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Results for “Alternative Splicing”

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PubMed2005

Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: implications for tauopathies.

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the gene encoding the microtubule-associated protein, tau. Some FTDP-17 mutations affect exon 10 splicing. To correct aberrant exon 10 splicing while retaining endogenous transcriptional control, we evaluated the feasibility of using spliceosome-mediated RNA trans-splicing (SMaRT) to reprogram tau mRNA. We designed a pre-trans-splicing molecule containing human tau exons 10 to 13 and a binding domain complementary to the 3' end of tau intron 9. A minigene comprising tau exons 9, 10, and 11 and minimal flanking intronic sequences was used as a target. RT-PCR analysis of SH-SY5Y cells or COS cells cotransfected with a minigene and a pre-trans-splicing molecule using primers to opposite sides of the predicted splice junction generated products containing exons 9 to 13. Sequencing of the chimeric products showed that an exact exon 9-exon 10 junction had been created, thus demonstrating that tau RNA can be reprogrammed by trans-splicing. Furthermore, by using the same paradigm with a minigene containing full-length intronic sequences, we show that cis-splicing exclusion of exon 10 can be by-passed by trans-splicing and that conversion of exon 10(-) tau RNA into exon 10(+) tau RNA could be achieved with approximately 34% efficiency. Our results demonstrate that an alternatively spliced exon can be replaced by trans-splicing and open the way to novel therapeutic applications of SMaRT for tauopathies and other disorders linked to aberrant alternative splicing.

Alternative Splicing↗

PubMed1995

The human high mobility group (HMG)-box transcription factor TCF-1: novel isoforms due to alternative splicing and usage of a new exon IXA.

The C-terminal peptide sequences of the human lymphocyte-specific high mobility group (HMG)-box transcription factor TCF-1 are determined by alternative splice mechanisms affecting the exons VIII to X. Here we report, in addition to four splice forms described previously (TCF-1A, B, C, D), the identification of three novel transcripts designated TCF-1E, F, G. Cloning and sequencing of the novel cDNAs revealed (i) joining of the exons VIII and IX to an internal exon X splice acceptor site resulting in a new open reading frame (ORF) of 99 amino acids derived from exon X sequences, (ii) the identification of an additional functional splice acceptor site within exon X, and (iii) a new 81-nucleotide insertion between exon VIII and exon X sequences in a novel transcript form. Genomic cloning and sequence analysis of this transcribed segment of 81 basepairs revealed that it was bordered by canonical splice consensus sites and located in a distance of some 400 bp from both the exons IX and X. It was therefore termed exon IXA. Novel ORFs were generated as a consequence of these alternative splice mechanisms resulting in TCF-1 gene products with significantly different C-terminal peptide sequences, which are prone to selective protein-protein interactions or transactivating functions.

Alternative Splicing↗

PubMed1994

Insulin receptor ribonucleic acid levels and alternative splicing in human liver, muscle, and adipose tissue: tissue specificity and relation to insulin action.

The absolute levels and alternative splicing of insulin receptor RNA molecules were determined in samples from liver, muscle, and adipose tissue from 17 nondiabetic individuals. Both the absolute levels and alternative splicing varied in a tissue-specific manner. In all tissues, a majority of the insulin receptor RNA molecules contained exon 11. Liver tissue had a lower percentage of RNA molecules without exon 11 (Ex 11-) than muscle and adipose tissue, but the absolute number of Ex 11- RNA copies was higher due to higher overall levels of insulin receptor RNA. Insulin receptor RNA levels in adipose tissue showed significant correlation with obesity, expressed as body mass index (kilograms per m2) as well as with in vivo insulin action, as measured by the insulin tolerance test. In this study, obesity and insulin action were not correlated with insulin receptor RNA expression in liver or muscle. Within individuals, no relation was detected between the number of insulin receptor RNA copies in a tissue and the number or percent Ex 11- RNA in the same tissue. Also, the absolute levels or Ex 11- percentages in one tissue could not predict corresponding measurements in the other two investigated tissues from the same individual.

Adipose Tissue↗

PubMed1993

A novel short isoform of the D3 dopamine receptor generated by alternative splicing in the third cytoplasmic loop.

The mouse D3 dopamine receptor has been cloned from olfactory tubercle cDNA using polymerase chain reaction and has been found to exist in two alternatively spliced forms. These two mRNA isoforms differ by the presence or absence of 63 base pairs (bp), which encode 21 amino acids in the putative third cytoplasmic loop of the receptor. The longer form corresponds to the previously reported rat D3 dopamine receptor, to which it bears sequence homology of 94%. Northern blot analysis shows the mouse D3 receptor to be most abundant in the olfactory tubercle. Expression studies show the novel short D3 isoform to bind dopaminergic ligands with a D3-like pharmacological profile. Polymerase chain reaction analysis on different mouse brain regions shows the long and short D3 receptors to be present in the same tissues, the longer form invariably being the predominant one. Analysis of the gene for the mouse D3 dopamine receptor shows that no separate exon encodes the 63-bp stretch and reveals the presence of a consensus sequence for an acceptor site at the 3' end of the 63-bp stretch. This suggests that an internal acceptor site in the exon coding for the distal part of the third cytoplasmic loop directs alternative splicing of the D3 dopamine receptor.

Alternative Splicing↗

PubMed1997

Alternative splicing of the FHIT gene in colorectal cancers.

In the present study, we examined the status of the FHIT gene in 112 colorectal cancer and 137 colorectal adenoma specimens. In a total of 5 specimens (4 colorectal cancers and 1 colorectal adenoma), a common smaller product was detected in addition to the normal size product. This smaller product had lost exon 4, the 5' noncoding region of the FHIT gene, owing to alternative splicing. Moreover, all of the 5 tumors with alternative splicing were located lower on the rectum than the anterior peritoneal reflection.

Acid Anhydride Hydrolases↗

PubMed2006

The Drosophila heterogeneous nuclear ribonucleoprotein M protein, HRP59, regulates alternative splicing and controls the production of its own mRNA.

The Drosophila heterogeneous nuclear ribonucleoprotein M, HRP59, is a nuclear protein that associates co-transcriptionally with pre-mRNA and is necessary for the correct expression of a subset of mRNAs. We show here that the hrp59 pre-mRNA is alternatively spliced to generate two different mRNAs that differ in the presence of exon 3. Exon 3-containing transcripts make up the majority of hrp59 transcripts and encode for the functional protein, HRP59-1. Transcripts that lack exon 3 contain a premature translation termination codon and are targeted to the nonsense mediated decay pathway. We show that exon 3 inclusion is itself inhibited by HRP59 and that changes in the HRP59 protein levels affect the splicing activity of the cell. We propose that the ability of HRP59 to regulate the alternative splicing of its own pre-mRNA serves in a negative feedback loop that controls the levels of the HRP59 protein and maintains the homeostasis of the splicing environment.

Alternative Splicing↗

PubMed1996

Alternative splicing gives rise to two novel long isoforms of Zn-alpha 2-glycoprotein, a member of the immunoglobulin superfamily.

We have isolated, from a rat liver cDNA library, two cDNAs encoding novel long isoforms of Zn-alpha2-glycoprotein (Zn-alpha2-gp), a member of the immunoglobulin superfamily with a high degree of sequence similarity to class-I major histocompatibility complex (MHC) antigens. Nucleotide (nt) sequence analysis of these two novel cDNAs has revealed that they contain insertions of 138 and 123 nt between the second and third exons of Zn-alpha2-gp, resulting in in-frame insertions of 46 and 41 amino acids (aa), respectively. Analysis of the mechanism of generation of both isoforms, named Zn-alpha2-gpA and Zn-alpha2-gpB, has shown that they result from a series of alternative splicing events, including alternative use of two additional exons, and of two different 3'-splice sites present in the first of these novel exons. The occurrence of these alternative splicing events in Zn-alpha2-gp could contribute to increasing the diversity of this nonpolymorphic and soluble class-I MHC antigen.

Alternative Splicing↗

PubMed1993

The type III-9 repeat of human fibronectin is encoded by a single exon which is not alternatively spliced.

Type III homologies of human fibronectin are generally encoded by two exons, with the exception of the ED-A and ED-B repeats which are encoded by a single exon undergoing alternative splicing. We report that also the type III-9 homology is encoded by a single exon. Further more, RT-PCR analysis, performed on mRNA purified from fetal and adult tissues and from normal and tumor-derived cell types, showed that the III-9 region is not undergoing alternative splicing in all samples tested.

Adult↗

PubMed1997

The murine C'-terminally alternatively spliced form of p53 induces attenuated apoptosis in myeloid cells.

The onset of p53-dependent apoptosis results from the accumulation of damaged DNA. Recently, it was shown that the C' terminus of the p53 protein plays a central role in sensing damaged DNA. In our present study, we examined the role of the C' terminus in the induction of apoptosis. A temperature-sensitive (ts) mutant of the alternatively spliced form of p53 (p53AS-ts) and the ts mutant of the regularly spliced form (p53RS-ts) were used to generate series of stable clones with increasing amounts of p53 protein. Apoptotic patterns induced by either the regularly spliced p53 product (p53RS) or a C'-terminally alternatively spliced p53 product (p53AS) were compared. We found that although both forms of p53 induced apoptosis following expression of the wild-type protein conformation, the kinetics were different. Apoptosis induced by the p53AS protein was attenuated compared to that induced by p53RS. The delay in the manifestation of the apoptotic features following p53AS expression was in agreement with a delay in the regulation of the expression of apoptosis-related genes. The observation that p53 with an altered C' terminus is still capable of inducing apoptosis suggests that the actual onset of the apoptotic process most probably involves structural domains other than the C' terminus of the p53 molecule. However, the fact that the apoptotic activity mediated by the p53AS product was slower than that mediated by the p53RS product suggests that the C' terminus indeed exerts a certain control on the apoptotic activity of the p53 molecule.

Alternative Splicing↗

PubMed2001

Nuclear translocation controlled by alternatively spliced isoforms inactivates the QUAKING apoptotic inducer.

The quaking viable mice have myelination defects and develop a characteristic tremor 10 d after birth. The quaking gene encodes at least five alternatively spliced QUAKING (QKI) isoforms that differ in their C-terminal 8--30-amino-acid sequence. The reason for the existence of the different QKI isoforms and their function are unknown. Here we show that only one QKI isoform, QKI-7, can induce apoptosis in fibroblasts and primary rat oligodendrocytes. Heterodimerization of the QKI isoforms results in the nuclear translocation of QKI-7 and the suppression of apoptosis. The unique C-terminal 14 amino acids of QKI-7 confers the ability to induce apoptosis to heterologous proteins such as the green fluorescent protein and a QKI-related protein, Caenorhabditis elegans GLD-1. Thus, the unique C-terminal sequences of QKI-7 may function as a life-or-death 'sensor' that monitors the balance between the alternatively spliced QKI isoforms. Moreover, our findings suggest that nuclear translocation is a novel mechanism of inactivating apoptotic inducers.

3T3 Cells↗

PubMed2005

Intronic CA-repeat and CA-rich elements: a new class of regulators of mammalian alternative splicing.

We have recently identified an intronic polymorphic CA-repeat region in the human endothelial nitric oxide synthase (eNOS) gene as an important determinant of the splicing efficiency, requiring specific binding of hnRNP L. Here, we analyzed the position requirements of this CA-repeat element, which revealed its potential role in alternative splicing. In addition, we defined the RNA binding specificity of hnRNP L by SELEX: not only regular CA repeats are recognized with high affinity but also certain CA-rich clusters. Therefore, we have systematically searched the human genome databases for CA-repeat and CA-rich elements associated with alternative 5' splice sites (5'ss), followed by minigene transfection assays. Surprisingly, in several specific human genes that we tested, intronic CA RNA elements could function either as splicing enhancers or silencers, depending on their proximity to the alternative 5'ss. HnRNP L was detected specifically bound to these diverse CA elements. These data demonstrated that intronic CA sequences constitute novel and widespread regulatory elements of alternative splicing.

Alternative Splicing↗

PubMed2002

MEC-8 regulates alternative splicing of unc-52 transcripts in C. elegans hypodermal cells.

Previous work has shown that C. elegans MEC-8 is a putative RNA-binding protein that promotes specific alternative splices of unc-52 transcripts. unc-52 encodes homologs of mammalian perlecan that are located extracellularly between muscle and hypodermis and are essential for muscle development in both embryos and larvae. We show that MEC-8 is a nuclear protein found in hypodermis at most stages of development and not in most late embryonic or larval body-wall muscle. We have also found that overexpression of MEC-8 in hypodermis but not muscle can suppress certain unc-52 mutant phenotypes. These are unexpected results because it has been proposed that UNC-52 is produced exclusively by muscle. We have constructed various tissue-specific unc-52 minigenes fused to a gene for green fluorescent protein that have allowed us to monitor tissue-specific mec-8-dependent alternative splicing; we show that mec-8 must be expressed in the same cell type as the unc-52 minigene in order to regulate its expression, supporting the view that MEC-8 acts directly on unc-52 transcripts and that UNC-52 must be synthesized primarily by the hypodermis. Indeed, our analysis of unc-52 genetic mosaics has shown that the focus of unc-52 action is not in body-wall muscle but most likely is in hypodermis.

Alternative Splicing↗

PubMed2003

Structural basis by which alternative splicing confers specificity in fibroblast growth factor receptors.

Binding specificity between fibroblast growth factors (FGFs) and their receptors (FGFRs) is essential for mammalian development and is regulated primarily by two alternatively spliced exons, IIIb ("b") and IIIc ("c"), that encode the second half of Ig-like domain 3 (D3) of FGFRs. FGF7 and FGF10 activate only the b isoform of FGFR2 (FGFR2b). Here, we report the crystal structure of the ligand-binding portion of FGFR2b bound to FGF10. Unique contacts between divergent regions in FGF10 and two b-specific loops in D3 reveal the structural basis by which alternative splicing provides FGF10-FGFR2b specificity. Structure-based mutagenesis of FGF10 confirms the importance of the observed contacts for FGF10 biological activity. Interestingly, FGF10 binding induces a previously unobserved rotation of receptor Ig domain 2 (D2) to introduce specific contacts with FGF10. Hence, both D2 and D3 of FGFR2b contribute to the exceptional specificity between FGF10 and FGFR2b. We propose that ligand-induced conformational change in FGFRs may also play an important role in determining specificity for other FGF-FGFR complexes.

Alternative Splicing↗

PubMed2003

Alternate forms of MHC class II-associated invariant chain are not produced by alternative splicing in rainbow trout (Oncorhynchus mykiss) but are encoded by separate genes.

A major limiting factor in understanding teleost major histocompatibility receptor function is the lack of knowledge about antigen presentation accessory molecules. We report here two cDNA clones encoding teleost versions of invariant chain and one encoding a related protein that may play a protease inhibition role in antigen presentation. The two invariant chain equivalents are similar to each other where they overlap, but differ in the presence or absence of a thyroglobulin domain. This domain is added to tetrapod invariant chain protein by alternative splicing but there was no evidence of alternative splicing of the two trout genes. Southern blotting confirmed that all three trout cDNAs are derived from single copy genes and Northern blotting indicated that they are expressed in antigen tissues. Thus the encoded proteins are probably involved in antigen presentation, but their expression is probably regulated in a manner different from tetrapods.

Alternative Splicing↗

PubMed1996

Alternative splicing of agrin alters its binding to heparin, dystroglycan, and the putative agrin receptor.

Agrin is a heparan sulfate proteoglycan that induces aggregation of acetylcholine receptors (AChRs) at the neuromuscular synapse. This aggregating activity is modulated by alternative splicing. Here, we compared binding of agrin isoforms to heparin, alpha-dystroglycan, and cultured myotubes. We find that the alternatively spliced 4 amino acids insert (KSRK) is required for heparin binding. The binding affinity of agrin isoforms to alpha-dystroglycan correlates neither with binding to heparin nor with their AChR-aggregating activities. Moreover, the minimal fragment sufficient to induce AChR aggregation does not bind to alpha-dystroglycan. Nevertheless, this fragment still binds to cultured muscle cells. Its binding is completed only by agrin isoforms that are active in AChR aggregation, and therefore this binding site is likely to represent the receptor that initiates AChR clustering.

Agrin↗

PubMed1994

Mice lacking Snrpn expression show normal regulation of neuronal alternative splicing events.

The SmN protein is closely related to the constitutively expressed RNA splicing protein SmB but is expressed only in brain and heart tissue. Mice which lack expression of SmN die shortly after birth suggesting a critical role for this protein possibly in the regulation of neuronal-specific alternative splicing events. We show here however that the neuronal-specific alternative splicing of the RNAs encoding several different classes of protein proceeds normally in mice lacking SmN expression. The potential role of SmN and the reasons for the lethal effect observed in non-expressing mice are discussed.

Alternative Splicing↗

PubMed2006

Regulation of the beta-hydroxyacyl ACP dehydratase gene of Picea mariana by alternative splicing.

The gene for beta-hydroxyacyl ACP dehydratase, a de novo fatty acid biosynthetic enzyme, was cloned from Picea mariana (black spruce) and consists of five exons and four introns. The first intron of the beta-hydroxyacyl ACP dehydratase mRNA is alternatively spliced. Retention of intron 1 in splice variants results in truncation of the beta-hydroxyacyl ACP dehydratase ORF at a premature termination codon. In addition, splicing of intron 1 was found to be associated with cold temperature. mRNAs retaining intron 1 increase with seed imbibition at 22 degrees C but not 4 degrees C, whereas, splicing of intron 1 increases in winter weeks with temperatures below freezing. These results provide evidence that alternative splicing may also contribute to regulation of lipid biosynthesis in Picea mariana.

Alternative Splicing↗

PubMed1998

Identification of four alternatively spliced isoforms of chicken casein kinase I alpha that are all expressed in diverse cell types.

Casein kinase I (CKI) is a family of widely expressed protein kinases. It is previously shown in mammalian tissues that CKIalpha exists as two or three alternatively spliced isoforms (Rowles et al.,1991; Zhang et al., 1996; Kuret et al., 1997). We now report that four alternatively spliced isoforms of CKIalpha are expressed in many chicken cells and tissues. A partial cDNA clone was isolated from a chicken brain library, using a probe derived from a bovine CKIalpha cDNA. The translated sequence of this clone was 100% identical to the bovine homolog containing the 'L' insert, with the addition of 12 amino acids just before the C terminus that had previously been reported in human and Xenopus CKIalpa. After completing the missing portion of the coding sequence by 5' RACE (rapid amplification of cDNA ends), full-length cDNA was PCR amplified from chicken brain cDNA, yielding four different products. These were cloned and sequenced and found to correspond to the four CKIalpha isoforms: CKIalpha, CKIalphaL, CKIalphaS and CKILalphaLS, where 'S' is the insert consisting of the 12 human/Xenopus C-terminal amino acids. Using reverse transcription and polymerase chain reaction (Rt-PCR), it was shown that the four isoforms are all expressed in neurons, fibroblasts and several tissues. This represents the first demonstration that four splice variants exist and are all expressed in a single type of cell.

Alternative Splicing↗