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PubMed1996

Analysis of alternative splicing patterns in the cystic fibrosis transmembrane conductance regulator gene using mRNA derived from lymphoblastoid cells of cystic fibrosis patients.

Using in vitro amplification of cDNA by the polymerase chain reaction, we analyzed alternatively spliced events of cystic fibrosis transmembrane conductance regulator gene in lymphoblastoid cells. Ten alternatively spliced transcripts were identified using analysis of 6 overlapping segments of amplified cDNA, 4 of which have not been described previously. These include transcripts lacking exon 16, 17b, 22 and a transcript resulting from the use of a cryptic acceptor and donor splice sites. Moreover, in 2 cystic fibrosis (CF) patients bearing nonsense mutations E60X or W1282X, we observed that nonsense mutations are associated with an alteration of splice site selection in vivo resulting in exon skipping of constitutive exons or in the use of cryptic splice sites. In addition, even though lymphoblastoid cells are not the relevant tissue to address the question of the relationship between clinical respiratory phenotype and genotype, our results concerning adult CF patients (delta F508/ delta F508) suggest that individual-specific RNA splicing patterns could influence the severity of the CF pulmonary disease. If this phenomenon of alternative splicing events proves to be significant in CF and to be a common feature of disease genes, the study of RNA splicing could become an important tool for the analysis of the genotype-phenotype relationship in many inherited disorders.

Adult↗
PubMed1997

Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy.

The mechanism of exon skipping induced by nonsense mutations has not been well elucidated. We now report results of in vitro splicing studies which disclosed that a particular example of exon skipping is due to disruption of a splicing enhancer sequence located within the exon. A nonsense mutation (E1211X) due to a G to T transversion at the 28th nucleotide of exon 27 (G3839T) was identified in the dystrophin gene of a Japanese Becker muscular dystrophy case. Partial skipping of the exon resulted in the production of truncated dystrophin mRNA, although the consensus sequences for splicing at both ends of exon 27 were unaltered. To determine how E1211X induced exon 27 skipping, the splicing enhancer activity of purine-rich region within exon 27 was examined in an in vitro splicing system using chimeric doublesex gene pre-mRNA. The mutant sequence containing G3839T abolished splicing enhancer activity of the wild-type purine-rich sequence for the upstream intron in this chimeric pre-mRNA. An artificial polypurine oligonucleotide mimicking the purine-rich sequence of exon 27 also showed enhancer activity that was suppressed by the introduction of a T nucleotide. Furthermore, the splicing enhancer activity was more markedly inhibited when a nonsense codon was created by the inserted T residue. This is the first evidence that partial skipping of an exon harboring a nonsense mutation is due to disruption of a splicing enhancer sequence.

Adult↗
PubMed2005

Intronic splicing of hyaluronan synthase 1 (HAS1): a biologically relevant indicator of poor outcome in multiple myeloma.

In this study, we show that the hyaluronan synthase 1 (HAS1) gene undergoes aberrant intronic splicing in multiple myeloma (MM). In addition to HAS1 full length (HAS1(FL)), we identify 3 novel splice variants of HAS1, HAS1Va, HAS1Vb, and HAS1Vc, detected in patients with MM or monoclonal gammopathy of undetermined significance (MGUS). HAS1Vb and HAS1Vc undergo intronic splicing with creation of a premature stop codon. MM cells expressing one or more HAS1 variants synthesize extracellular and/or intracellular hyaluronan (HA). Expression of the HAS1Vb splice variant was significantly correlated with reduced survival (P = .001). Together, alternative HAS1 gene splicing, the correlations between HAS1 splicing and HA synthesis, and the correlations between HAS1 splicing and reduced survival of MM patients support the hypothesis that the family of HAS1 protein plays a significant role in disease progression. Further, expression of HAS1Vb, in conjunction with HAS1(FL) and/or other HAS1 variants, may lead to accumulation of intracellular HA molecules and an impact on receptor for HA-mediated motility (RHAMM)-mediated mitotic abnormalities in MM. This study highlights the potential importance of HAS1 and its alternative splicing in pathophysiology of MGUS and MM.

Alternative Splicing↗
PubMed2002

Functional role of alternatively spliced deoxycytidine kinase in sensitivity to cytarabine of acute myeloid leukemic cells.

Development of resistance to cytarabine (AraC) is a major problem in the treatment of patients with acute myeloid leukemia (AML). Inactivation of deoxycytidine kinase (dCK) plays an important role in AraC resistance in vitro. We have identified inactive, alternatively spliced dCK forms in leukemic blasts from patients with resistant AML. Because these dCK-spliced variants were only detectable in resistant AML, it was hypothesized that they might play a role in AraC resistance in vivo. In the current study, the biologic role of the alternatively spliced dCK forms in AraC resistance was further investigated by retroviral transductions in rat leukemic cells. Introduction of inactive, alternatively spliced dCK forms into AraC-resistant K7 cells, with no endogenous wild-type (wt) dCK activity, could not restore AraC sensitivity, whereas wt dCK fully restored the AraC-sensitive phenotype. Transfection of alternatively spliced dCK forms into AraC-sensitive KA cells, as well as in human leukemic U937 cells and in phytohemagglutinin-stimulated T cells, did not significantly change sensitivity toward AraC. In addition, cotransduction of wt dCK with alternatively spliced dCK in K7 cells did not result in altered sensitivity to AraC compared with K7 cells only transduced with wt dCK. These data indicate that the alternatively spliced dCK forms cannot act as a dominant-negative inhibitor on dCK wt activity when they are coexpressed in a single cell. However, a cell expressing alternatively spliced dCK forms that has lost wt dCK expression is resistant to the cytotoxic effects of AraC.

Acute Disease↗
PubMed2006

Assessing the number of ancestral alternatively spliced exons in the human genome.

BACKGROUND: It is estimated that between 35% and 74% of all human genes undergo alternative splicing. However, as a gene that undergoes alternative splicing can have between one and dozens of alternative exons, the number of alternatively spliced genes by itself is not informative enough. An additional parameter, which was not addressed so far, is therefore the number of human exons that undergo alternative splicing. We have previously described an accurate machine-learning method allowing the detection of conserved alternatively spliced exons without using ESTs, which relies on specific features of the exon and its genomic vicinity that distinguish alternatively spliced exons from constitutive ones. RESULTS: In this study we use the above-described approach to calculate that 7.2% (+/- 1.1%) of all human exons that are conserved in mouse are alternatively spliced in both species. CONCLUSION: This number is the first estimation for the extent of ancestral alternatively spliced exons in the human genome.

Algorithms↗
PubMed2004

Analysis of Maxi-K alpha subunit splice variants in human myometrium.

BACKGROUND: Large-conductance, calcium-activated potassium (Maxi-K) channels are implicated in the modulation of human uterine contractions and myometrial Ca2+ homeostasis. However, the regulatory mechanism(s) governing the expression of Maxi-K channels with decreased calcium sensitivity at parturition are unclear. The objectives of this study were to investigate mRNA expression of the Maxi-K alpha subunit, and that of its splice variants, in human non-pregnant and pregnant myometrium, prior to and after labour onset, to determine whether altered expression of these splice variants is associated with decreased calcium sensitivity observed at labour onset. METHODS: Myometrial biopsies were obtained at hysterectomy (non-pregnant, NP), and at Caesarean section, at elective (pregnant not-in-labour, PNL) and intrapartum (pregnant in-labour, PL) procedures. RNA was extracted from all biopsies and quantitative real-time RT-PCR was used to investigate for possible differential expression of the Maxi-K alpha subunit, and that of its splice variants, between these functionally-distinct myometrial tissue sets. RESULTS: RT-PCR analysis identified the presence of a 132 bp and an 87 bp spliced exon of the Maxi-K alpha subunit in all three myometrial tissue sets. Quantitative real-time PCR indicated a decrease in the expression of the Maxi-K alpha subunit with labour onset. While there was no change in the proportion of Maxi-K alpha subunits expressing the 87 bp spliced exon, the proportion of alpha subunits expressing the 132 bp spliced exon was significantly increased with labour onset, compared to both non-pregnant and pregnant not-in-labour tissues. An increased proportion of 132 bp exon-containing alpha subunit variants with labour onset is of interest, as channels expressing this spliced exon have decreased calcium and voltage sensitivities. CONCLUSIONS: Our findings suggest that decreased Maxi-K alpha subunit mRNA expression in human myometrium at labour onset, coupled to an increased proportion of Maxi-K channels expressing the 132 bp spliced exon, may be linked to decreased Maxi-K channel calcium and voltage sensitivity, thereby promoting enhanced uterine activity at the time of labour.

Adult↗
PubMed2006

A statistical method for predicting splice variants between two groups of samples using GeneChip expression array data.

BACKGROUND: Alternative splicing of pre-messenger RNA results in RNA variants with combinations of selected exons. It is one of the essential biological functions and regulatory components in higher eukaryotic cells. Some of these variants are detectable with the Affymetrix GeneChip that uses multiple oligonucleotide probes (i.e. probe set), since the target sequences for the multiple probes are adjacent within each gene. Hybridization intensity from a probe correlates with abundance of the corresponding transcript. Although the multiple-probe feature in the current GeneChip was designed to assess expression values of individual genes, it also measures transcriptional abundance for a sub-region of a gene sequence. This additional capacity motivated us to develop a method to predict alternative splicing, taking advance of extensive repositories of GeneChip gene expression array data. RESULTS: We developed a two-step approach to predict alternative splicing from GeneChip data. First, we clustered the probes from a probe set into pseudo-exons based on similarity of probe intensities and physical adjacency. A pseudo-exon is defined as a sequence in the gene within which multiple probes have comparable probe intensity values. Second, for each pseudo-exon, we assessed the statistical significance of the difference in probe intensity between two groups of samples. Differentially expressed pseudo-exons are predicted to be alternatively spliced. We applied our method to empirical data generated from GeneChip Hu6800 arrays, which include 7129 probe sets and twenty probes per probe set. The dataset consists of sixty-nine medulloblastoma (27 metastatic and 42 non-metastatic) samples and four cerebellum samples as normal controls. We predicted that 577 genes would be alternatively spliced when we compared normal cerebellum samples to medulloblastomas, and predicted that thirteen genes would be alternatively spliced when we compared metastatic medulloblastomas to non-metastatic ones. We checked the consistency of some of our findings with information in UCSC Human Genome Browser. CONCLUSION: The two-step approach described in this paper is capable of predicting some alternative splicing from multiple oligonucleotide-based gene expression array data with GeneChip technology. Our method employs the extensive repositories of gene expression array data available and generates alternative splicing hypotheses, which can be further validated by experimental studies.

Algorithms↗
PubMed1994

Characterization and relative abundance of alternatively spliced luteinizing hormone receptor messenger ribonucleic acid in the ovine ovary.

Complementary DNA (cDNA) clones encoding the LH receptor (LHR) were recently isolated from pig, rat, mouse, and human testes or ovaries. Many of the LHR cDNAs isolated from these species encoded incomplete and, therefore, possibly inactive forms of the LHR. The four major incomplete cDNAs, designated B, C, D, and E, were due to alternative splicing of the full-length cDNA, designated the A form. Northern analyses of messenger RNA (mRNA) encoding LHR in these species and in sheep revealed multiple mRNA species in ovarian tissue, but were unable to distinguish between the full-length (functional) form and the splice variants. We have used reverse transcription of mRNA, amplification via the polymerase chain reaction, and cDNA sequencing to determine which alternatively spliced mRNA species were present in ovine ovarian follicles and corpora lutea, and ribonuclease protection assays to confirm these results and determine the relative abundance of these splice variants. Ovine LHR cDNAs of the full-length A form, B form, and two novel splice forms, designated F and G, were isolated and sequenced. By using LHR cDNAs that spanned the regions of the gene in which the majority of splicing variation occurred, ribonuclease-protected fragments of different sizes were generated depending on which mRNA species (A-G) were present. It is estimated that the ratios of the steady state mRNA levels of the splice variant B form/full-length A form/G form/F form were 5-3.5:1:1:0.3. The E, C, and D forms were not detected, even when using the sensitive method of reverse transcription-polymerase chain reaction for the latter two forms. The overall level of expression of LHR mRNA was greater in corpora lutea than follicles, but the relative abundance of the splice variants was similar in follicles and corpora lutea.

Alternative Splicing↗
PubMed1995

An alternatively spliced human insulin-like growth factor-I transcript with hepatic tissue expression that diverts away from the mitogenic IBE1 peptide.

An alternatively spliced transcript of the human insulin-like growth factor-I (IGF-I) gene is described. The transcript was identified in human liver RNA by reverse transcriptase-polymerase chain reaction, cloning, and sequencing. It contained IGF-I exons 3 and 4, 49 basepairs of exon 5, then exon 6 (exon 4-5-6). The 5'-donor site at the exon 5-6 junction was a cryptic 5'-donor splice site (IGF633). The 3'-acceptor site of the splice was the usual intron-exon 6 junction. A second pair of primers across the exon 5-exon 6 junction was used to confirm the presence of the transcript by reverse transcriptase-polymerase chain reaction. Cloning and sequencing this second fragment confirmed the presence of this splice in human liver. The exon 4-5-6 transcript was quantified at about 10% relative to the exon 4-6 transcript in human livers (n = 7 subjects), but was not detected in other tissues. The exon 4-5-6 transcript was found in cultured human hepatoma HepG2 cells and increased, relative to exon 4-6 transcripts, in response to GH, but not in cultured human lymphoblast IM-9 cells. The exon 4-5-6 splice predicts a prepro-IGF-I of 158 amino acid residues, with an E-peptide sequence of 24 residues (Ec). The deduced Ec peptide sequence is 73% homologous to the rat Eb-peptide sequence. The predicted final residues of the Ec peptide are frameshifted exon 6 codons ending in an in-frame stop codon. The predicted peptide sequences of Ec and Eb differ at the cleavage site of the Eb-peptide fragment (IBE1), which has been shown to have mitogenic activity. These data suggest that 1) the exon 4-5-6 splice has hepatic tissue expression and occurs by the use of a cryptic 5'-donor consensus splice site (IGF633) in exon 5; 2) exon 4-5-6 can be hormonally regulated in cultured human HepG2 cells; 3) exon 4-5-6 is the human counterpart of the rat IGF-IEb, because the complementary DNA and predicted sequences are homologous; and 4) the production of IBE1 is potentially regulated by alternative splicing.

Alternative Splicing↗
PubMed1992

Regulation of alternative splicing in the IIICS region of human fibronectin pre-mRNA encoding cell binding sites CS1 and CS5.

The cell binding sites CS1 and CS5 in the IIICS region of human fibronectin (FN) mediate the adhesion of specific cell types by interacting with the integrin alpha 4 beta 1. IIICS pre-mRNA is alternatively spliced via the use of three alternative splice acceptor sites and one alternative splice donor site. These alternative splicing pathways can potentially give rise to variant FN molecules which are CS1+,CS5+; CS1+,CS5-; CS1-,CS5+ or CS1-,CS5-. Here we show that selection of the acceptor site which incorporates mRNA encoding CS1 and CS5 is more frequent in foetal tissues compared to adult liver, whereas an alternative acceptor site and the alternative donor site, which exclude CS1 and CS5, are used at a higher level in adult liver compared to foetal tissue. All possible splice junctions were accurately processed, and selected at different levels in mRNA expressed from a IIICS minigene transiently transfected into a HeLa cell line which does not express FN, suggesting that all the cellular factors required for alternative processing of IIICS are present in this system. Furthermore, pre-mRNA expressed from a mutant construct lacking IIICS-1 intron sequence, was correctly processed in HeLa cells via selection of all possible splice sites. On the basis of our results we propose that regulation of splice site selection in IIICS and thus expression of CS1 and CS5 is achieved by subtle tuning of splicing systems involving the interaction of local cis elements and cellular factors which are not necessarily restricted developmentally or tissue-specifically, and that expression of CS1 and CS5 is independently regulated.

Base Sequence↗
PubMed2006

A subset of Mer1p-dependent introns requires Bud13p for splicing activation and nuclear retention.

In the yeast Saccharomyces cerevisiae, Mer1p is expressed only during meiosis, and its expression is linked to the splicing of at least three mRNAs: MER2, MER3, and AMA1. Previous evidence suggests that Mer1p activates splicing by directly recruiting snRNPs or stabilizing intermediate splicing complexes formed on pre-mRNA that contains an intronic Mer1p enhancer element. However, some splicing factors, especially accessory/non-snRNP factors, have critical roles in retaining unspliced pre-mRNAs in the nucleus. We tested if Mer1p may indirectly regulate splicing by preventing the export of pre-mRNAs to the cytoplasm and also demonstrated that a second subunit of the Retention and Splicing (RES) complex, Bud13p, has transcript-specific effects on Mer1p-activated splicing. The results indicated that Mer1p can retain unspliced pre-mRNA in the nucleus; however, nuclear retention could not be uncoupled from splicing activation. In the absence of Mer1p, the AMA1 pre-mRNA is exported to the cytoplasm, translated, but not subjected to nonsense-mediated decay (NMD) despite a premature stop codon in the intron. These data imply that Mer1p can retain pre-mRNAs in the nucleus only by facilitating their interaction with the spliceosome and that two subunits of the RES complex modulate Mer1p function on two of the three Mer1p-dependent introns. The results also support models for cytoplasmic degradation of unspliced pre-mRNAs that fail to assemble into spliceosomes in yeast.

Cell Nucleus↗
PubMed2004

Both U2 snRNA and U12 snRNA are required for accurate splicing of exon 5 of the rat calcitonin/CGRP gene.

Two classes of spliceosome are present in eukaryotic cells. Most introns in nuclear pre-mRNAs are removed by a spliceosome that requires U1, U2, U4, U5, and U6 small nuclear ribonucleoprotein particles (snRNPs). A minor class of introns are removed by a spliceosome containing U11, U12, U5, U4atac, and U6 atac snRNPs. We describe experiments that demonstrate that splicing of exon 5 of the rat calcitonin/CGRP gene requires both U2 snRNA and U12 snRNA. In vitro, splicing to calcitonin/ CGRP exon 5 RNA was dependent on U2 snRNA, as preincubation of nuclear extract with an oligonucleotide complementary to U2 snRNA abolished exon 5 splicing. Addition of an oligonucleotide complementary to U12 snRNA increased splicing at a cryptic splice site in exon 5 from <5% to 50% of total spliced RNA. Point mutations in a candidate U12 branch sequence in calcitonin/CGRP intron 4, predicted to decrease U12-pre-mRNA base-pairing, also significantly increased cryptic splicing in vitro. Calcitonin/CGRP genes containing base changes disrupting the U12 branch sequence expressed significantly decreased CGRP mRNA levels when expressed in cultured cells. Coexpression of U12 snRNAs containing base changes predicted to restore U12-pre-mRNA base pairing increased CGRP mRNA synthesis to the level of the wild-type gene. These observations indicate that accurate, efficient splicing of calcitonin/CGRP exon 5 is dependent upon both U2 and U12 snRNAs.

Animals↗
PubMed2004

Nonsense-associated alternative splicing of T-cell receptor beta genes: no evidence for frame dependence.

Mutations that generate premature translation-termination codons (PTCs) often result in production of alternatively spliced mRNAs. While in many cases, the PTC-causing mutation was found to affect splicing directly by disrupting an exonic splicing enhancer, induction of alternative splicing of TCR-beta pre-mRNA has been reported to be specific for mutations that prematurely terminate the open reading frame. During testing of a cyto-nuclear feedback model that would have explained how cytoplasmic translation could influence nuclear splicing of TCR-beta transcripts, control experiments questioned the frame dependence of the nonsense-associated altered splicing (NAS) of TCR-beta pre-mRNA. A subsequent detailed analysis of alternatively spliced TCR-beta mRNA expressed from different minigene constructs with nonsense, silent, or frame-shift mutations at various positions revealed no correlation between truncation of the reading frame and production of alternatively spliced mRNA. Our study thus contradicts the previously reported PTC specificity of TCR-beta NAS and points out the need for systematically testing the PTC specificity in other cases where NAS has been observed.

Alternative Splicing↗
PubMed2004

Alternative splicing induced by nonsense mutations in the immunoglobulin mu VDJ exon is independent of truncation of the open reading frame.

In addition to triggering nonsense-mediated mRNA decay (NMD), premature translation-termination codons (PTCs) frequently induce alternative splicing, an observation referred to as nonsense-associated alternative splicing (NAS). In many cases, NAS is induced because the nonsense mutation alters a splicing signal, such as inactivating an exonic splicing enhancer. However, for a few genes, NAS was reported to be PTC specific, implying that a translation signal could influence splicing. Here, we investigated whether production of a previously undetected alternatively spliced transcript from immunoglobulin mu (Ig-mu) depends on premature termination of the open reading frame. We show that PTCs at different positions in the VDJ exon of an Ig-mu minigene activate usage of an alternative 3' splice site, generating an alternative transcript that lacks the initial PTC and a previously identified NMD-promoting element (NPE), but contains new PTCs because of a frame shift. Corroborating the importance of the NPE for maximal NMD response, the alternative transcript is only moderately down-regulated by NMD. We further demonstrate that NAS of Ig-mu minigene transcripts is not PTC specific. This finding, together with our results that contradict the previously reported frame dependence of TCR-beta NAS, challenges the idea that cells might possess mechanisms that would allow regulation of splice site selection in response to premature termination of the ORF.

Alternative Splicing↗
PubMed2006

Unusual intron conservation near tissue-regulated exons found by splicing microarrays.

Alternative splicing contributes to both gene regulation and protein diversity. To discover broad relationships between regulation of alternative splicing and sequence conservation, we applied a systems approach, using oligonucleotide microarrays designed to capture splicing information across the mouse genome. In a set of 22 adult tissues, we observe differential expression of RNA containing at least two alternative splice junctions for about 40% of the 6,216 alternative events we could detect. Statistical comparisons identify 171 cassette exons whose inclusion or skipping is different in brain relative to other tissues and another 28 exons whose splicing is different in muscle. A subset of these exons is associated with unusual blocks of intron sequence whose conservation in vertebrates rivals that of protein-coding exons. By focusing on sets of exons with similar regulatory patterns, we have identified new sequence motifs implicated in brain and muscle splicing regulation. Of note is a motif that is strikingly similar to the branchpoint consensus but is located downstream of the 5' splice site of exons included in muscle. Analysis of three paralogous membrane-associated guanylate kinase genes reveals that each contains a paralogous tissue-regulated exon with a similar tissue inclusion pattern. While the intron sequences flanking these exons remain highly conserved among mammalian orthologs, the paralogous flanking intron sequences have diverged considerably, suggesting unusually complex evolution of the regulation of alternative splicing in multigene families.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Intron retention: a common splicing event within the human kallikrein gene family.

BACKGROUND: All human kallikrein (KLK) genes have at least one splice variant, some of which possess clinical utility in cancer diagnostics/prognostics. Given that introns <100 bp in length are retained in 95% of human genes and that splice variants of KLK3 and KLK4 retain intron III, we hypothesized that other proteins in this family, with a small intron III, may also retain it. METHODS: Variant-specific reverse transcription-PCRs (RT-PCRs) for KLK1, KLK2, KLK5, and KLK15 were used to identify and clone the full coding sequence of intron III-containing splice variants. In addition, variant-specific RT-PCRs for the cloned KLK3 and KLK4 variants as well as for the "classical" forms of the six genes were used to determine their expression profiles in healthy tissues, their regulation by steroids, and their differential expression in prostate cancer. RESULTS: KLK1, KLK2, KLK3, KLK4, KLK5, and KLK15 showed a common type of splice variant in which intron III is retained. Expression profiling of these splice variants revealed expression profiles similar to those of the classical mRNA forms, although the pattern of hormonal regulation was different. The KLK15 splice variant was up-regulated in 8 of 12 cancerous prostate tissues. All encoded variant proteins were predicted to be truncated and catalytically inactive because of a lack of the serine residue of the catalytic triad. CONCLUSIONS: The first six centromeric members of the KLK gene family have splice variants that retain intron III. Some variants show tissue-specific expression. The KLK15 splice variant appears to be a candidate biomarker for prostate cancer.

Alternative Splicing↗
PubMed2006

Mammalian mRNA splice-isoform selection is tightly controlled.

Post-transcriptional RNA processing is an important regulatory control mechanism for determining the phenotype of eukaryotic cells. The processing of a transcribed RNA species into alternative splice isoforms yields products that can perform different functions. Each type of cell in a multi-cellular organism is presumed to actively control the relative quantities of alternative splice isoforms. In this study, the alternatively spliced isoforms of five mRNA transcription units were examined by quantitative reverse transcription-PCR amplification. We show that interindividual variation in splice-isoform selection is very highly constrained when measured in a large population of genetically diverse mice (i.e., full siblings; N = 150). Remarkably, splice-isoform ratios are among the most invariant phenotypes measured in this population and are confirmed in a second, genetically distinct population. In addition, the patterns of splice-isoform selection show tissue-specific and age-related changes. We propose that splice-isoform selection is exceptionally robust to genetic and environmental variability and may provide a control point for cellular homeostasis. As a consequence, splice-isoform ratios may be useful as a practical quantitative measure of the physiological status of cells and tissues.

Age Factors↗
PubMed2003

Triiodothyronine affects the alternative splicing of thyroid hormone receptor alpha mRNA.

The c-erbAalpha gene encodes two thyroid hormone receptors, TRalpha1 and TRalpha2, that arise from alternative splicing of the TRalpha pre-mRNA. TRalpha2 is not able to bind triiodothyronine (T(3)) and acts as a weak antagonist of TRs. It has been suggested that the balance of TRalpha1 to TRalpha2 is important in maintaining homeostasis. Here, we study the effect of thyroid hormone on the splicing of TRalpha under various conditions in HepG2 cells. First, T(3) was added to HepG2 cells that endogenously express TRalpha. This resulted in a decrease in the TRalpha1:TRalpha2 mRNA ratio after the addition of 10(-)(8 )M or 10(-)(7 )M T(3). Then, HepG2 cells were incubated with sera from hypothyroid or hyperthyroid patients. Sera from hyperthyroid patients (n=6) decreased the TRalpha1:TRalpha2 ratio compared with HepG2 cells incubated with sera from euthyroid patients (n=8). Sera from hypothyroid patients (n=6) had no effect on the TRalpha1:TRalpha2 ratio but supplementation with T(3) caused a decrease in the ratio. Finally, we tested sera from patients with nonthyroidal illness (NTI; n=17) which showed no effect on TRalpha splicing when compared with controls. Free thyroxine levels in sera from hypo-, eu-, and hyperthyroid patients, but not that of NTI patients, were negatively correlated (P<0.01) to the TRalpha1:TRalpha2 ratio. We next studied the expression of the splicing factors hnRNP A1 and ASF/SF2 (SF2) in relation to the splicing of the TRalpha gene. In HepG2 cells incubated with NTI sera a negative relationship was found between the ratio of hnRNP A1:SF2 and the TRalpha1:TRalpha2 ratio. A high hnRNP A1:SF2 ratio is associated with the use of the distal 5'-splice site. The splicing direction should then change towards TRalpha2, which is indeed the case. Rev-ErbA, which is partly complementary to TRalpha2 and could therefore interfere in the splicing process, did not relate to the TRalpha1:TRalpha2 ratio. In conclusion, high T(3) levels induce a low TRalpha1:TRalpha2 ratio which could protect the cell from excessive T(3)-induced gene expression. In vivo, this might be a mechanism to keep tIssues relatively euthyroid during high serum T(3) levels.

Alternative Splicing↗