Evidence For Alternative Splicing Research Articles

Pattern Formation in Mosquitos

IMMUNOLOGY Like the innate immune systems of vertebrates, those of the insect world possess pattern recognition receptors that detect the broad signatures displayed by different classes of pathogens. In contrast, the narrow immune receptor specificity afforded by mechanisms of genetic recombination has been considered a feature unique to adaptive immunity in higher vertebrates. This view has recently undergone some revision, however, with the observation that lower vertebrates and invertebrates are also adept at manufacturing diverse immune receptors. For example, Drosophila use alternative splicing of transcripts from an immunoglobulin domain-containing locus—the Down syndrome cell adhesion molecule gene Dscam —to generate recognition receptors that assist in the phagocytosis of bacteria. Dong et al. observe that in the mosquito Anopheles gambiae (the vector for malaria), the large number of exons in AgDscam could yield as many as 31,000 alternatively spliced products, a range similar to that calculated for Drosophila. Challenging mosquito cell lines with different pathogens resulted in a varied representation of these exons via alternative splicing and AgDscam molecules with distinct specificities. Evidence for alternative splicing of AgDscam was also demonstrated in adult mosquitos, and RNA interference-mediated silencing decreased the resistance of mosquitos to bacterial infection and to oocytes of the malaria parasite carried in the insect midgut. As in Drosophila , the AgDscam forms appeared to enhance phagocytosis of bacteria by hemocytes, although it is likely that the mechanism of Dscam action extends to other modes of immune defense. A further series of experiments revealed that the repertoires of AgDscam molecules could be tailored, in terms of binding affinity, to the infecting pathogens, underscoring the degree to which specificity provided by the Dscam system might help refine pathogen pattern recognition in insects. — SJS PLoS Biol. 4 , e229 (2006).

FHR-4A: a new factor H-related protein is encoded by the human FHR-4 gene

We describe a new member of the human Factor H protein family, termed Factor H-related protein 4A (FHR-4A). The corresponding cDNA sequence was isolated and encodes a secreted protein of 559 amino acids, with a predicted molecular weight of 63.2 kDa. Apparently, this novel cDNA is derived from the human FHR-4 gene. Genetic analysis shows that the human FHR-4 gene is composed of 10 coding exons, and two distinct mRNA transcripts are derived from this gene by alternative splicing. The short FHR-4B form represents a truncated variant and encodes a secreted protein of five domains (previously termed FHR-4). The long transcript encodes the novel FHR-4A protein that is composed of nine complement control protein (CCP) domains. A unique feature of FHR-4A is the tandem arrangement of four CCP domains forming a 'natural dimer' of the short isoform. The FHR-4A protein is identified in human plasma as a 86 kDa protein. The difference between the predicted and observed molecular masses is explained by glycosylation. Comparison of the deduced protein sequence of FHR-4A with peptides from a 86 kDa apolipoprotein described by us earlier suggests that the long form, FHR-4A, represents this apoprotein. In summary, FHR-4A is a new Factor H-related protein with a unique domain composition, that is, an internal duplication of four CCP domains. To our knowledge, FHR-4A provides the first evidence for alternative splicing among Factor H-related genes.

Comparative analysis and characterization of expressed sequence tags in gilthead sea bream ( Sparus aurata) liver and embryos

The gilthead sea bream ( Sparus aurata) is one of the main European aquaculture products and a prospective model species for the Sparidae, which includes several other commercially important species. Future selective breeding of aquaculture stocks will be heavily underpinned by molecular genetic techniques, especially by marker-assisted selection (MAS). Gene marker resources in marine fish species, however, lag behind those of other agricultural animals, and only scanty information exists about the genetic source of phenotypic variation and the identity of quantitative trait loci (QTL). In order to develop molecular resources in gilthead sea bream, complementary DNA libraries were constructed from liver and mixed embryo and larval stages by unidirectional cloning. A long-read expressed sequence tag (EST) database was generated, containing 1394 cDNA clones representing 852 unique cDNA sequence-reads. Tissue-specific patterns of gene expression were determined when grouped using the proposal for characterizing cellular component put forward by the Gene Ontology consortium. Transcripts encoding cytoskeletal proteins were most abundant in the embryonic/larval library, while the most abundant transcripts in the liver library encoded secreted and extracellular proteins. Of both libraries, 505 clones were sequenced in both orientations (5′ and 3′ end sequencing), where 226 clones were determined as full-length sequence reads. Cluster analysis of 3′end-sequenced clones from both libraries revealed that alternative polyadenylation signals were utilized, although no evidence of alternative splicing was found. We report for the first time for gilthead sea bream or any sparid a transcriptional analysis of two tissues and briefly consider the utility of ESTs for characterizing tissue-specific expression profiles.

Molecular genetic responses to lysergic acid diethylamide include transcriptional activation of MAP kinase phosphatase-1, C/EBP-beta and ILAD-1, a novel gene with homology to arrestins.

We recently demonstrated that the potent hallucinogenic drug lysergic acid diethylamide (LSD) dynamically influences the expression of a small collection of genes within the mammalian prefrontal cortex. Towards generating a greater understanding of the molecular genetic effects of hallucinogens and how they may relate to alterations in behavior, we have identified and characterized expression patterns of a new collection of three genes increased in expression by acute LSD administration. These genes were identified through additional screens of Affymetrix DNA microarrays and examined in experiments to assess dose-response, time course and the receptor mediating the expression changes. The first induced gene, C/EBP-beta, is a transcription factor. The second gene, MKP-1, suggests that LSD activates the MAP (mitogen activated protein) kinase pathway. The third gene, ILAD-1, demonstrates sequence similarity to the arrestins. The increase in expression of each gene was partially mediated through LSD interactions at 5-HT2A (serotonin) receptors. There is evidence of alternative splicing at the ILAD-1 locus. Furthermore, data suggests that various splice isoforms of ILAD-1 respond differently at the transcriptional level to LSD. The genes thus far found to be responsive to LSD are beginning to give a more complete picture of the complex intracellular events initiated by hallucinogens.

Evidence for alternative splicing and developmental regulation of the Drosophila melanogaster Mgat2 ( N-acetylglucosaminyltransferase II) gene

We present a molecular study of the Drosophila melanogaster Mgat2 gene that codes for the N-acetylglucosaminyltransferase II. Isolation and analysis of two cDNA clones indicated that the gene is alternatively spliced, generating two transcripts of 3.3 and 2.7 kb. Developmental specificity was observed between the two alternative transcripts during the major developmental stages of D. melanogaster. The deduced amino acid sequences of the two proteins show significant homology to the equivalent mammalian proteins, especially in the carboxyterminal region. In situ hybridizations in embryos and embryonic imaginal discs showed that the gene is expressed mainly but not exclusively in the brain.

Alternatively spliced products of the human kinesin light chain 1 (KNS2) gene.

Conventional kinesin is a microtubule-based molecular motor involved in the transport of membranous and non-membranous cargoes. The kinesin holoenzyme exists as a heterotetramer, consisting of two heavy chain and two light chain subunits. It is thought that one function of the light chains is to interact with the cargo. Alternative splicing of kinesin light chain pre-mRNA has been observed in lower organisms, although evidence for alternative splicing of the human gene has not been reported. We have identified 19 variants of the human KNS2 gene (KLC1) that are generated by alternative splicing of downstream exons, but calculate that KNS2 has the potential to produce 285 919 spliceforms. Corresponding spliceforms of the mouse KLC1 gene were also identified. The alternative exons are all located 3' of exon 12 and the novel spliceforms produce both alternative carboxy termini and alternative 3' untranslated regions. The observation of multiple light chain isoforms is consistent with their proposed role in specific cargo attachment.

Class XIII myosins from the green alga Acetabularia: driving force in organelle transport and tip growth?

The green alga Acetabularia cliftonii (Dasycladales) contains at least two myosin genes, which already have been assigned class XIII of the myosin superfamily (Cope et al., 1996, Structure 4: 969-987). Here we report a complete analysis of their gene structure and their corresponding transcripts Aclmyo1 and Aclmyo2. Despite promising Northern blot data no evidence for alternative splicing could be found. Dissecting the primary structure at complementary deoxyribonucleic acid (cDNA) level we found a myosin typical organization in head, neck and variable tail region. Most striking is the extremely short tail region of Aclmyo1 with only 18 residues and the maximum number of 7 IQ motifs in Aclmyo2. Probing Acetabularia protein extracts with an antibody raised to a synthetic peptide derived from the amino terminal region in Alcmyo1 showed cross-reactivity to a polypeptide with a molecular mass of approximately 100 kD. This corresponds to the predicted molecular weight of Aclmyo1, which is 106 kD as deduced from the amino acid sequence. Additionally, the same cross-reactive protein is capable of binding F-actin as indicated by a co-sedimentation assay. Confocal laser scanning microscopy with raised antibody revealed co-localization with organelles, the budding region of lateral whorls and the cell apex suggesting involvement of putative Acetabularia myosin in organelle transport and tip growth.

Osteopontin--a molecule for all seasons.

Osteopontin (OPN) is a multifunctional protein, and although highly expressed in bone, it is also expressed by various cell types including macrophages, endothelial cells, smooth muscle cells and epithelial cells.1,,2 OPN is involved in diverse biological processes and the aim of this review is to give a broad outline of some important aspects of the biology of OPN. Although this review is written primarily from a renal perspective, it is hoped that the reader will appreciate that OPN is involved in both physiological and pathological processes in multiple organs and tissues including biomineralization, inflammation, leukocyte recruitment and cell survival. OPN is a negatively‐charged acidic hydrophilic protein of approximately 300 amino acid residues, and is secreted into all body fluids. The OPN cDNA from various mammalian species exhibits a high degree of sequence homology. There is evidence of alternative splicing, although the functional significance of this is unclear at present. The molecule undergoes considerable post‐translational modification, and is phosphorylated and glycosylated. OPN has an arginine‐glycine‐aspartic acid (RGD) cell binding sequence, a calcium binding site and two heparin binding domains. Cells may bind OPN via multiple integrin receptors including the vitronectin receptor ( α v β 3) as well as various β 1 and β 5 integrins. Integrin binding may be RGD‐dependent or ‐independent (e.g. via the motif SVVYGLR). OPN does not bind the standard form of CD44 (hyaluronic acid receptor) but does bind various isoforms of CD44. These CD44 isoforms bind OPN via multiple sites. OPN may be cleaved by thrombin, resulting in the exposure of additional cryptic binding sites as well as the production of functional chemotactic fragments.3 The regulation of OPN expression is incompletely understood at present and may differ between various cell types. Studies indicate that the OPN promoter contains various motifs including a purine‐rich …

Evidence for alternate splicing within the mRNA transcript encoding the DNA damage response kinase ATR

Proper cellular response to genotoxic insult often requires the activity of one or more members of a family of high-molecular weight protein kinases referred to as phosphatidylinositol-3 kinase (PIK)-like proteins. While catalytic activity is an indispensable part of PIK-like protein function, little is currently known about factors that control their activity and/or functions. This deficiency stems, in large part, from our lack of knowledge concerning functionally significant subdomains within the large non-catalytic domain of these proteins. We have determined that the transcript encoding the PIK-like protein ATR undergoes alternate splicing within the region of the mRNA encoding its non-catalytic domain. This conclusion is based on the sequencing of a human expressed sequence tag clone encoding a portion of the ATR cDNA, and is supported by the results of reverse transcriptase-polymerase chain reaction (RT-PCR) assays conducted on total and polyA+ RNA, as well as sequencing of cloned RT-PCR products. Cloning and sequencing of a segment of human genomic DNA indicated that this event arises from splicing of a single 192 bp exon within the ATR gene. Analysis of several human tissues indicated that alternate ATR transcripts are differentially expressed, suggesting that this region of the ATR protein may be of functional importance.

Expression and alternative splicing of c-ret RNA in papillary thyroid carcinomas.

Somatic rearrangements of the ret receptor tyrosine kinase have been consistently reported in papillary thyroid carcinomas (PTC). It is unclear whether the expression of wild-type c-ret may also be implicated in thyroid tumorigenesis. We studied ret mRNA expression in PTC from Norwegian patients. Using RT-PCR, wild-type ret mRNA was detected in all of 22 PTC and in a PTC cell line. c-ret mRNA was clearly overexpressed in PTC as compared to non-neoplastic thyroid tissue. Hybridization using ret exon DNA dot blot arrays and complex cDNA probes confirmed expression of ret RNA in thyroid biopsies. In accordance with the RNA data, Western immunoblotting showed evidence of wild-type Ret protein in PTC. Rearrangements generating the ret/PTC oncogenes co-existed with c-ret mRNA in PTC. Multiple alternative ret splicing variants were detected in PTC. Four novel ret splicing events were found in the region encoding the extracellular domain. The open reading frames of these transcripts were all in-frame with the Ret tyrosine kinase domain. In the central ret mRNA region encoding the cysteine-rich, transmembrane, and main tyrosine kinase domains, no evidence of alternative splicing was detected. Two alternative splice events were detected in the ret mRNA encoding the C-terminal part of Ret protein harboring tyrosine residues important for Ret signaling, excluding exon 19, or retaining intron 19, respectively. Ribonuclease protection assays confirmed the presence of ret alternative splicing events in thyroid biopsies. We conclude that in addition to ret/PTC rearrangements, wild-type c-ret mRNA and alternatively spliced ret transcripts are present in PTC. Transcriptional up-regulation and post-transcriptional mechanisms of c-ret RNA processing may contribute to differences in expression of Ret protein observed in PTC compared to non-neoplastic thyroid tissue.

The 33-kDa Platelet α-granule Membrane Protein (GMP-33) Is an N-terminal Proteolytic Fragment of Thrombospondin

GMP-33 is a platelet membrane associated protein that is recognised by RUU-SP 1.77, an antibody raised against activated platelets. GMP-33 is predominantly associated with the membrane of platelet alpha-granules and it is translocated to the plasma membrane upon platelet activation (Metzelaar et al. Blood 1992; 79: 372-9). In this study we have isolated the protein by immunoaffinity chromatography. The N-terminus was sequenced and was identical to the N-terminal sequence of human thrombospondin. The protein was N-glycosylated and bound to heparin as would be expected of the N-terminal part of thrombospondin. RUU-SP 1.77 reacted only with reduced thrombospondin. Plasmin and trypsin digestion of thrombospondin yielded fragments of approximately the same size as GMP 33 that reacted with RUU-SP 1.77 after reduction. No evidence for alternative splicing was found. We postulate that GMP 33 is an N-terminal proteolytic fragment of thrombospondin that is membrane associated.

Identification of a Novel Gene on Chromosome 7q31 That Is Interrupted by a Translocation Breakpoint in an Autistic Individual

The results of genetic linkage studies for autism have suggested that a susceptibility locus for the disease is located on the long arm of chromosome 7 (7q). An autistic individual carrying a translocation, t(7;13)(q31.3;q21), with the chromosome 7 breakpoint located in the region of 7q implicated by genetic studies was identified. A novel gene known as "RAY1" (or "FAM4A1") was found to be directly interrupted by the translocation breakpoint. The gene, which was found to be encoded by 16 exons with evidence of alternative splicing, spanned > or =220 kb of DNA at 7q31.3. Mutation screening of the entire coding region in a set of 27 unrelated autistic individuals failed to identify phenotype-specific variants, suggesting that coding region mutations are unlikely to be involved in the etiology of autism. Apparent homologues of RAY1 have also been identified in mouse, rat, pig, chicken, fruit fly, and nematode. The human and mouse genes share similar splicing patterns, and their predicted protein products are 98% identical.

Frequent alternative splicing of human genes.

Alternative splicing can produce variant proteins and expression patterns as different as the products of different genes, yet the prevalence of alternative splicing has not been quantified. Here the spliced alignment algorithm was used to make a first inventory of exon-intron structures of known human genes using EST contigs from the TIGR Human Gene Index. The results on any one gene may be incomplete and will require verification, yet the overall trends are significant. Evidence of alternative splicing was shown in 35% of genes and the majority of splicing events occurred in 5' untranslated regions, suggesting wide occurrence of alternative regulation. Most of the alternative splices of coding regions generated additional protein domains rather than alternating domains.

Evidence for alternative splicing of ecto-ATPase associated with termination of purinergic transmission

Ectonucleotidases provide the signal termination mechanism for purinergic transmission, including fast excitatory neurotransmission by ATP in the CNS. This study provides evidence for ectonucleotidase expression in the rat cochlea, brain and other tissues. In addition to detection of rat ecto-ATPase and ecto-ATPDase in these tissues, we identify a novel ecto-ATPase splice variant arising from the loss of a putative exon (193 bp) in the C-terminal coding region. This is the first evidence of alternative splicing in the ecto-ATPase gene family. Splicing of the 193-bp putative exon containing a stop codon extends the open reading frame and provides translation of an additional 50 amino acids compared with the isoform isolated earlier from the rat brain (rEATPase A; GenBank accession #Y11835). The splice variant (rEATPase B; GenBank accession #AF129103) encodes 545 amino acids with a predicted protein molecular mass of 60 kDa. rEATPase B contains a long cytoplasmic tail (62 amino acids) with three potential protein kinase CK2 phosphorylation sites not present in rEATPase A. Co-expression of two ecto-ATPase isoforms with different regulatory sites suggests that the extracellular ATP signal levels may be differently influenced by intracellular feedback pathways.

Structure of the cDNA encoding the precursor for the neuropeptide FMRFamide in the bivalve mollusc Mytilus edulis.

FMRFamide immunoreactivity is widespread in the tissues of bivalve molluscs, but some of this immunoreactivity may represent distinct related peptides (FaRPs) rather than the exact tetrapeptide FMRFamide. We have cloned the first full-length cDNA encoding the precursor protein for FMRFamide from this class of molluscs to investigate the possibility that additional peptides may be produced. The precursor contains one copy each of NFLRFamide, FLRFamide, ALAGDHFFRFamide and 16 copies of FMRFamide. This precursor is expressed in all three ganglia of the central nervous system. Since the gene encoding the FMRFamide precursor in pulmonate molluscs is alternatively spliced to give two distinct messages, we searched for evidence that the FMRFamide gene of Mytilus is also alternatively spliced. No evidence of alternative splicing was found.

Increased Mdm2 expression in rat brain after transient middle cerebral artery occlusion.

The negative regulator of p53 transactivation, Mdm2, increased in the ischemic territory after 90 minutes of transient middle cerebral artery occlusion in spontaneously hypertensive rats compared to sham controls. Increased mdm2 mRNA was detected by semiquantitative reverse transcriptase polymerase chain reaction by 6 hours of reperfusion in the ipsilateral hemisphere. In situ hybridization histochemistry was used to localize increases in mdm2 mRNA which occurred in neurons of ischemic cortex and dorsolateral striatum. The number of labeled neurons increased by approximately 20-fold and the cells displayed five-fold increases of mdm2 mRNA in the cortex. Immunohistochemical staining for Mdm2 revealed that its mRNA was efficiently translated in the ischemic cortex, but not striatum, by 8 to 24 hours of reperfusion. Western blotting confirmed 30- to 40-fold increases in the full-length protein of 90 kd at these time points without evidence of alternative splicing. Because Mdm2 is a negative regulator of the apoptosis promoting activity of p53, increased expression of Mdm2 may be a component of a repair response in injured neurons, and supports Mdm2 being an indicator of DNA damage in the brain early after an ischemic insult in a similar way to Gadd45.

Disruption of the 5' and 3' splice sites flanking the major latency-associated transcripts of herpes simplex virus type 1: evidence for alternate splicing in lytic and latent infections.

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) are the only viral gene products expressed within latently infected neurones. The most abundant (major) LATs consist of two collinear nuclear polyA- RNAs of 2 kb and 1.5 kb which it has been suggested represent stable introns derived from a less abundant primary transcript (minor LAT). Consistent with this proposition is the identification of consensus splice donor and acceptor sites flanking major LATs which are conserved between HSV types 1 and 2. Here we test the functionality of the predicted splice sites within the context of the virus genome during productive infection in vitro and latent infection in vivo. To this end viruses in which the LAT splicing signals were disrupted by site-directed mutagenesis were constructed. We report that mutation of the splice acceptor site abrogates 2 kb major LAT generation during productive infection but does not significantly influence major LAT synthesis during neuronal latency. Similarly, mutation of the splice donor site significantly reduces levels of 2 kb major LAT during productive infection but has no detectable effect on the generation of 2 kb major LAT during neuronal latency as assessed by Northern and in situ hybridization analyses of latently infected neuronal tissue. From these data it can be concluded that the proposed splice sites flanking the major LAT region are dispensable for 2 kb major LAT production in neurones latently infected with HSV-1 but constitute functional splicing signals in productively infected non-neuronal cells.

Evidence for Alternative Splicing in Hepatic α1B-Adrenergic Receptor Gene Expression

The interactions of catecholamines with α1B-adrenergic receptors (α1B-AR) located on the surface of many cell types are responsible for physiologic and pathologic functions in mammalian systems. Transcription of the α1B-AR gene leads to the expression of multiple α1B-AR mRNAs that are distributed in a tissue-specific fashion. The purpose of this study was to define the 5′-untranslated regions of the multiple α1B-AR gene transcripts. Evidence for a previously unidentified intron in the α1B-AR gene upstream of the receptor open reading frame was obtained via rapid amplification of cDNA ends. A product was amplified and was found to be missing the nucleotide interval from -708 to -194, (+1 is the start of translation). Evidence for tissue-specific alternative intron splicing was obtained from ribonuclease protection assays and RT-PCR experiments. Using an RNA probe extending from -240 to +93 and including 45 nucleotides into the putative intron, a single protected fragment was detected in heart RNA while two protected fragments were detected in liver RNA. RT-PCR amplification of the region spanning the intron resulted in detection of two PCR products in liver RNA and no detectable product in heart RNA. These findings emphasize the complexity of α1B-AR gene regulation and suggest that multiple α1B-AR mRNAs with different 5′-UTRs may play a role in regulating α1B-adrenergic responsiveness.

Characterization of the transcription start point of the trout estrogen receptor-encoding gene: evidence for alternative splicing in the 5′ untranslated region

The estrogen receptor (ER)-encoding gene ( ER) regulates many genes implicated in the reproductive functions. Moreover, rainbow trout ER (rtER) is itself up-regulated by its own product. We have used Northern blot, RNase protection, primer extension and reverse transcription-polymerase chain reaction (RT-PCR) to study the position of the rtER mRNA transcription start point ( tsp) in liver. This analysis has revealed the presence of a tsp positioned at the beginning of the cloned rtER cDNA. Functionality of this tsp was tested in transient transfections in CHO-K1 cells. The characterization of the rtER 5′ untranslated region ( UTR) showed that two transcripts exist in liver which differ in their 5′-UTR. The first one is 100% homologous to the cloned rtER cDNA sequence. The other one contains a 41-bp insertion. The isolation and sequencing of the first intron showed that this insertion arises from alternative splicing, due to the use of a splicing site internal to the first intron.

The structure of the presenilin 1 (S182) gene and identification of six novel mutations in early onset AD families

Genetic linkage studies place a gene causing early onset familial Alzheimer's disease (FAD) on chromosome 14q24.3 (refs 1-4). Five mutations within the S182 (Presenilin 1: PS-1) gene, which maps to this region, have recently been reported in several early onset FAD kindreds. We have localized the PS-1 gene to a 75 kb region and present the structure of this gene, evidence for alternative splicing and describe six novel mutations in early onset FAD pedigrees all of which alter residues conserved in the STM2 (Presenilin 2: PS-2) gene.