Extrasomatic targeting of MAP2, vasopressin and oxytocin mRNAs in mammalian neurons.
Explore the source record for details and available documents.
3' Untranslated Regions↗
Biomedical subjects
M Rehbein
Publications and source records attributed to M Rehbein.
Identification of a cis-acting dendritic targeting element in the mRNA encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II.
In mammalian neurons a selected group of mRNAs, including the transcript encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II, is found in dendrites. The molecular mechanisms underlying extrasomatic RNA trafficking are not well described. It is thought that dendritic transcripts contain cis-acting elements that direct their selective subcellular sorting. Here we report the identification of an extrasomatic targeting element in the 3' untranslated region of the mRNA encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II. In primary hippocampal neurons, this 1200-nucleotide-spanning, cis-acting element is sufficient to mediate dendritic localization of chimeric reporter transcripts. The trafficking signal does not share any striking sequence similarity with a previously characterized dendritic targeting element in transcripts encoding the microtubule-associated protein 2. In dendrites of transfected primary neurons, recombinant RNAs form granules with an average diameter of 0.45 microm that may represent preferential RNA docking sites or multimolecular transport units. These findings imply that extrasomatic sorting of individual dendritic mRNAs involves at least partially distinct molecular mechanisms, as well as large trafficking complexes.
3' Untranslated Regions↗
Two rat brain staufen isoforms differentially bind RNA.
In neurones, a limited number of mRNAs is found in dendrites, including transcripts encoding the microtubule-associated protein 2 (MAP2). Recently, we identified a cis-acting dendritic targeting element (DTE) in MAP2 mRNAs. Here we used the yeast tri-hybrid system to identify potential trans-acting RNA-binding factors of the DTE. A cDNA clone was isolated that encodes a member of a mammalian protein family that is highly homologous to the Drosophila RNA-binding protein Staufen. Mammalian Staufen appears to be expressed in most tissues and brain areas. Two distinct rat brain Staufen isoforms, rStau+I6 and rStau-I6, are encoded by alternatively spliced mRNAs. Both isoforms contain four double-stranded RNA-binding domains (dsRBD). In the larger rStau+I6 isoform, six additional amino acids are inserted in the second dsRBD. Although both isoforms interacted with the MAP2-DTE and various additional RNA fragments in an in vitro north-western assay, rStau-I6 exhibited a stronger signal of bound radioactively labelled RNAs as compared with rStau+I6. Using an antibody directed against mammalian Staufen, the protein was detected in somata and dendrites of neurones of the adult rat hippocampus and cerebral cortex. Ultrastructural studies revealed that in dendrites, rat Staufen accumulates along microtubules. Thus in neurones, rat Staufen may serve to link RNAs to the dendritic microtubular cytoskeleton and may thereby regulate their subcellular localization.
Alternative Splicing↗
Two trans-acting rat-brain proteins, MARTA1 and MARTA2, interact specifically with the dendritic targeting element in MAP2 mRNAs.
Different isoforms of the microtubule-associated protein 2 (MAP2) are somatodendritic components of neurons that seem to regulate the stability of the dendritic cytoskeleton. MAP2 localization into dendrites appears to be a complex multicausal mechanism that involves the specific recruitment of MAP2 mRNAs into dendritic compartments. Recently, we have functionally characterized a 640-nucleotide dendritic targeting element (DTE) in the 3' untranslated region (3' UTR) of MAP2 transcripts that mediates extrasomatic mRNA localization in primary neurons (Blichenberg et al. , 1999). In analogy to molecular mechanisms regulating cytoplasmic RNA translocation in other cell systems, we propose that, in vivo, the cis-acting MAP2-DTE interacts with specific protein factors present in neurons. To identify putative trans-acting DTE-binding proteins, we performed in vitro ultraviolet crosslinking assays. Using this experimental system, two 90-kDa and 65-kDa MAP2-RNA trans-acting proteins, MARTA1 and MARTA2, were identified in rat-brain extracts. Both MARTAs bind with high affinity to the MAP2-DTE, but not to other investigated regions of MAP2 transcripts or the somatically restricted alpha-tubulin mRNA. Moreover, MARTA1 and MARTA2 do not bind significantly to other dendritically localized transcripts encoding vasopressin and arg3.1, nor to a dendritic trafficking element from the mRNA encoding the alpha-subunit of the Ca(2+)/calmodulin-dependent protein kinase II. Binding of MARTA1 and MARTA2 to the MAP2-DTE occurs with an affinity in the nanomolar range. Whereas MARTA1 is clearly detectable in crude lysates, cytosolic and ribosomal salt-wash fractions, and in nuclear extracts, MARTA2 is preferentially found in the ribosomal salt-wash preparation. Neither MARTA is restricted to rat brain, and both are present in a number of other rat tissues. Thus, both proteins may be involved in a variety of nuclear and cytoplasmic events that regulate RNA metabolism in different cell types.
3' Untranslated Regions↗
Identification of a cis-acting dendritic targeting element in MAP2 mRNAs.
In neurons, a limited number of mRNAs have been identified in dendritic processes, whereas other transcripts are restricted to the cell soma. Here we have investigated the molecular mechanisms underlying extrasomatic localization of mRNAs encoding microtubule-associated protein 2 (MAP2) in primary neuronal cultures. Vectors expressing recombinant mRNAs were introduced into hippocampal and sympathetic neurons using DNA transfection and microinjection protocols, respectively. Chimeric mRNAs containing the entire 3' untranslated region of MAP2 transcripts fused to a nondendritic reporter mRNA are detected in dendrites. In contrast, RNAs containing MAP2 coding and 5' untranslated regions or tubulin sequences are restricted to the cell soma. Moreover, 640 nucleotides from the MAP2 3' untranslated region (UTR) are both sufficient and essential for extrasomatic localization of chimeric mRNAs in hippocampal and sympathetic neurons. Thus, a cis-acting dendritic targeting element that is effective in two distinct neuronal cell types is contained in the 3' UTR of MAP2 transcripts. The observation of RNA granules in dendrites implies that extrasomatic transcripts seem to assemble into multimolecular complexes that may function as transport units.
3' Untranslated Regions↗
The rat insulin-degrading enzyme. Molecular cloning and characterization of tissue-specific transcripts.
The primary structure of the rat insulin-degrading enzyme (IDE) was determined by cDNA analysis. Rat IDE, as well as the previously characterized homologs from human and Drosophila, contain the carboxyl-terminal consensus sequence A/S-K-L, for peroxisome targeting. A stretch of 43 bp surrounding an alternatively used polyadenylation site is highly conserved between rat and human, suggesting that it may contain important regulatory information. Northern blot analysis revealed two IDE transcripts of 3.7 and 5.5 kb in various tissues. Testis was found to be exceptional in having three different RNAs (3.7, 4.1 and 6.1 kb) at a relatively high abundance. The expression of the IDE gene in testis is correlated with sexual maturation.
Amino Acid Sequence↗
Molecular characterization of a novel rat protein structurally related to poly(A) binding proteins and the 70K protein of the U1 small nuclear ribonucleoprotein particle (snRNP)
A cDNA has been isolated from a rat testis library which encodes a novel protein of 100 kDa that contains domains found in two different proteins involved in the processing of pre-mRNAs. Computer-assisted comparison reveals that one sequence motif of 30 amino-acid residues is very similar to a region conserved in the C-terminal part of eukaryotic poly(A) binding proteins (PABP). A second region of the rat 100 kDa protein, containing alternating basic, mostly arginine, and acidic amino-acid residues, is structurally related to sequence motifs found in the 70K protein of the U1 small nuclear ribonucleoprotein particle (snRNP), which is involved in RNA splicing. Northern blot analysis shows that a corresponding 9.5 kb transcript is highly expressed in rat testis; lower mRNA levels are found in other tissues such as liver, kidney, lung and brain. Ontogenic studies reveal that the expression of the 100 kDa protein-encoding gene and sexual maturation are correlated, being barely detectable during early post-natal life but reaching maximal levels around the first month after birth.
Amino Acid Sequence↗
Poly(A) tail length of oxytocin- and lysine vasopressin-encoding mRNAs increases during development in the porcine hypothalamus.
Lysine vasopressin- and oxytocin-encoding mRNAs have been analysed in the developing hypothalamus of the pig. The two hormone-encoding mRNAs were first detectable on fetal day 49 by Northern blot analysis. Whereas RNase mapping revealed identical transcripts throughout the developmental stages studied, Northern blots showed that the early transcripts appeared to be shorter (by 100-200 nucleotides) and more heterogeneous in size than those of later stages. This developmentally related length polymorphism was shown to be due to different poly(A) lengths and was abolished by removal of the poly(A) tails with RNase H. These results indicate that maturation of neurones in the developing porcine hypothalamus is accompanied by an increase in length of the poly(A) tail of vasopressin and oxytocin mRNAs.
Amino Acid Sequence↗
The neurohypophyseal hormones vasopressin and oxytocin. Precursor structure, synthesis and regulation.
Complete cDNA sequences for the vasopressin and oxytocin precursor polyproteins have been determined for the rat, calf, human and pig (vasopressin only), indicating the essential conservation of the precursor structures throughout mammals. DNA probes specific for vasopressin or oxytocin mRNAs have been used to identify both classic (hypothalamic) and novel (thymus, corpus luteum, phaeochromocytoma) sites of hormone expression. Semiquantitative DNA/RNA hybridization suggests that in rats expression of the vasopressin and oxytocin genes is positively effected by osmotic stress, negatively by a systemically applied excess of vasopressin; in the latter experiment a reduction in the hypothalamic levels of vasopressin and oxytocin mRNAs in normal and Brattleboro rats have been observed. This suggests a feedback regulation by the hormone as a possible element in controlling the transcription of the vasopressin gene.
Animals↗
Deduced amino acid sequence from the bovine oxytocin-neurophysin I precursor cDNA.
The nonapeptide hormone oxytocin-like arginine-vasopressin (AVP) is synthesized as part of a larger precursor polypeptide. The precursor also includes the neurophysin molecule with which the hormone is associated in the neurosecretory granules of the hypothalamo-pituitary tract. A protein of molecular weight (Mr) approximately 20,000 has been isolated from supraoptic nuclei of rat hypothalami which, after tryptic cleavage, released a neurophysin-like molecule of Mr approximately 10,000 and an oligopeptide related to oxytocin. This result was complemented by in vitro translation of bovine hypothalamic mRNA. Among the primary translation products a single polypeptide of Mr approximately 16,500 was shown to contain antigenic determinants recognized by specific antisera against bovine neurophysin I and oxytocin. Here we report the amino acid sequence of the bovine oxytocin-neurophysin I (OT-NpI) precursor which was derived from sequence analysis of the cloned cDNA. As is the case for the bovine arginine-vasopressin-neurophysin II (AVP-NpII) precursor, the signal sequence of the OT-NpI precursor is immediately followed by the nonapeptide hormone which is connected to neurophysin I by a Gly-Lys-Arg sequence. A striking feature of the nucleic acid sequence is the 197-nucleotide long perfect homology with the AVP-NpII precursor mRNA sequence encoding the conserved middle part of neurophysins I and II.
Amino Acid Sequence↗