Messenger RNA transcripts coding for progesterone receptor A and B isoforms are expressed in human osteoblast cells.
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Animals↗
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Messenger RNA expression of granulocyte colony-stimulating factor receptor isoforms in the fetus.
Granulocyte colony-stimulating factor (G-CSF) is a growth factor known to support the proliferation, differentiation, and survival of cells of the neutrophil lineage. G-CSF affects these cells after binding to its specific receptor, G-CSF-R, which exists in seven isoforms. While information exists about the distribution of these isoforms in hematopoietic cells and placenta, G-CSF-R isoforms on non-hematopoietic fetal tissues have not been described. Using RT-PCR, we analyzed a variety of human fetal tissues ranging from 6 to 18 weeks gestation. Isoforms I and III were present in all tissues, and the expression of isoform III varied with gestational age. The remaining isoforms were variably expressed in relation to tissue type and gestational age. Thus in the human fetus, G-CSF-R isoform I is the predominant form expressed on non-hematopoietic and hematopoietic tissues.
Fetus↗
Effects of alternate RNA splicing on glucokinase isoform activities in the pancreatic islet, liver, and pituitary.
Different glucokinase isoforms are produced by tissue-specific alternative RNA splicing in the liver and pancreatic islet, the only tissues in which glucokinase activity has been detected. To determine whether differences in protein structure brought about by alternative RNA splicing have an effect on glucose phosphorylating activity, we expressed cDNAs encoding four different hepatic and islet glucokinase isoforms and determined the Km and Vmax of each. When the glucokinase B1 and L1 isoforms were expressed in eukaryotic cells, both high Km glucose phosphorylating activity and immunoreactive protein were detected. However, when the glucokinase B2 and L2 isoforms were expressed, both of which differ by deletion of 17 amino acids in a region between the putative glucose and ATP-binding domains, no high Km glucose phosphorylating activity and much less immunoreactive protein were detected. When the glucokinase B1 and B2 isoforms were expressed in Escherichia coli as fusion proteins with glutathione S-transferase, affinity-purified B1 fusion protein was able to phosphorylate glucose whereas the B2 fusion protein was not, thus indicating that the lack of glucose phosphorylating activity from both the B2 and L2 isoforms is due to lack of intrinsic activity in addition to accumulation of less protein. The Km values of the B1 and L1 isoforms, which differ from each other by 15 amino acids at the NH2 terminus, were similar, but the Vmax of the B1 isoform was 2.8-fold higher than that of the L1 isoform. Mutagenesis of the first two potential initiation codons in the glucokinase B1 cDNA from ATG to GTC (methionine to valine) indicated that the first ATG was crucial for activity and is, therefore, the likely translation initiation codon. Messenger RNAs encoding both the B2 and L2 isoforms of glucokinase were detected in islet and liver by polymerase chain reaction amplification of total cDNA, indicating that mRNAs utilizing this weak alternate splice acceptor site in the fourth exon are normally present in both the liver and islet but as minor components. A regulatory role for weak alternate splice acceptor and donor sites in the glucokinase gene was suggested by examining the expression of the gene in the pituitary and in AtT-20 cells. Interestingly, although glucokinase mRNAs of appropriate sizes were detected in both the AtT-20 cells and rat pituitaries, neither exhibited any detectable high Km glucose phosphorylating activity.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Fourth isoform of preprotachykinin messenger RNA encoding for substance P in the rat intestine.
Three different isoforms of preprotachykinin mRNA (PPT mRNA) encode for substance P and related neuropeptides (1). Here we report a fourth isoform of PPT mRNA which is generated by alternative exclusion of exon-7 and exon-6 from the PPT mRNA. It was present mainly in ileal smooth muscle and mucosa, colon, heart and brain and low level of this mRNA was detected in the jejunal smooth muscle and mucosa. This was not detected in the kidney or uterus. The level of this PPT mRNA was enhanced significantly by 60% during colitis in rat induced by trinitro benzene sulphonic acid.
Alternative Splicing↗
Alterations in uterine sodium pump abundance may contribute to the onset and progression of term and preterm labor in mice.
OBJECTIVE: Other investigators have shown that reductions in active sodium pump units increase uterine contractility. Therefore, our goal was to determine whether uterine sodium pump abundance is decreased in mouse models of term and preterm labor. STUDY DESIGN: Mice were studied during the final one-third of pregnancy. Other pregnant mice had preterm labor induced with lipopolysaccharide and were studied at timed intervals thereafter. Uterine sodium pump alpha3-isoform messenger RNA and protein were measured. Data were analyzed by analysis of variance. RESULTS: Uterine sodium pump alpha3-isoform messenger RNA fell significantly from day 14 to day 18 and remained low on the day of birth. Uterine sodium pump alpha3-isoform protein levels decreased significantly also. In lipopolysaccharide-induced preterm labor, uterine sodium pump alpha3-isoform protein, but not messenger RNA, decreased significantly. CONCLUSION: Sodium pump alpha3-isoform protein levels decreased in uterus before term labor and lipopolysaccharide-induced preterm labor. These findings are similar to those in humans, which suggests that this mouse model may be useful in the study of the sodium pump in human pregnancy. Reductions in sodium pump number can increase uterine contractile force and may contribute to labor.
Animals↗
Mutually exclusive exon splicing of type III brain sodium channel alpha subunit RNA generates developmentally regulated isoforms in rat brain.
We have identified two exons of the type III rat brain sodium channel alpha subunit gene that undergo mutually exclusive alternative RNA splicing to produce mRNAs coding either for an isoform predominant in neonatal brain (IIIN) or a different isoform (IIIA) predominant in the adult. These exons are 92 base pairs in length and encode amino acids 203-232, which correspond to part of the S3 and most of the S4 transmembrane segments within domain I and the extracellular loop between them. Despite 21 nucleotide differences between the exons, only a single amino acid at position 209 is altered, specifying either aspartic acid (IIIA) or serine (IIIN). As evidence that these isoforms are generated via alternative splicing, we demonstrate that both exons are encoded within the type III gene. The nucleotide sequences of the neonatal and adult type III exons and the intervening intron as well as the developmental regulation of this splicing are nearly identical in the type II sodium channel gene. The conservation of the exon/intron structure and of the developmentally regulated patterns of expression of the type II and III sodium channel genes suggests that alternative mRNA splicing of this exon may play a substantial role in modulating sodium channel function during brain development by alteration of a single amino acid.
Animals↗
Phylogenetic diversity of the expression of the microtubule-associated protein tau: implications for neurodegenerative disorders.
The microtubule-associated protein tau regulates the dynamic stability of the neuronal cytoskeleton by interacting with microtubules. It is encoded by a single gene, but expressed in a variety of isoforms due to differential RNA splicing. Six isoforms can be found in the human central nervous system. These isoforms differ in their ability to promote the assembly of microtubules as well as in their capacity to stabilize existing microtubule structures. Furthermore, some of the isoforms of tau are specifically involved in the pathogenesis of neurodegenerative disorders. Thus, splicing of tau might critically influence the physiological functions of tau protein as well as the pathogenesis of neurodegenerative diseases with tauopathy. The present study addresses the differential expression of the six isoforms of tau in the central nervous system of 12 mammalian species including Homo sapiens. The occurrence of each of the six tau isoforms was highly variable. However, species that were phylogenetically related expressed a similar pattern of tau isoforms. These results suggest a phylogenetic descent of splicing paradigms, which can be matched with known phylogenetic concepts based on morphological and molecular genetical studies. Especially, the unique expression pattern of tau isoforms in the human central nervous system implicates a possible link to the particular vulnerability of humans to neurodegenerative disorders with tauopathy, namely Alzheimer's disease, frontotemporal dementia and Pick's disease.
Animals↗
Cloning and characterization of fiber type-specific ryanodine receptor isoforms in skeletal muscles of fish.
We have cloned a group of cDNAs that encodes the skeletal ryanodine receptor isoform (RyR1) of fish from a blue marlin extraocular muscle library. The cDNAs encode a protein of 5,081 amino acids with a calculated molecular mass of 576,302 Da. The deduced amino acid sequence shows strong sequence identity to previously characterized RyR1 isoforms. An RNA probe derived from a clone of the full-length marlin RyR1 isoform hybridizes to RNA preparations from extraocular muscle and slow-twitch skeletal muscle but not to RNA preparations from fast-twitch skeletal or cardiac muscle. We have also isolated a partial RyR clone from marlin and toadfish fast-twitch muscles that shares 80% sequence identity with the corresponding region of the full-length RyR1 isoform, and a RNA probe derived from this clone hybridizes to RNA preparations from fast-twitch muscle but not to slow-twitch muscle preparations. Western blot analysis of slow-twitch muscles in fish indicates the presence of only a single high-molecular-mass RyR protein corresponding to RyR1. [3H]ryanodine binding assays revealed the fish slow-twitch muscle RyR1 had a greater sensitivity for Ca2+ than the fast-twitch muscle RyR1. The results indicate that, in fish muscle, fiber type-specific RyR1 isoforms are expressed and the two proteins are physiologically distinct.
Amino Acid Sequence↗
Biochemical properties of a novel U2AF65 protein isoform generated by alternative RNA splicing.
A variety of RNA binding proteins with one or more RNA recognition (RNP-CS) motifs play essential roles in the pre-mRNA splicing process. One such factor, the U2 snRNP auxiliary factor large subunit (U2AF65), contains three RNP-CS motifs each of which is required for high affinity binding to polypyrimidine tracts. Here we report the isolation of a natural cDNA variant of human U2AF65, U2AF65 (S), which is shortened by a 12 nucleotide in frame deletion between RNP-CS2 and -CS3 motifs. A portion of the U2AF65 (S) cDNA was reported previously but was not characterized further. We observe that the U2AF65 (S) variant predominates in a variety of tissues and cell lines, and is generated together with the U2AF65 (L) form (2) by alternative 5' splice site selection from a single gene. The corresponding histidine-tagged recombinant proteins bind with similar affinities to model RNA substrates containing strong or weak polypyrimidine tracts. Both U2AF65 (S) and (L) protein isoforms reconstitute splicing activity with similar kinetic profiles in U2AF-depleted (splicing-deficient) HeLa nuclear extracts. Finally, the thermal stabilities of the protein isoforms are essentially equivalent. Thus, the presence or absence of the peptide segment, VSPP (residues 345-348), in the linker region between RNP-CS2 and -CS3 does not detract from the intrinsic RNA binding and splicing properties of the U2AF65 protein. The biological implications of alternative splicing for the function and evolution of RNA binding proteins are discussed.
Alternative Splicing↗
RNA-editing of the 5-HT(2C) receptor alters agonist-receptor-effector coupling specificity.
1. The serotonin(2C) (5-HT(2C)) receptor couples to both phospholipase C (PLC)-inositol phosphate (IP) and phospholipase A(2) (PLA(2))-arachidonic acid (AA) signalling cascades. Agonists can differentially activate these effectors (i.e. agonist-directed trafficking of receptor stimulus) perhaps due to agonist-specific receptor conformations which differentially couple to/activate transducer molecules (e.g. G proteins). Since editing of RNA transcripts of the human 5-HT(2C) receptor leads to substitution of amino acids at positions 156, 158 and 160 of the putative second intracellular loop, a region important for G protein coupling, we examined the capacity of agonists to activate both the PLC-IP and PLA(2)-AA pathways in CHO cells stably expressing two major, fully RNA-edited isoforms (5-HT(2C-VSV), 5-HT(2C-VGV)) of the h5-HT(2C) receptor. 2. 5-HT increased AA release and IP accumulation in both 5-HT(2C-VSV) and 5-HT(2C-VGV) expressing cells. As expected, the potency of 5-HT for both RNA-edited isoforms for both responses was 10 fold lower relative to that of the non-edited receptor (5-HT(2C-INI)) when receptors were expressed at similar levels. 3. Consistent with our previous report, the efficacy order of two 5-HT receptor agonists (TFMPP and bufotenin) was reversed for AA release and IP accumulation at the non-edited receptor thus demonstrating agonist trafficking of receptor stimulus. However, with the RNA-edited receptor isoforms there was no difference in the relative efficacies of TFMPP or bufotenin for AA release and IP accumulation suggesting that the capacity for 5-HT(2C) agonists to traffic receptor stimulus is lost as a result of RNA editing. 4. These results suggest an important role for the second intracellular loop in transmitting agonist-specific information to signalling molecules.
Amphetamines↗
Rescuing qkV dysmyelination by a single isoform of the selective RNA-binding protein QKI.
Alternative splicing of the qkI transcript generates multiple isoforms of the selective RNA-binding protein QKI, which play key roles in controlling the homeostasis of their mRNA targets. QKI deficiency in oligodendrocytes of homozygous quakingviable (qkV/qkV) mutant mice results in severe hypomyelination, indicating the essential function of QKI in myelinogenesis. However, the molecular mechanisms by which QKI controls myelination remain elusive. We report here that QKI-6 is the most abundant isoform in brain and is preferentially reduced in the qkV/qkV mutant during normal myelinogenesis. To test whether QKI-6 is the predominant isoform responsible for advancing CNS myelination, we developed transgenic mice that express Flag-QKI-6 specifically in the oligodendroglia lineage, driven by the proteolipid protein (PLP) promoter. When introduced into the qkV/qkV mutant, the QKI-6 transgene rescues the severe tremor and hypomyelination phenotype. Electron microscopic studies further revealed that the Flag-QKI-6 transgene is sufficient for restoring compact myelin formation with normal lamellar periodicity and thickness. Interestingly, Flag-QKI-6 preferentially associates with the mRNA encoding the myelin basic protein (MBP) and rescues MBP expression from the beginning of myelinogenesis. In contrast, Flag-QKI-6 binds the PLP mRNA with lower efficiency and has a minimal impact on PLP expression until much later, when the expression level of QKI-6 in the transgenic animal significantly exceeds what is needed for normal myelination. Together, our results demonstrate that QKI-6 is the major isoform responsible for CNS myelination, which preferentially promotes MBP expression in oligodendrocytes.
Animals↗
Expression of mRNA for a newly identified Pax5 exon is reduced in multiple myeloma.
Pax5 is a transcription factor that is critical in the bone marrow for differentiation and proliferation of B cells until the plasma cell stage. In Pax5(-/-) mice, B-cell development stalls at the pro-B-cell stage. Messenger RNA profiles of alternatively spliced isoforms of Pax5 in bone marrow frequently differ between multiple myeloma (MM) patients and healthy donors. We sought to determine if Pax5 mRNA profiles also differed in blood and unexpectedly detected the presence of a previously unreported exon that alters the amino acid code for the transactivating domain of Pax5 in CD138(-) B cells. This unique exon escapes detection by conventional analyses of RT-PCR products and may serve as a prototype for other exons, in other genes, that escape RT-PCR detection. Eight percent of tested human subjects were heterozygous for an allele with a nonsynonymous nucleotide substitution in the new exon, and one MM patient was homozygotic for this base difference. Subsequent analysis of plasma and B-cell populations from bone marrow revealed a markedly reduced mRNA expression of the new isoform in cells from MM patients when compared to cells from normal subjects.
Alternative Splicing↗
Lost in translation: translational interference from a recurrent mutation in exon 1 of MECP2.
BACKGROUND: Rett syndrome (RTT) is an X linked neuro-developmental disorder affecting mostly girls. Mutations in the coding region of MECP2 are found in 80% of classic RTT patients. Until recently, the region encoding MECP2 was believed to comprise exons 2, 3, and 4 with the ATG start site located at the end of exon 2 (MeCP2_e2). METHODS: Recent reports of another mRNA transcript transcribed from exon 1 (MeCP2_e1) prompted us to screen exon 1 among RNA samples from 20 females with classic or atypical RTT. RESULTS: A previously reported 11 base pair deletion in exon 1 was detected in one subject with a milder phenotype. Although RNA expression for both protein isoforms was detected from the mutant allele, evaluation of MeCP2 protein in uncultured patient lymphocytes by immunocytochemistry revealed that MeCP2 protein production was restricted to only 74-76% of lymphocytes. X chromosome inactivation studies of genomic DNA revealed similar XCI ratios at the HUMARA locus (73:27 with HpaII and 74:26 with McrBC). We have demonstrated that translation but not transcription of the MeCP2_e2 isoform is ablated by the 11 nucleotide deletion, 103 nucleotides upstream of the e2 translation start site. CONCLUSIONS: These findings reveal that nucleotides within the deleted sequence in the 5'-UTR of the MeCP2_e2 transcript, while not required for transcription, are essential for translation.
Adolescent↗
The circadian regulation of Presenilin-2 gene expression.
Circadian rhythms are generated by a molecular clock composed of clock genes and their protein products. Other genes are regulated in a rhythmic way by this molecular clockwork, but are not themselves constituents of the clock. This study shows that one of these clock-controlled genes encodes the signalling protein Presenilin-2. Indeed, evidence is presented that the promoter of the mouse Presenilin-2 gene is bound and activated by CLOCK and BMAL1, transcription factors of the mammalian circadian clock. Quantification of Presenilin-2 RNA shows that its expression is non-rhythmic in many peripheral tissues (heart, muscle, kidney, spleen, and thymus). Note, though, that careful analysis of the liver data shows that Presenilin-2 RNA exists in distinct isoforms in this tissue, and that rhythmicity is restricted to only a subset of these RNA isoforms. These data indicate a unique mode of regulation of Presenilin-2 transcripts, the circadian control of which appears to happen at the transcriptional and post-transcriptional levels.
ARNTL Transcription Factors↗
Polymorphic expression of CD46 protein isoforms due to tissue-specific RNA splicing.
CD46 is a member of the regulators of complement activation (RCA) family and serves to protect autologous cells from complement mediated lysis. The CD46 gene consists of 14 exons and extensive RNA splicing produces protein isoforms of different molecular weight. Predominant protein isoforms of 66 and 56 kDa arise from splicing in or out of exon 8 which encodes a region rich in serine, threonine and proline residues known to be heavily O-glycosylated. An inherited allelic polymorphism controls the relative expression of these isoforms in PBL and other tissues. This study has analysed an independent and overriding tissue specific regulation of CD46 splicing. Salivary gland and kidney produce RNA transcripts that preferentially include exon 8, giving rise to the 66 kDa protein species, while exon 8 is spliced out in brain tissue to give the 56 kDa protein. The cytoplasmic tail of CD46 is encoded by either exon 13 (CYT 1) or exon 14 (CYT 2). There is a preferential deletion of exon 13 from transcripts in salivary gland, kidney and brain to encode a protein containing cytoplasmic tail CYT 2. This preferential production of the CYT 2 tail is contrary to that seen on peripheral blood lymphocytes where equivalent expression of both CYT 1 and CYT 2 is observed. Our results suggest that while the splicing of exons within most cells is controlled by nucleotide sequences within or close to the CD46 gene (i.e. cis-regulation), splicing in tissues such as salivary gland, kidney and brain is regulated by trans-splicing factors encoded by another gene(s).
Alternative Splicing↗
Characterization of progesterone receptor isoform expression in fetal membranes.
OBJECTIVE: To quantify expression of progesterone receptor (PR) messenger RNA (mRNA) isoforms in fetal membranes, and to determine whether these levels change in culture. STUDY DESIGN: Placentas from women undergoing term cesarean delivery before labor were collected. Layers of amnion, chorion, and decidua were separated manually, enzymatically digested, and separated further with the use of a density gradient. RNA was extracted immediately and after culture for 48 hours, then analyzed by quantitative reverse transcription polymerase chain reaction for PR-A, PR-B, and beta-2 microglobulin mRNA expression. Separation of cell types was confirmed by immunohistochemistry. RESULTS: PR isoform expression was identified in fetal membranes, with levels highest in decidua and below the limits of detection in amnion. The ratio of PR-A/PR-B mRNA was not significantly different between cell layers. PR mRNA isoform levels did not differ significantly in fresh versus cultured cells. CONCLUSION: Quantitative reverse transcription polymerase chain reaction was used to quantitate expression of PR mRNA isoforms in cells of fetal membranes and to validate systems for further study of PR with respect to inflammation, infection, and preterm delivery.
Extraembryonic Membranes↗
Distinct functional profiles of aripiprazole and olanzapine at RNA edited human 5-HT2C receptor isoforms.
In this study we have functionally characterized aripiprazole (OPC-14597; 7-(4-[4-(2,3-dichlorophenyl)-1-piperazinyl]butyloxy-3,4-dihydro-2-(1H)-quinolinone), the prototype of a new generation antipsychotic drug termed dopamine-serotonin-system stabilizer, in cells expressing 5-hydroxytryptamine2 (5-HT2) receptor subtypes in comparison with olanzapine. In Chinese hamster ovary (CHO) cells stably expressing 5-HT2 receptors, aripiprazole displayed a dual agonist/antagonist profile for 5-HT2C receptor (VNI isoform) mediated calcium signaling (EC50 1070 nM, IC50 281 nM). It exhibited no appreciable 5-HT2A or 5-HT2B agonism, whereas it antagonized 5-HT-stimulated calcium increase at either 5-HT2A or 5-HT2B receptor expressed in CHO cells (IC50s of 369 and 0.46 nM, respectively). In comparison, olanzapine was devoid of agonism but was an antagonist at all three subtypes, with a potency rank order of 5-HT2A (IC50, 2.5 nM)>5-HT2B (47 nM)>5-HT2C (69 nM). In human embryonic kidney (HEK) cells transiently expressing 5-HT2C receptor isoforms, aripiprazole exhibited full agonism at the unedited INI, but partial agonism at the partially edited VNI and fully edited VSV isoforms (EC50s of 571, 1086 and 2099 nM, respectively). A partial antagonism was also observed for aripiprazole at the two edited isoforms (IC50s of 1138 and 1000 nM, respectively). In contrast, while lacking agonist activity at the VNI and VSV, olanzapine showed inverse agonism at the INI isoform (IC50 594 nM), reaching a maximal attenuation of 20%. In addition, olanzapine was a full antagonist at all three isoforms, with a rank order of potency of VNI (IC50, 79 nM)>VSV (101 nM)>INI (3856 nM). The modest 5-HT2A antagonism and 5-HT2C partial agonism, along with reported D2 and 5-HT1A partial agonism, may allow aripiprazole to stabilize the disturbed dopamine-serotonin interplay in schizophrenia with a moderate yet adequate pharmacological intervention. 5-HT2C agonism may also underlie the minimal weight gain seen with aripiprazole.
Animals↗
Two mitofusin proteins, mammalian homologues of FZO, with distinct functions are both required for mitochondrial fusion.
Mitochondria are dynamic organelles that undergo frequent fission and fusion or branching. Although these morphologic changes are considered crucial for cellular functions, the underlying mechanisms remain elusive, especially in mammalian cells. We characterized two rat mitochondrial outer membrane proteins, Mfn1 and Mfn2, with distinct tissue expressions, that are homologous to Drosophila Fzo, a GTPase involved in mitochondrial fusion. Expression of the GTPase-domain mutant of Mfn2 (Mfn2(K109T)) in HeLa cells induced mitochondrial fragmentation in which Mfn2(K109T) localized at the restricted domains. Immuno-electronmicroscopy revealed that Mfn2(K109T) was concentrated at the contact domains between adjacent mitochondria, suggesting that fusion of the outer membrane was arrested at some intermediate step. Mfn1 expression induced highly connected tubular network structures depending on the functional GTPase domain. The Mfn1-induced tubular networks were suppressed by co-expression with Mfn2. In vivo depletion of either isoform by RNA interference revealed that both are required to maintain normal mitochondrial morphology. The fusion of differentially-labeled mitochondria in HeLa cells subjected to depletion of either Mfn isoform and subsequent cell fusion by hemagglutinating virus of Japan revealed that both proteins have distinct functions in mitochondrial fusion. We conclude that the two Mfn isoforms cooperate in mitochondrial fusion in mammalian cells.
Animals↗