-transcriptional regulation of alternative splicing. Exon 3 of the alpha-tropomyosin gene is specifically repressed in smooth muscle cells. By delaying synthesis of an essential downstream inhibitory element, we show that the decision to splice or repress exon 3 occurs during a limited window of opportunity following transcription, indicating that splice site selection proceeds rapidly after transcription.

Alternative Splicing↗
PubMed1996

An alternatively spliced polycistronic mRNA encoding cyclic adenosine 3',5'-monophosphate (cAMP)-responsive transcription factor CREB (cAMP response element-binding protein) in human testis extinguishes expression of an internally translated inhibitor CREB isoform.

Cyclic AMP response element-binding protein (CREB) regulates the expression of cAMP-responsive genes. In the rat testis, several isoforms of CREB arise from alternative exon splicing that occurs cyclically during the 12-day cell association cycles of spermatogenesis. Insertion of alternatively spliced exon W into CREB mRNA during spermatogenesis results in a polycistronic RNA that encodes two novel internally translated CREB repressor isoforms called I-CREBs, consisting of the carboxy-terminal DNA-binding domain devoid of the transactivation domains. Here we report the alternative splicing of an additional novel exon Z in CREB mRNA expressed in human but not in mouse or rat testis. Insertion of exon Z abolishes the synthesis of one of the two inhibitor CREBs due to the introduction of an inframe stop codon within exon Z. We show that exon Z is not spliced into mRNAs in mouse and rat testes due to the evolution of mutations in the splice signals flanking exon Z. These findings suggest that the splicing in of exon Z may be part of a human-specific mechanism to regulate cAMP-dependent regulatory pathways in spermatogenesis by extinguishing the expression of a CREB repressor.

Alternative Splicing↗