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PubMed2006

Misregulation of tau alternative splicing in neurodegeneration and dementia.

Tau is a microtubule-associated protein that fulfills several functions critical for neuronal formation and health. Tau discharges its functions by producing multiple isoforms via intricately regulated alternative splicing. These isoforms modulate tau function in normal brain by altering the domains of the protein, thereby influencing its conformation and post-translational modifications and hence its affinity for microtubules and other ligands. Disturbances in tau expression result in disruption of the neuronal cytoskeleton and formation of pathological tau structures (neurofibrillary tangles) found in brains of dementia sufferers. More specifically, aberrations in tau splicing regulation directly cause several neurodegenerative diseases that lead to dementia. This review briefly presents our cumulative knowledge of tau splicing regulation in connection with the alterations in tau splicing seen in neurodegeneration.

Alternative Splicing↗

PubMed2006

Novel IL-15 isoforms generated by alternative splicing are expressed in the intestinal epithelium.

Previous studies have identified mRNA three isoforms encoding interleukin-15 (IL-15) that are produced through differential splicing and encode for the same mature IL-15 protein with two different signal peptides. Our analysis of mouse intestinal epithelial cells revealed two new IL-15 mRNA isoforms generated by different alternative splicing events. In one form (IL-15DeltaE6), exon 6 is absent, and in the second form the first 48 nt of exon 7 are absent (IL-15DeltaE7) through usage of an alternative 5' splicing site within exon 7. These mRNA isoforms encoded in-frame IL-15 protein variants lacking either 15aa (IL-15DeltaE6) or 16aa (IL-15DeltaE7) both utilizing the normal long signal peptide. Significant structural changes were predicted for these new IL-15 isoforms. RNAse protection assays revealed the highest expression of isoform mRNA in the intestinal epithelium and functional analysis of recombinant IL-15 isoform proteins suggested possible regulatory functions.

Alternative Splicing↗

PubMed1990

Alternative splicing of the transcript encoding the human muscle isoenzyme of phosphofructokinase.

The genomic DNA encoding human muscle phosphofructokinase (HPFK-M) exons VII to X has been cloned and the coding regions have been sequenced. The intron/exon boundaries are located at the same positions as those identified for the rabbit phosphofructokinase-M gene (Lee, C. -P., Kao, M. -C., French, B. A., Putney, S. D., and Chang, S. H. (1987) J. Biol. Chem. 262, 4195-4199). A HPFK-M cDNA clone lacking the sequences corresponding to exon IX was isolated from a human fibroblast (IMR-90) library, suggesting that the HPFK-M transcript may be alternatively spliced. Exon IX is 93 nucleotides in length, and the absence of this sequence from the HPFK-M transcript would generate an RNA coding for a HPFK-M-related polypeptide lacking 31 amino acids compared with the HPFK-M polypeptide. HPFK-M transcripts approximately 3.0 kilobases in length are expressed in a tissue-specific manner with high levels in cell lines and skeltal muscle tissue and very low levels in peripheral blood mononuclear cells and liver tissue. Characterization of the structure of these HPFK-M transcripts by nuclease S1 and polymerase chain reaction analysis demonstrated that all the cell lines and tissues examined expressed the alternatively spliced transcript in addition to the transcript coding for the enzymatically functional HPFK-M polypeptide.

Amino Acid Sequence↗

PubMed1999

Regulation of alternative splicing by RNA editing.

The enzyme ADAR2 is a double-stranded RNA-specific adenosine deaminase which is involved in the editing of mammalian messenger RNAs by the site-specific conversion of adenosine to inosine. Here we identify several rat ADAR2 mRNAs produced as a result of two distinct alternative splicing events. One such splicing event uses a proximal 3' acceptor site, adding 47 nucleotides to the ADAR2 coding region, changing the predicted reading frame of the mature ADAR2 transcript. Nucleotide-sequence analysis of ADAR2 genomic DNA revealed the presence of adenosine-adenosine (AA) and adenosine-guanosine (AG) dinucleotides at these proximal and distal alternative 3' acceptor sites, respectively. Use of the proximal 3' acceptor depends upon the ability of ADAR2 to edit its own pre-mRNA, converting the intronic AA to an adenosine-inosine (AI) dinucleotide which effectively mimics the highly conserved AG sequence normally found at 3' splice junctions. Our observations indicate that RNA editing can serve as a mechanism for regulating alternative splicing and they suggest a novel strategy by which ADAR2 can modulate its own expression.

Adenosine↗

PubMed1997

Sequences of exon 6 and the adjacent intron boundaries of the rat platelet-derived growth factor A-chain gene: implications for alternative splicing.

Platelet-derived growth factor (PDGF) is a potent stimulator of vascular smooth muscle cell growth. Two isoforms of PDGF A-chain mRNA that either include (long form) or exclude (short form) exon 6 are produced as a result of alternative splicing in mouse, rabbit, and human. The short form of PDGF A-chain is expressed in both resting and activated cells, while the long form is present predominantly in activated cells. Reverse transcription-polymerase chain reaction analysis with primers encompassing exon 6 revealed the presence of both long- and short-form PDGF A-chain transcripts in rat vascular smooth muscle cells. The nucleotide sequences of exon 6 and its intron boundaries were determined from rat vascular smooth muscle cell cDNA and rat leukocyte genomic DNA. Translation of the long form of PDGF A-chain mRNA was shown to terminate in the 70-base pair exon 6. Conserved sequences that may contribute to the regulation of alternative RNA splicing were identified in intron 5.

Alternative Splicing↗

PubMed2005

Characterisation of the mulibrey nanism-associated TRIM37 gene: transcription initiation, promoter region and alternative splicing.

The TRIM37 gene encodes a peroxisomal protein of unknown function. Mutations in TRIM37 underlie mulibrey nanism, a rare autosomal recessively inherited disorder with severe growth failure of prenatal onset, constrictive pericardium, hepatomegaly and characteristic dysmorphic features. Eleven mulibrey nanism-associated mutations have been identified. We here characterised TRIM37 further by mapping the transcription initiation site and promoter region as well as by analysing splice variants. By primer extension analysis, several transcription initiation sites were localised to a region between -246 and -373 relative to the ATG codon for translation initiation. Basal promoter activity was mapped within 600 nucleotides upstream from the translation initiation site using promoter-luciferase reporter constructs. Several alternative splice variants of TRIM37 exist in databases. Most of these predict non-functional protein products, are expressed at low levels and are thus likely to be targets for nonsense-mediated mRNA decay. A novel splice variant, TRIM37b, with an alternative termination codon and 3'untranslated region (UTR) transcribed from an exon 16 kb downstream from exon 24, predicts an identical protein product with the previously identified transcript, TRIM37a. As seen by Northern blot analysis and quantitative real-time PCR, both transcripts are highly expressed in testis, whereas in other tissues TRIM37a is prominent. The 3'UTR of the PPM1E gene in the opposite strand overlaps TRIM37b. These data suggest that TRIM37 expression is regulated by several mechanisms: through nonsense surveillance of non-functional transcripts, as well as through 3'UTR regulatory sequences and/or naturally occurring antisense RNAs especially in testis.

3' Untranslated Regions↗

PubMed2003

The circadian clock regulated RNA-binding protein AtGRP7 autoregulates its expression by influencing alternative splicing of its own pre-mRNA.

The clock-regulated RNA-binding protein AtGRP7 is part of a negative feedback circuit through which the protein influences circadian oscillations of its own transcript. Constitutive overexpression of AtGRP7 in transgenic plants leads to the appearance of a low amount of an alternatively spliced Atgrp7 transcript with a premature stop codon. It is generated by the use of a 5' cryptic splice site in the middle of the intron at the expense of the fully spliced mRNA, indicating a role for AtGRP7 in splice site selection. Accelerated decay of this transcript accounts for its low steady state abundance. This implicates a mechanism for the AtGRP7 feedback loop: Atgrp7 expression is downregulated, as AtGRP7 protein accumulates over the circadian cycle, partly by the generation of an alternate transcript that due to its instability does not accumulate to high levels and does not produce a functional protein. Recombinant AtGRP7 protein specifically interacts with the 3' untranslated region and the intron of its transcript, suggesting that the shift in splice site selection and downregulation involves binding of AtGRP7 to its pre-mRNA. AtGRP7 also influences the choice of splice sites in the Atgrp8 transcript encoding a related RNA-binding protein, favoring the production of an alternatively spliced, unstable Atgrp8 transcript. This conservation points to the importance of this regulatory mechanism to control the level of the clock-regulated glycine-rich RNA-binding proteins and shows how AtGRP7 can control abundance of target transcripts.

Alternative Splicing↗

PubMed1991

Alternative splicing of human VCAM-1 in activated vascular endothelium.

Vascular cell adhesion molecule 1 (VCAM-1)/inducible cell adhesion molecule 110 is a mononuclear leukocyte-selective adhesion molecule, expressed on vascular endothelium following activation by certain cytokines or endotoxin. This inducible transmembrane protein and member of the immunoglobulin gene superfamily was previously reported to contain six immunoglobulinlike domains. Using the polymerase chain reaction, a VCAM-1 cDNA was obtained from mRNA of interleukin-1 (IL-1)-treated cultured human umbilical vein endothelial cells (HUVEC). The cDNA clone contained an additional 276 base-pair (bp) domain, located between domains 3 and 4. This new domain is most homologous to the existing N-terminal domain (domain 1). The internal 276-bp region is encoded by a single exon of the human VCAM-1 gene, indicating that the two forms of mRNA arise by alternative splicing. Both forms of VCAM-1 mRNA were detected by polymerase chain reaction in IL-1-stimulated HUVEC, although the seven-domain form appeared predominant. On the surface of HUVEC only a 110-kd polypeptide, consistent with the seven-immunoglobulinlike domain form of VCAM-1, was detectable by immunoprecipitation. Alternative splicing of the VCAM-1 gene in cytokine-activated endothelium may generate functionally distinct cell-surface adhesion molecules.

Amino Acid Sequence↗

PubMed1995

Alternative splicing restricts translation of rearrangements at the T-cell receptor delta/alpha locus.

Lymphocytes expressing alpha beta or gamma delta T-cell receptors (TCR) represent distinct T-cell populations. Because TCR delta genes lie within the TCR alpha locus, the rearrangement processes, transcription, and translation of TCR delta or TCR alpha variable domain exons require tight regulation. Human precursor B-cell leukemias (eg, the REH cell line) constitute an interesting model to study TCR delta/alpha recombination because they rearrange TCR delta/alpha loci along a hierarchically ordered pathway in which V delta 2D delta 3 segments are joined to the J alpha cluster. We now show for REH cells that chimeric TCR delta/alpha variable domain exons are posttranscriptionally modified by alternative splicing resulting in truncated V delta 2C alpha transcripts. This process also takes place during thymic differentiation. CD7+/CD3- T-cell precursors exhibit V delta 2D delta 3 rearrangements. Further differentiation into CD7+/CD3+ thymocytes is associated with the expression of a truncated V delta 2C alpha RNA species. In contrast, chimeric TCR delta/alpha rearrangements containing a V delta 1 segment (but no D delta sequences) are predominantly expressed as full-length V delta 1J alpha C alpha transcripts. These data suggest that alternative splicing constitutes a mechanism that restricts the production of distinct chimeric TCR alpha chains.

Antigens, CD↗

PubMed2002

Alternative splicing modulates the frequency-dependent response of CaMKII to Ca(2+) oscillations.

Ca(2+) oscillations are required in various signal trans duction pathways, and contain information both in their amplitude and frequency. Remarkably, the Ca(2+)/calmodulin(CaM)-dependent protein kinase II (CaMKII) can decode such frequencies. A Ca(2+)/CaM-stimulated autophosphorylation leads to Ca(2+)/CaM-independent (autonomous) activity of the kinase that outlasts the initial stimulation. This autonomous activity increases exponentially with the frequency of Ca(2+) oscillations. Here we show that three beta-CaMKII splice variants (beta(M), beta and beta(e)') have very similar specific activity and maximal autonomy. However, their autonomy generated by Ca(2+) oscillations differs significantly. A mechanistic basis was found in alterations of the CaM activation constant and of the initial rate of autophosphorylation. Structurally, the splice variants differ only in a variable 'linker' region between the kinase and association domains. Therefore, we propose that differences in relative positioning of kinase domains within multimeric holoenzymes are responsible for the observed effects. Notably, the beta-CaMKII splice variants are differentially expressed, even among individual hippocampal neurons. Taken together, our results suggest that alternative splicing provides cells with a mechanism to modulate their sensitivity to Ca(2+) oscillations.

Alternative Splicing↗

PubMed1988

Thyroid hormone receptor alpha isoforms generated by alternative splicing differentially activate myosin HC gene transcription.

Thyroid hormones are thought to modulate gene expression positively or negatively through interactions with chromatin-associated receptors. Recently, the c-erb A proto-oncogene products have been shown to be nuclear thyroid hormone (T3) receptors (TR) by sequence similarity with other steroid receptors and by their ability to bind thyroid hormone. But it has not been shown that these receptors directly activate transcription of the responsive genes in vivo. In addition, the rat TR alpha gene encodes several messenger RNA (mRNA) species, generated by differential processing of its transcripts (ref. 22). For these reasons we investigated the ability of two major isoforms of the rat TR alpha gene products to activate transcription of a sarcomeric myosin heavy chain (mHC) gene, because expression of all members of this gene family is responsive to T3. We show here that the rTR alpha 1 receptor is a thyroid hormone-dependent transcriptional factor, which upon binding the T3 responsive element of the alpha-mHC gene, activates expression of this gene in vivo. The rTR alpha 2 isoform, which is identical to rTR alpha 1 except for its carboxyl terminal portion, is generated by alternative splicing of the rTR alpha gene transcript. This peptide, when produced in vitro and in vivo failed to bind T3 or other hormones or to trans-activate alpha-mHC gene expression. Thus, alternative splicing can produce marked differences in the functional properties of a transcriptional factor.

Acetyltransferases↗

PubMed1990

Alternative splicing of mRNA encoding rat liver cytochrome P450e (P450IIB2).

Cytochrome P450e (P450IIB2) is a phenobarbital(PB)-inducible member of the rat liver P450IIB subfamily. Among P450 cDNA clones previously isolated from a cDNA library made from the liver of a single rat were several that contained P450e inserts, including PB13, PB16, and PB22. By nucleotide sequence analysis, the PB16 and PB22 inserts have now been found to contain an additional 24-bp segment not present in the PB13 insert or in previously reported P450e-coding sequences. According to the published P450e genomic sequence, the 24-bp segment is exactly at the junction of the fifth and the sixth exons and its sequence is identical to the first 24 bp of the fifth intron. Translation of this segment would add 8 amino acid residues to the P450e protein. To detect the alternatively spliced P450e mRNA, a synthetic oligodeoxyribonucleotide (oligo) corresponding to 18 of the 24 bp of the intronic sequence found in the PB16 and PB22 inserts was made. This oligo hybridized with a 2.1-kb RNA on Northern blots of liver RNA from PB- or Aroclor 1254-treated rats. Taken together, these results indicate that individual rats can possess both forms of P450e mRNA and that an alternative splicing mechanism is responsible for their formation.

Amino Acid Sequence↗

PubMed1998

Genomic architecture and transcriptional activation of the mouse and human tumor susceptibility gene TSG101: common types of shorter transcripts are true alternative splice variants.

The functional inactivation of the tumor susceptibility gene tsg101 in mouse NIH3T3 cells leads to cell transformation and the formation of metastatic tumors in nude mice. We cloned, mapped and sequenced the mouse tsg101 gene and further identified a processed pseudogene that is 98% identical to the tsg101 cDNA. Based on Northern blot analysis, tsg101 is expressed ubiquitously in mouse tissues. A comparison of the coding region of the mouse tsg101 gene with the human TSG101 cDNA revealed that both the mouse and human gene encode ten additional highly conserved amino acids at the N-terminus. Based on the mouse tsg101 genomic structure, we predicted four additional introns within the human TSG101 gene. Their location was confirmed using PCR and sequencing analysis. The presence of these so far unidentified introns now explains published data on aberrantly spliced mRNA products that were frequently observed in primary breast tumors. We show that a majority of shorter TSG101 transcripts are not the result of aberrant splicing events, but represent a fraction of true alternative splice variants. Finally, we examined tsg101 expression patterns during different stages of mammary gland development and in different transgenic mouse models for breast tumorigenesis.

3T3 Cells↗

PubMed1991

Complex pattern of alternative splicing generates unusual diversity in the leader sequence of the chicken link protein mRNA.

We report here the isolation of the 5' end and the promoter region of the gene for chicken cartilage link protein, and demonstrate extensive heterogeneity of the leader sequence arising from differential utilization of multiple splice sites within the 5'-most exon. The 500-base pairs (bp) exon 1 consists of solely untranslated sequence and is followed by an intron greater than 33 kilobase pairs (kb). Together, the five exons predict a gene size longer than 100 kb. Multiple transcription initiation sites were mapped 34, 46, 56, 66 and 76 bp downstream of a TATA-like motif. Sequence analysis revealed that in addition to the non-spliced variant, multiple mRNA species were generated by alternative splicing resulting in the exclusion of 92, 166, 170, 174 and 263 nucleotides (nt), respectively, from exon 1. Polymerase chain reaction confirmed the existence of various splice forms, and showed cell type- and developmental stage-specific expression for one group of them. Secondary structure predictions indicated that the leaders of the splice forms could form stable hairpin structures with different free energies of formation (up to delta G = -110 kcal/mol), suggesting translational control. The splice variant detected in the largest amount had the least stable predicted hairpin (delta G = -31.7 kcal/mol).

Animals↗

PubMed1992

Molecular cloning and sequencing of an alternatively spliced form of the human thyrotropin receptor transcript.

In the present study, we report the molecular cloning and sequencing of an alternatively spliced form of the human thyrotropin receptor (hTSHR) mRNA transcript, which has previously been detected on Northern blot analysis of human thyroid cells. The smaller hTSHR cDNA, designated hTSHR cDNA-I, is approximately 1 kb in size and encodes a protein of 253 amino acids. Comparison of the nucleotide sequence of hTSHR cDNA-I with available hTSHR genomic sequence data reveals that the cDNA-I contains exons 1-8 and unidentified DNA tract, presumably an intron. Thus, the hTSHR cDNA-I encodes for the N-terminal half of the extracellular domain of the hTSHR (approximately 60%). The truncated TSHR-I may be secreted and function as a TSH binding protein.

Alternative Splicing↗

PubMed2004

Multifocal transitional cell carcinoma of the bladder and upper urinary tract: molecular screening of clonal origin by characterizing CD44 alternative splicing patterns.

PURPOSE: CD44 is a widely expressed cell surface adhesion molecule in which various isoforms arise from alternative RNA splicing mechanism during cancer initiation. We assessed whether multifocal transitional cell carcinoma of the urothelium is due to field change and/or intraluminal seeding and implantation. MATERIALS AND METHODS: In a series of 24 patients with synchronous and/or metachronous multiple urothelial cancers we performed reverse transcription-polymerase chain reaction analysis using a set of primers capable of amplifying all CD44 splice variant isoforms. After polymerase chain reaction products were electrophoresed band intensities with areas corresponding to the major isoforms (that is CD44s, CD44v10 and CD44v8-10) were quantified, and CD44v10-to-CD44s and CD44v8-10-to-CD44s ratios were calculated. Moreover, p53 gene mutations in exons 4 to 11 were screened by direct DNA sequencing. RESULTS: Of these 24 cases 18 showed similar CD44v10-to-CD44s and CD44v8-10-to-CD44s ratios in among multiple urothelial cancers in each case. However, in the remaining 6 cases these ratios were quite different among multiple cancer lesions. Furthermore, different types of p53 mutation were detected among multiple cancer lesions in only 2 of 24 cases, which also indicated different patterns of CD44 alternative splicing. CONCLUSIONS: These findings suggest that at least some multiple transitional cell carcinomas of the urothelium seem to be of independent origin based on the analysis of alternative RNA splicing of CD44. Moreover, this hypothesis was further supported by the evaluation of p53 gene mutation.

Alternative Splicing↗

PubMed1991

Human liver glucokinase gene: cloning and sequence determination of two alternatively spliced cDNAs.

A human liver glucokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) cDNA was isolated from a liver cDNA library. This cDNA (hLGLK1) appeared to be full length [2548 base pairs (bp) plus additional poly(A) residues], as its size was consistent with a single 2.8-kilobase (kb) glucokinase mRNA on Northern blot analysis of liver poly(A)+ RNA. The cDNA contained an open reading frame of 1392 bp that predicted a protein of 464 amino acids and a molecular mass of 52 kDa; this protein has 97% identity to rat liver glucokinase. Fourteen residues on the amino terminus of the predicted human liver glucokinase, however, differed completely from those of the predicted rat liver enzyme and could be explained by alternative splicing of a 124-bp cassette exon in human cDNA. A second glucokinase cDNA (hLGLK2), missing the 124-bp cassette exon, was isolated by PCR amplification of human liver cDNA. The hLGLK2 cDNA contained an open reading frame of 1398 bp from an ATG codon at position 164, encoding a predicted protein of 466 residues, 98% identical to the rat enzyme, but different from the predicted protein of hLGLK1 cDNA by 16 amino-terminal residues. In contrast, hLGLK1 cDNA contains multiple initiator codons upstream of the predicted initiator codon at position 294 within the cassette exon. Translation of the two mRNAs in vitro by a reticulocyte lysate system resulted in proteins of the expected size (52 kDa) for both mRNAs; yet hLGLK2 mRNA was translated four to six times more efficiently. These results suggested that the alternative splicing of a cassette exon in hLGLK1 resulted in an mRNA with an upstream initiator codon and reduced function. The relative biological activity of the two isoforms of human glucokinase and their possible developmental and/or metabolic regulation remain to be determined.

Amino Acid Sequence↗

PubMed1999

Characterization of the human and mouse ETV1/ER81 transcription factor genes: role of the two alternatively spliced isoforms in the human.

The Ets transcription factors of the PEA3 group--E1AF/PEA3, ETV1/ER81 and ERM--are almost identical in the ETS DNA-binding and the transcriptional acidic domains. To accelerate our understanding of the molecular basis of putative diseases linked to ETV1 such as Ewing's sarcoma we characterized the human ETV1 and the mouse ER81 genes. We showed that these genes are both encoded by 13 exons in more than 90 kbp genomic DNA, and that the classical acceptor and donor splicing sites are present in each junction except for the 5' donor site of intron 9 where GT is replaced by TT. The genomic organization of the ETS and acidic domains in the human ETV1 and mouse ER81 (localized to chromosome 12) genes is similar to that observed in human ERM and human E1AF/PEA3 genes. Moreover, as in human ERM and human E1AF/PEA3 genes, a first untranslated exon is upstream from the first methionine, and the mouse ER81 gene transcription is regulated by a 1.8 kbp of genomic DNA upstream from this exon. In human, the alternative splicing of the ETV1 gene leads to the presence (ETV1 alpha) or the absence (ETV1 beta) of exon 5 encoding the C-terminal part of the transcriptional acidic domain, but without affecting the alpha helix previously described as crucial for transactivation. We demonstrated here that the truncated isoform (human ETV1 beta) and the full-length isoform (human ETV1 alpha) bind similarly specific DNA Ets binding sites. Moreover, they both activate transcription similarly through the PKA-transduction pathway, so suggesting that this alternative splicing is not crucial for the function of this protein as a transcription factor. The comparison of human ETV1 alpha and human ETV1 beta expression in the same tissues, such as the adrenal gland or the bladder, showed no clear-cut differences. Altogether, these data open a new avenue of investigation leading to a better understanding of the functional role of this transcription factor.

Amino Acid Sequence↗