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At least 253 records · Page 14Linked to original sources
PubMed2004

RNA editing and short variant of serotonin 2C receptor mRNA in neuronally differentiated NG108-15 cells.

Two types of serotonin 2C subtype receptor mRNA, receptor-type and short variant, has been reported. The expression of the receptor-type mRNA could be detected as well as the short variant in NG108-15 cells by using a high temperature stable reverse transcriptase and the expression of the receptor-type mRNA was enhanced in drug-induced neuronal differentiated cells. The deleted sequence of the short variant include the RNA editing site by adenosine deaminase. Analysis of the sequence at the editing site revealed that the mRNA of undifferentiated cells was highly edited at sites A and B and that cytosine deaminase activity may also be involved in neuronal differentiation.

Animals↗
PubMed2001

Coordinated expression of muscarinic receptor messenger RNAs in striatal medium spiny neurons.

The postsynaptic effects of acetylcholine in the striatum are largely mediated by muscarinic receptors. Two of the five cloned muscarinic receptors (M1 and M4) are expressed at high levels by the medium spiny neurons-the principal projection neurons of the striatum. Previous studies have suggested that M4 muscarinic receptors are found primarily in medium spiny neurons that express substance P and participate in the "direct" striatonigral pathway. This view is difficult to reconcile with electrophysiological studies suggesting that nearly all medium spiny neurons exhibit responses characteristic of M4 receptors. To explore this apparent discrepancy, the coordinated expression of M1-M5 receptor messenger RNAs in identified medium spiny neurons was assayed using single-cell reverse transcription-polymerase chain reaction techniques. Nearly all medium spiny neurons had detectable levels of M1 receptor messenger RNA. Although M4 receptor messenger RNA was detected more frequently in substance P-expressing neurons (70%), it was readily seen in a substantial population of enkephalin-expressing neurons (50%). To provide a quantitative estimate of transcript abundance, quantitative reverse transcription-polymerase chain reaction experiments were performed. These studies revealed that M4 messenger RNA was expressed by both substance P and enkephalin neurons, but was roughly five-fold higher in abundance in substance P-expressing neurons. This quantitative difference provides a means of reconciling previous estimates of M4 receptor distribution and function.

Animals↗
PubMed1999

Iron-regulatory proteins, iron-responsive elements and ferritin mRNA translation.

Iron plays a central role in the metabolism of all cells. This is evident by its major contribution to many diverse functions, such as DNA replication, bacterial pathogenicity, photosynthesis, oxidative stress control and cell proliferation. In mammalian systems, control of intracellular iron homeostasis is largely due to posttranscriptional regulation of binding by iron-regulatory RNA-binding proteins (IRPs) to iron-responsive elements (IREs) within ferritin and transferrin receptor (TfR) mRNAs. the TfR transports iron into cells and the iron is subsequently stored within ferritin. IRP binding is under tight control so that it responds to changes in intracellular iron requirements in a coordinate manner by differentially regulating ferritin mRNA translational efficiency and TfR mRNA stability. Several different stimuli, as well as intracellular iron levels and oxidative stress, are capable of regulating these RNA-protein interactions. In this mini-review, we shall concentrate on the mechanisms underlying modulation of the interaction of IRPs and the ferritin IRE and its role in regulating ferritin gene expression.

Animals↗
PubMed2005

RNA-targeted suppression of stress-induced allostasis in primate spinal cord neurons.

Peripheral acetylcholine levels notably control the synthesis in macrophages of pro-inflammatory cytokines; however, it remains unclear whether this peripheral regulatory pathway affects central nervous system neurons. To explore the interrelationship between neuronal cholinergic homeostasis and peripheral inflammatory responses in primates, we used spinal cord sections from cynomolgus monkeys after 7 days oral or intravenous treatment with Monarsen oligonucleotide. Monarsen is an antisense oligonucleotide 3'-protected by 2'-oxymethylation, which was proved to induce selective destruction of the stress-induced acetylcholinesterase splice variant AChE-R mRNA. Handling stress predictably suppressed neuronal choline acetyl transferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in all treated monkeys. In Monarsen-treated animals, we further observed suppression of stress-induced increases in plasma AChE activities. Corresponding decreases in AChE-R mRNA were seen in spinal cord neurons, associated with parallel decline patterns in the mRNA encoding for the splice factor SC35 (the levels of which co-increase with those of AChE-R) as well as in the neuronal pro-inflammatory interleukins IL-1beta and IL-6. The antisense effects showed direct dose dependence and were inversely associated with neuronal cell size. These findings suggest a causal association between neuronal cholinergic allostasis and inflammatory reactions in primates and support the peripheral use of RNA-targeted intervention with AChE-R accumulation for the management of both stress and inflammatory responses.

Acetylcholine↗
PubMed2005

Effect of the [CCTG]n repeat expansion on ZNF9 expression in myotonic dystrophy type II (DM2).

Myotonic dystrophy is caused by two different mutations: a (CTG)n expansion in 3' UTR region of the DMPK gene (DM1) and a (CCTG)n expansion in intron 1 of the ZNF9 gene (DM2). The most accredited mechanism for DM pathogenesis is an RNA gain-of-function. Other findings suggest a contributory role of DMPK-insufficiency in DM1. To address the issue of ZNF9 role in DM2, we have analyzed the effects of (CCTG)n expansion on ZNF9 expression in lymphoblastoid cell lines (n=4) from DM2 patients. We did not observe any significant alteration in ZNF9 mRNA and protein levels, as shown by QRT-PCR and Western blot analyses. Additional RT-PCR experiments demonstrated that ZNF9 pre-mRNA splicing pattern, which includes two isoforms, is unmodified in DM2 cells. Our results indicate that the (CCTG)n expansion in the ZNF9 intron does not appear to have a direct consequence on the expression of the gene itself.

Base Sequence↗
PubMed2006

Flies deficient in Muscleblind protein model features of myotonic dystrophy with altered splice forms of Z-band associated transcripts.

Myotonic dystrophy (DM) is a dominantly inherited neuromuscular disorder characterised by muscle weakness and wasting. There are two forms of DM; both of which are caused by the expansion of repeated DNA sequences. DM1 is associated with a CTG repeat located in the 3' untranslated region of a gene, DMPK and DM2 with a tetranucleotide repeat expansion, CCTG, located in the first intron of a different gene, ZNF9. Recent data suggest a dominant RNA gain-of-function mechanism underlying DM, as transcripts containing either CUG or CCUG repeat expansions accumulate as foci in the nuclei of DM1 and DM2 cells respectively, where they exert a toxic effect, sequestering specific RNA binding proteins such as Muscleblind, which leads to splicing defects and the disruption of normal cellular functions. Z-band disruption is a well-known histological feature of DM1 muscle, which has also been reported in Muscleblind deficient flies. In order to determine whether there is a common molecular basis for this abnormality we have examined the alternative splicing pattern of transcripts that encode proteins associated with the Z-band in both organisms. Our results demonstrate that the missplicing of ZASP/LDB3 leads to the expression of an isoform in DM1 patient muscle, which is not present in normal controls, nor in other myopathies. Furthermore the Drosophila homologue, CG30084, is also misspliced, in Muscleblind deficient flies. Another Z-band transcript, alpha actinin, is misspliced in mbl mutant flies, but not in DM1 patient samples. These results point to similarities but subtle differences in the molecular breakdown of Z-band structures in flies and DM patients and emphasise the relevance of Muscleblind proteins in DM pathophysiology.

Actinin↗
PubMed2000

Developmental regulation of the L-type calcium channel alpha1C subunit expression in heart.

We used Northern analyses, RNase protection assays and immunoblot analyses to examine the relationship among developmental age of the heart, abundance of mRNA and L-type calcium channel alpha1C subunit protein, and to establish the size of the native protein in heart. Northern analysis, RNase protection assays, and immunoblots were used to study RNA and protein from rat heart of various ages. In fetal and adult ventricles there was a predominant 8.3-kb transcript for the alpha1C subunit with no change in transcript size during development. RNase protection assays demonstrated a 2-fold increase in abundance of the DHP receptor message during postnatal development. Immunoblots identified a 240 kD protein, corresponding to the predicted molecular mass of the full length alpha1C subunit. No change in size of protein for the alpha1C subunit was observed at any developmental stage and there was no evidence for a truncated isoform. There was an approximate 2-fold increase in alpha1C subunit protein in ventricular homogenates during postnatal development. Thus, in the developing rat heart, alterations in calcium channel properties during development appear to result neither from alternative splicing that produces a smaller transcript for the alpha1C subunit nor from expression of a truncated protein, but at least in part from transcriptionally-regulated expression of the 240 kDa polypepde.

Age Factors↗
PubMed2003

Polypyrimidine tract-binding protein down-regulates fibroblast growth factor receptor 1 alpha-exon inclusion.

Exclusion of the alpha-exon by alternative RNA splicing of the fibroblast growth factor receptor 1 (FGFR1) primary transcript leads to the production of FGFR1beta. Glial cell transformation is associated with a progressive increase in FGFR1beta expression that coincides with a dramatic increase in the expression of the splicing factor polypyrimidine tract-binding protein (PTB). Cell-specific overexpression of PTB increased alpha-exon skipping, and a reduction in PTB increased alpha-exon inclusion. Targeted disruption of PTB interaction with FGFR1 precursor RNA in vivo by an antisense oligonucleotide also increased alpha-exon inclusion. These results suggest that PTB plays a direct role in alpha-exon splicing.

Alternative Splicing↗
PubMed2006

Novel noncoding RNA from human Y distal heterochromatic block (Yq12) generates testis-specific chimeric CDC2L2.

The human Y chromosome, because it is enriched in repetitive DNA, has been very intractable to genetic and molecular analyses. There is no previous evidence for developmental stage- and testis-specific transcription from the male-specific region of the Y (MSY). Here, we present evidence for the first time for a developmental stage- and testis-specific transcription from MSY distal heterochromatic block. We isolated two novel RNAs, which localize to Yq12 in multiple copies, show testis-specific expression, and lack active X-homologs. Experimental evidence shows that one of the above Yq12 noncoding RNAs (ncRNAs) trans-splices with CDC2L2 mRNA from chromosome 1p36.3 locus to generate a testis-specific chimeric beta sv13 isoform. This 67-nt 5'UTR provided by the Yq12 transcript contains within it a Y box protein-binding CCAAT motif, indicating translational regulation of the beta sv13 isoform in testis. This is also the first report of trans-splicing between a Y chromosomal and an autosomal transcript.

Adult↗
PubMed2003

Steroid receptor RNA activator stimulates proliferation as well as apoptosis in vivo.

Steroid receptor RNA activator (SRA) is an RNA that coactivates steroid hormone receptor-mediated transcription in vitro. Its expression is strongly up-regulated in many human tumors of the breast, uterus, and ovary, suggesting a potential role in pathogenesis. To assess SRA function in vivo, a transgenic-mouse model was generated to enable robust human SRA expression by using the transcriptional activity of the mouse mammary tumor virus long terminal repeat. Transgenic SRA was expressed in the nuclei of luminal epithelial cells of the mammary gland and tissues of the male accessory sex glands. Distinctive evidence for SRA function in vivo was obtained from the elevated levels of estrogen-controlled expression of progesterone receptor in transgenic mammary glands. Although overexpression of SRA showed strong promoting activities on cellular proliferation and differentiation, no alterations progressed to malignancy. Epithelial hyperplasia was accompanied by increased apoptosis, and preneoplastic lesions were cleared by focal degenerative transformations. In bitransgenic mice, SRA also antagonized ras-induced tumor formation. This work indicates that although coactivation of steroid-dependent transcription by SRA is accompanied by a proliferative response, overexpression is not in itself sufficient to induce turmorigenesis. Our results underline an intricate relationship between the different physiological roles of steroid receptors in conjunction with the RNA activator in the regulation of development, tissue homeostasis, and reproduction.

Animals↗
PubMed2001

Creatine transporter protein content, localization, and gene expression in rat skeletal muscle.

The present study examined the gene expression and cellular localization of the creatine transporter (CreaT) protein in rat skeletal muscle. Soleus (SOL) and red (RG) and white gastrocnemius (WG) muscles were analyzed for CreaT mRNA, CreaT protein, and total creatine (TCr) content. Cellular location of the CreaT protein was visualized with immunohistochemical analysis of muscle cross sections. TCr was higher (P < or = 0.05) in WG than in both RG and SOL, and was higher in RG than in SOL. Total CreaT protein content was greater (P < or = 0.05) in SOL and RG than in WG. Two bands (55 and 70 kDa) of the CreaT protein were found in all muscle types. Both the 55-kDa (CreaT-55) and the 70-kDa (CreaT-70) bands were present in greater (P < or = 0.05) amounts in SOL and RG than in WG. SOL and RG had a greater amount (P < or = 0.05) of CreaT-55 than CreaT-70. Immunohistochemical analysis revealed that the CreaT was mainly associated with the sarcolemmal membrane in all muscle types. CreaT mRNA expression per microgram of total RNA was similar across the three muscle types. These data indicate that rat SOL and RG have an enhanced potential to transport Cr compared with WG, despite a higher TCr in the latter.

Animals↗
PubMed2005

Dark matter in the genome: evidence of widespread transcription detected by microarray tiling experiments.

Microarrays provide the opportunity to measure transcription from regions of the genome without bias towards the location of known genes. This technology thus offers an important source of genomic sequence annotation that is complementary to cDNA sequencing and computational gene-finding methods. Recent "tiling" microarray experiments that assay transcription at regular intervals throughout the genome have shown evidence of large amounts of transcription outside the boundaries of known genes. This transcription is observed in polyadenylated RNA samples and appears to be derived from intergenic regions, from introns of known genes and from sequences antisense to known transcripts. In this article, we discuss different explanations for this phenomenon.

Animals↗
PubMed2003

A muscleblind knockout model for myotonic dystrophy.

The neuromuscular disease myotonic dystrophy (DM) is caused by microsatellite repeat expansions at two different genomic loci. Mutant DM transcripts are retained in the nucleus together with the muscleblind (Mbnl) proteins, and these abnormal RNAs somehow interfere with pre-mRNA splicing regulation. Here, we show that disruption of the mouse Mbnl1 gene leads to muscle, eye, and RNA splicing abnormalities that are characteristic of DM disease. Our results support the hypothesis that manifestations of DM can result from sequestration of specific RNA binding proteins by a repetitive element expansion in a mutant RNA.

Alternative Splicing↗
PubMed2001

Human GABA(B)R genomic structure: evidence for splice variants in GABA(B)R1 but not GABA(B)R2.

The type B gamma-aminobutryic acid receptor (GABA(B)R) is a G protein coupled receptor that mediates slow pre- and post-synaptic inhibition in the nervous system. We find that the human GABA(B)R2 gene spans greater than 350 kb and contains 2.8 kb of coding region in 19 exons. The overall similarity in genomic structure with regard to conservation of intron position and exon size between human or Drosophila GABA(B)R1 and GABA(B)R2 genes suggests a common ancestral origin. Multiple transcripts GABA(B)R1a-c and GABA(B)R2a-c have been described and alternative splicing has been proposed to result in GABA(B)R1c, GABA(B)R2b and GABA(B)R2c. The results described here provide support for the existence of GABA(B)R1c but not for GABA(B)R2b and GABA(B)R2c. Splice junctions present in the GABA(B)R1 gene sequence are consistent with the formation of GABA(B)R1c by exon skipping of one sushi domain module. The GABA(B)R2 gene lacks canonical splice junctions for the reported variants. Consistent with this, RNA analysis demonstrates the presence of GABA(B)R1c and GABA(B)R2 transcripts in fetal and adult human brain RNA but GABA(B)R2b and GABA(B)R2c transcripts are not detected. These results provide insight into the evolution and transcript diversity of the mammalian GABA(B)R genes.

Alternative Splicing↗
PubMed2000

Identification and characterization of a new member of the placental prolactin-like protein-C (PLP-C) subfamily, PLP-Cbeta.

We have isolated a complementary DNA (cDNA) clone that encodes a new member of the PRL-like protein-C (PLP-C) subfamily of the PRL gene family. The clone was amplified from a 13.5-day-old mouse conceptus cDNA library by PCR using primers based on conserved regions of PLP-C sequences. The full-length cDNA encodes a predicted protein of 241 residues, which contains a putative signal sequence and 2 putative N-linked glycosylation sites. The predicted protein shares 55-66% amino acid identity with mouse PLP-Calpha and rat PLP-D, PLP-H, PLP-Cv, and PLP-C and also contains 6 homologously positioned cysteine residues. Thus, we named this protein PLP-Cbeta for consistency. We have also isolated rat PLP-Cbeta from rat placenta cDNA library. Surprisingly, two messenger RNA (mRNA) isoforms of rat PLP-Cbeta were isolated: one mRNA (rPLP-Cbeta) encodes a 241-amino acid product, but another mRNA (rPLP-Cbetadelta39) lacks 39 bases that encode for a region rich in aromatic amino acids. The 39-bp region corresponds to exon 3 of other PLP-C subfamily members, such as PLP-Calpha, PLP-Cv, and d/tPRP. It suggests that the two isoforms are probably generated by an alternative splicing from a single gene. RT-PCR analysis revealed that the rPLP-Cbeta form was dominantly expressed in placenta, although both isoforms are coexpressed during placentation. The mouse PLP-Cbeta mRNA expression, which was specific to the placenta, was first detected by Northern analysis on embryonic day 11.5 (E 11.5) and persisted until birth. However, in situ hybridization analysis revealed mPLP-Cbeta expression on E 10.5 in specific trophoblast subsets, such as giant cells and spongiotrophoblast cells. mPLP-Cbeta mRNA was detected in the labyrinthine zone on E 18.5, suggesting that spongiotrophoblast cells had penetrated the labyrinthotrophoblast zone. Consistent with the observed expression in trophoblast giant cells, PLP-Cbeta expression was also detected in in vitro differentiated Rcho-1 cells, which express the trophoblast giant cell phenotype. In summary, overall high amino acid identity (79%), the locations of cysteine residues, and consensus sites for N-linked glycosylation between mouse and rat PLP-Cbeta clearly indicate that PLP-Cbeta is a bona fide member of the PLP-C subfamily. The conservation between mouse and rat, the presence of alternative isoforms, and the pattern of expression during gestation suggest the biological significance of PLP-Cbeta during pregnancy.

Amino Acid Sequence↗
PubMed1997

Differential distribution of mRNAs encoding phosphatidylinositol transfer proteins alpha and beta in the central nervous system of the rat.

The expression of the alpha and beta isoforms of phosphatidylinositol transfer protein (PI-TP alpha and PI-TP beta) in the adult rat brain was examined by in situ hybridization analysis with isoform-specific RNA probes. PI-TP alpha mRNA was detected in rather restricted regions of the brain whereas PI-TP beta mRNA was widely distributed in the brain. PI-TP alpha mRNA signals were remarkable in neocortex layers II/III and V/VI, Purkinje cell layer, deep cerebellar nuclei of the cerebellum, red nucleus and most part of brain stem. Low levels of PI-TP alpha transcript were present in CA3 of the hippocampus, ventral and dorsal thalamic nuclei, and motoneurons of spinal cord. No hybridization signals was obtained in the olfactory bulb, basal ganglia, amygdala, hypothalamus, and pituitary gland. In contrast, strong signals of PI-TP beta mRNA were detected in the dentate gyrus. The beta isoform mRNA was moderately expressed in olfactory bulb, layers II/III of the neocortex, striatum, CA1-CA4 regions of the hippocampus, medial habenula, cerebellum, amygdala, hypothalamus, spinal cord, and pituitary gland. Thalamus and brain stem contained relatively low, but significant levels of PI-TP beta transcript. The distinct distribution of PI-TP alpha and PI-TP beta mRNAs suggests different functional roles for each of the gene products in the mature nervous system.

Animals↗
PubMed2001

Five novel alternatively spliced transcripts of DNA (cytosine-5) methyltransferase 2 in human peripheral blood leukocytes.

Alternative splicing of RNA molecules transcribed from DNA (cytosine-5) methyltransferases has been proposed as a mechanism by which methylation is able to effect diverse biological processes in higher eukaryotes. This study has investigated transcriptional versatility of DNA (cytosine-5) methyltransferase 2, which may methylate cytosine residues within 5'-CCTGG-3' pentanucleotides in regions of the human genome devoid of 5'-CG-3' methylation. Five novel splice variants of DNA (cytosine-5) methyltransferase 2 were identified in the peripheral blood leukocytes of healthy subjects following cloning and sequencing of RT-PCR products amplified using gene specific oligodeoxyribonucleotide primers. The generation of some of these splice variants may be influenced by the formation of secondary structures within pre-mRNA due to the repetition of sequences flanking alternatively spliced exons in a reverse and complementary orientation on the same strand. These findings enable novel approaches to investigate the role of RNA secondary structures in alternative splicing. The DNA (cytosine-5) methyltransferase 2 splice variants are generated in all the major cell types of peripheral blood, as well as in neoplastic lymphoid cells indicating that they are unlikely to generate proteins involved in control of the cell cycle or cellular differentiation. Interestingly, the gene products generated by some splice variants completely or partially lack highly conserved amino acid motifs shown to be important for the catalysis of cytosine methylation. The possibility cannot be excluded, therefore, that alternative splicing of DNA (cytosine-5) methyltransferase 2 pre-mRNA may generate protein isoforms which have different methylating capabilities or which are involved in biological processes other than the catalysis of cytosine methylation.

Alternative Splicing↗
PubMed2001

Age-related loss of skeletal muscle function and the inability to express the autocrine form of insulin-like growth factor-1 (MGF) in response to mechanical overload.

The response of insulin-like growth factor-1 (IGF-1) signalling and the capacity of skeletal muscle to adapt to mechanical overload was studied using synergistic muscle ablation. Overload of the plantaris and soleus resulted in marked hypertrophy and activation of satellite cells (as indicated by MyoD expression), particularly in young rats. Two muscle IGF-1 splice variants were measured and found to be differentially regulated at the RNA level. The significant changes associated with the inability of the older muscles to respond to mechanical overload included the considerably lower expression of the local splice variant mechano growth factor, and the failure to up-regulate IGF-1 receptor and MyoD mRNA.

Aging↗