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PubMed1998

Alternative RNA splicing of the NMDA receptor NR1 mRNA in the neurons of the teleost electrosensory system.

The sequence for cDNA encoding the NMDA receptor subunit 1 (aptNR1) of the weakly electric fish Apteronotus leptorhynchus has been determined. The deduced amino acid sequence is approximately 88% identical to other vertebrate NR1 proteins, with sequence homology extending to the alternatively spliced cassettes N1 and C1. The fish and mammalian N1 and C1 splice cassettes are identical at 20 of 21 and 30 of 37 amino acid positions, respectively. We did not detect a C2 splice cassette in aptNR1 mRNA, but we did find two novel C-terminal alternative splice cassettes labeled C1' and C1". The relative levels of NR1 transcripts containing the N1 and C1 splice cassettes were determined by using RNase protection and in situ hybridization analysis. N1-containing mRNAs are more abundant in caudal brain regions, similar to the patterns reported for mammalian brain. In contrast, the relative levels of transcripts containing the C1 splice cassette are much lower in fish than in mammals, averaging only 9% for the whole brain. The levels of C1 splicing increased in more rostral brain regions. In situ hybridizations with N1- and C1-specific probes demonstrated that N1 cassette splicing occurs in most neurons but that C1 splicing is heterogeneous and is restricted to a subset of neuronal types in the electrosensory system.

Alternative Splicing↗
PubMed2026

An endothelial RNA splicing atlas catalogs effects of IL-1β and identifies an alternative PROCR isoform with genetic links to pleiotropic vascular disease.

Alternative splicing (AS) alters the sequence and dynamics of mRNA but has not been comprehensively annotated in human endothelial cells (ECs). EC dysfunction is a hallmark of complex inflammatory diseases, including cancer and atherosclerosis. Therefore, we modeled inflammation in vitro using 53 genetically distinct human aortic EC lines exposed to interleukin-1β (IL-1β) or control media. This identified 1,224 differentially spliced transcripts (DSTs). DSTs were enriched for alternative first (AF) exon usage, including isoforms of several disease-associated and metabolic genes. To confirm that IL-1β-dependent AF exons were produced by alternative promoters, a quantitative measure of promoter activity was defined using epigenetic data. Ratios of histone 3 lysine 27 acetylation and binding of ERG and RELA between alternative promoters correlated RNA levels of AF exons, confirming our hypothesis. Finally, the effects of genetic variation on AS were investigated by mapping splicing quantitative trait loci (sQTLs). Significant sQTLs were tested for genetic colocalization with cardiovascular risk loci from genome-wide association studies. This identified 66 colocalized signals corresponding to 30 genes and 39 lead variants. The sQTLs identified here provide testable mechanisms explaining some of the genetic risk for vascular disease. For example, genetic association for a previously undescribed isoform of endothelial protein C receptor (PROCR) colocalized with genetic risk for deep vein thrombosis and coronary artery disease with opposing risk alleles. This study demonstrates the prevalence of inducible promoters upon inflammatory stimuli and shows that genetic risk for vascular disease may be in part governed through AS in ECs.

Humans↗
PubMed2000

The mouse Nkx-1.2 homeobox gene: alternative RNA splicing at canonical and noncanonical splice sites.

A mouse homeobox gene, Nkx-1.2, (previously termed Sax-1) that is closely related to the Drosophila NK-1/S59 gene was cloned, and genomic DNA and cDNA were sequenced. Nine Nkx-1.2 cDNA clones were found that correspond to three species of Nkx-1.2 mRNA that are formed by alternative splicing at conventional 5' donor and 3' acceptor splice sites; however, seven cDNA clones were found that correspond to three species of Nkx-1.2 mRNA from testes that have novel TG/AC 5' and 3' splice sites. The consensus splice sequences are: 5' donor, CC downward arrowTGGAAG; 3' acceptor, ACTTAC downward arrow. Predicted amino acid sequences suggest that some transcripts may be translated into proteins that lack part or all of the homeodomain. At least three bands of Nkx-1.2 mRNA were found in RNA from the testes. Nkx-1.2 mRNA was shown to be present in postmeiotic germ cells of the testis and in mature spermatozoa. Nkx-1.2 mRNA also was found in regions of the adult cerebral cortex, hippocampus, diencephalon, pons/medulla, and cerebellum. Nkx-1.2 mRNA was found in embryos in highest abundance in 10-day embryos; the mRNA levels decrease during further development. Nkx-1.2 mRNA also was found in discrete zones of the embryonic mesencephalon and myelencephalon.

Alternative Splicing↗
PubMed2005

Delocalization of the multifunctional RNA splicing factor TLS/FUS in hippocampal neurones: exclusion from the nucleus and accumulation in dendritic granules and spine heads.

Long-term synaptic change in the cortex and the hippocampus is believed to require the highly localized delivery and translation of mRNAs in the dendritic shafts and spines. The molecular interactions that underlie local signalling between synapses and mRNAs are still largely undefined. After purification from total brain extracts, the NMDA receptor is known to be associated with numerous proteins, including the multifunctional RNA-binding factor TLS (also called FUS). In non-neural tissue, TLS is a vital nuclear protein with roles in DNA repair, homologous recombination, transcriptional regulation and pre-mRNA processing. We have examined the distribution of TLS in hippocampal neurones, both in the adult brain and in mature primary cultures, using subcellular fractionation and immunofluorescence techniques. TLS immunoreactivity is largely excluded from the neuronal nucleus and is found in the cytosol and in somatodendritic particles. In some of these particles, TLS colocalizes with Sam68, a nuclear RNA-binding protein that we previously showed is incorporated into dendritic RNA granules. Some of the TLS clusters also colocalize with NMDA receptor clusters. Finally, TLS clusters are occasionally seen within spine heads. The apparent removal of TLS from the nucleus might result in specific patterns of mRNA transcription or splicing in hippocampal neurones. TLS may also contribute to steering, anchoring or regulating mRNAs at synaptic sites.

Actins↗
PubMed1994

Cell-specific regulation of agrin RNA splicing in the chick ciliary ganglion.

Alternative splicing results in production of four agrin proteins (agrin0, agrin8, agrin11, and agrin19) with different AChR aggregating activities. However, the cellular origin of mRNAs encoding each agrin isoform remains unknown. Using single-cell PCR, we demonstrate that in the chick ciliary ganglion, nonneuronal cells express only mRNA encoding agrin0, whereas neurons express one or any combination of agrin mRNAs. Moreover, significant differences were observed between the agrin mRNA profiles of ciliary and choroid neurons in the ganglion. The abundance of each agrin mRNA, the fraction of neurons expressing each transcript, and the combinations of transcripts expressed by neurons also change during development. Our results demonstrate that transcripts encoding agrin proteins with high AChR aggregating activity are expressed exclusively by neurons in the ciliary ganglion and that alternative splicing of agrin mRNA is regulated during development and in a cell-specific manner.

Agrin↗
PubMed2006

A profile of alternative RNA splicing and transcript variation of CACNA1H, a human T-channel gene candidate for idiopathic generalized epilepsies.

Highly alternatively spliced genes may provide complex targets for disease mutations. Structural changes created by missense mutations may differentially affect the activity of alternative gene products, whereas missense, silent and non-coding mutations may alter developmental regulation of splice variant expression. CACNA1H is a human gene encoding Ca(v)3.2 low-voltage-activated, T-type calcium channels associated with bursting behavior in neurons and has been linked to more than 30 mutations apparently predisposing to childhood absence epilepsy (CAE) and other idiopathic generalized epilepsies (IGEs). Biophysical properties, including the effects of missense mutations, have been evaluated previously for a single splice form of Ca(v)3.2 expressed in transformed cell lines. We here show that CACNA1H is alternatively spliced at 12-14 sites, capable of generating both functional and non-functional transcripts. Variable cytoplasmic and extracellular protein domains point to likely differences in gating behavior, sensitivity to neuromodulation and interactions with extracellular matrix. Biophysical profiles of selected physiological Ca(v)3.2 forms reveal variations in kinetics and steady-state gating parameters, most likely to affect membrane firing. These were comparable to or larger than changes reported for previously studied mutations. Missense CAE and IGE mutations were clustered near segments associated with anomalous splicing. Missense and silent mutations were found to destroy, create or change the regulatory specificity of predicted exonic splicing enhancer sequences that may control splicing regulation. We discuss a paradigm for CACNA1H expression of Ca(v)3.2 subunits, which may influence future basic and clinical studies.

Alternative Splicing↗
PubMed1999

Missense and silent tau gene mutations cause frontotemporal dementia with parkinsonism-chromosome 17 type, by affecting multiple alternative RNA splicing regulatory elements.

Frontotemporal dementia with parkinsonism, chromosome 17 type (FTDP-17) is caused by mutations in the tau gene, and the signature lesions of FTDP-17 are filamentous tau inclusions. Tau mutations may be pathogenic either by altering protein function or gene regulation. Here we show that missense, silent, and intronic tau mutations can increase or decrease splicing of tau exon 10 (E10) by acting on 3 different cis-acting regulatory elements. These elements include an exon splicing enhancer that can either be strengthened (mutation N279(K)) or destroyed (mutation Delta280(K)), resulting in either constitutive E10 inclusion or the exclusion of E10 from tau transcripts. E10 contains a second regulatory element that is an exon splicing silencer, the function of which is abolished by a silent FTDP-17 mutation (L284(L)), resulting in excess E10 inclusion. A third element inhibiting E10 splicing is contained in the intronic sequences directly flanking the 5' splice site of E10 and intronic FTDP-17 mutations in this element enhance E10 inclusion. Thus, tau mutations cause FTDP-17 by multiple pathological mechanisms, which may explain the phenotypic heterogeneity observed in FTDP-17, as exemplified by an unusual family described here with tau pathology as well as amyloid and neuritic plaques.

Aged↗
PubMed1993

Gene structure of human mitochondrial ATP synthase gamma-subunit. Tissue specificity produced by alternative RNA splicing.

We completely sequenced the human gene for ATP synthase gamma-subunit, which was approximately 23 kilobases long and was composed of 10 exons. Exons 1 and 2 encoded the N-terminal presequence required for mitochondrial import, while exons 9 and 10 encoded the C-terminal portions of mature protein. Enzymatic amplification of human heart and liver cDNAs using the polymerase chain reaction revealed two mRNA transcripts that were predicted to encode two 30-kDa isoforms of the gamma-subunit, which differed by the addition of a single amino acid (Asp273) at the C terminus of the liver type isoform. These two mRNA transcripts of the heart (H) type and liver (L) type were generated by alternative splicing of an exon. The same alternative splicing event was observed in bovine tissue. In human tissues, the H type mRNA devoid of exon 9 was expressed specifically in the heart and skeletal muscle, which require rapid energy supply. The L type mRNA was expressed in the brain, liver, kidney etc. Both transcripts were expressed in the skin, intestine, stomach, and aorta. This tissue specificity of transcript heterogeneity suggests the distinct functional or regulatory roles of the gamma-subunit isoforms in the catalysis of ATP synthase. This is the first report on tissue-specific isoforms generated by alternative splicing in an energy transducing mitochondrial protein.

Alternative Splicing↗
PubMed1997

An alternative gonadotropin-releasing hormone (GnRH) RNA splicing product found in cultured GnRH neurons and mouse hypothalamus.

Gonadotropin-releasing hormone (GnRH) is encoded by the proGnRH gene which contains four exons and three introns. In this study, two immortalized GnRH-expressing cell lines (Gn11 and NLT) were characterized. The NLT and Gn11 cells, derived from a same brain tumor in a transgenic mouse, display neuronal morphology and neuron-specific markers. However, NLT cells secrete much higher levels of GnRH than Gn11 cells. To delineate the mechanism underlying this difference, reverse transcriptase-polymerase chain reaction and RNase protection assays were performed to examine proGnRH gene expression. While the mature proGnRH mRNA was predominately expressed in NLT cells, Gn11 cells express an abundant short transcript. Sequence analysis revealed that this short transcript contains exons 1, 3, and 4, but not exon 2, which encodes the GnRH decapeptide. RNase protection assays demonstrated that NLT cells express much higher levels of mature proGnRH mRNA than Gn11 cells. The lower level of GnRH secreting capacity in Gn11 cells is due, in part, to decreased expression of mature proGnRH mRNA. When proGnRH gene expression in the mouse brain was examined, the same short splicing variant was observed in the olfactory area and preoptic area-anterior hypothalamus. But the prevalent transcript in these regions was the mature proGnRH mRNA. In contrast, only the mature proGnRH mRNA was found in the caudal hypothalamus. These results suggest that alternative splicing may be one of the mechanisms regulating proGnRH gene expression in the animal brain.

Alternative Splicing↗
PubMed1994

Regulation of adenovirus alternative RNA splicing at the level of commitment complex formation.

The adenovirus late region 1 (L1) represents an example of an alternatively spliced gene where one 5' splice site is spliced to two alternative 3' splice sites, to produce two mRNAs; the 52,55K and IIIa mRNAs, respectively. Accumulation of the L1 mRNAs is temporally regulated during the infectious cycle. Thus, the proximal 3' splice site (52,55K mRNA) is used at all times during the infectious cycle whereas the distal 3' splice site (IIIa mRNA) is used exclusively late in infection. Here we show that in vitro splicing extracts prepared from late adenovirus-infected cells reproduces the virus-induced temporal shift from proximal to distal 3' splice site selection in L1 pre-mRNA splicing. Two stable intermediates in spliceosome assembly have been identified; the commitment complex and the pre-spliceosome (or A complex). We show that the transition in splice site activity in L1 alternative splicing results from an increase in the efficiency of commitment complex formation using the distal 3' splice site in extracts prepared from late virus-infected cells combined with a reduction of the efficiency of proximal 3' splice site splicing. The increase in commitment activity on the distal 3' splice site is paralleled by a virus-induced increase in A complex formation on the distal 3' splice site. Importantly, the virus-induced shift from proximal to distal L1 3' splice site usage does not require cis competition between the 52,55K and the IIIa 3' splice sites, but rather results from the intrinsic property of the two 3' splice sites which make them respond differently to factors in extracts prepared from virus-infected cells.

Adenoviruses, Human↗
PubMed1998

Expression of a new RNA-splice isoform of WT1 in developing kidney-gonadal complexes of the turtle, Trachemys scripta.

WT1 is a tumor suppressor gene encoding a zinc finger DNA-binding protein required for normal vertebrate kidney and gonad development. Although the sequence and function of this gene has been studied mostly in mammals, comparative analysis in other vertebrates may suggest regions of conservation of function as well as evolution of function. We have initiated a study of this gene in the freshwater turtle, Trachemys scripta, a species that demonstrates temperature dependent sex determination. The turtle WT1 amino acid sequence (GenBank Accession No. AF019779) is over 85% identical to that of other species overall, but there are some major differences. The greatest differences are in the N-terminal portion of the peptide which is thought to mediate transcriptional repression by interaction with other proteins. Turtle WT1, like those of the alligator, chicken, and Xenopus lacks the proline- and glycine-rich stretches that are present in mammalian WT1. Exon 5, which is alternatively spliced in mammals, is altogether absent in the non-mammalian vertebrates. In addition, turtle WT1 is alternatively spliced so that exon 4 is either present or absent. These differences suggest that the interaction of reptilian WT1 with other factor required for mediation of activity may be different than the interaction of mammalian WT1. It also suggests that alternative splicing is a conserved regulatory mechanism of vertebrate WT1. Expression of WT1 in turtle embryonic kidney-gonadal complexes begins after the mesonephroi have formed and continues at least until the bipotential gonad begins to differentiate. Although the proportions of the different splice isoforms are relatively constant during these stages of kidney development, the level of steady state expression is increased in embryos incubated at 26 degrees C, the testis-producing temperature.

Alternative Splicing↗
PubMed1981

Controls of RNA splicing and termination in the major late adenovirus transcription unit.

The major late adenovirus promoter is active early after infection, selectively producing messenger RNAs coding for polypeptides with molecular weights of 55,000, 52,000 and 14,000. This selective expression suggests that a differential splicing pattern occurs at the transition from early to late viral gene expression. Activation of the late promoter and splicing of the 55, 52K mRNAs does not require newly synthesized virus polypeptides.

Adenoviruses, Human↗
PubMed2002

Novel mutation and RNA splice variant of fibroblast growth factor receptor 3 in multiple myeloma patients at diagnosis.

BACKGROUND AND OBJECTIVES: The karyotypically silent t(4;14)(p16.3;q32) translocation can be found in approximately 15-20% of multiple myeloma (MM) patients and results in the ectopic expression of fibroblast growth factor receptor 3 (FGFR3) from der4. Point mutations in specific FGFR3 domains can be found in the translocated allele, and have been recently proven to be oncogenic. These mutations produce a constitutively activated receptor, which shows dimerization and autophosphorylation even in the absence of ligand. We investigated the presence of FGFR3 expression and activating mutations in a series of newly diagnosed MM patients. DESIGN AND METHODS: We validated a new sensitive and specific Taqman real-time reverse-transcription polymerase-chain-reaction (RT-PCR) set up to evaluate FGFR3 mRNA expression, and applied it to 78 newly diagnosed patients; in positive cases, FGFR3 mRNA transcripts were sequenced. Fluorescence in situ hybridization (FISH) was done in 32 cases with sufficient material. RESULTS: Real-time RT-PCR revealed FGFR3 mRNA expression in 10/78 (13%) patients. In two cases, sequence analysis revealed novel FGFR3 mutations. In a patient with FISH evidence of the t(4;14), a CGC to TGC transition was detected in codon 248. In a patient without the t(4;14), three additional, abnormal-sized transcripts were detected, corresponding to truncated transcripts originating from cryptic splice donor sites located within exon 7. INTERPRETATION AND CONCLUSIONS: We describe a novel FGFR3 mutation (with a demonstrated deregulatory mechanism), as well as a case of alternative splicing in the absence of t(4;14), detected in newly diagnosed MM patients overexpressing FGFR3. This implies that FGFR3 mutation can occur at an early stage of myelomagenesis and even in the absence of the t(4;14).

Alternative Splicing↗