Mohr Evita, Kächele Iris, Mullin Carola, Richter Dietmar
Universität Hamburg, Institut für Zellbiochemie und Klinische Neurobiologie, Martinistrasse 52, 20246 Hamburg, Germany.
Prog Brain Res. 2002;139:211-24. doi: 10.1016/s0079-6123(02)39018-6.
The genes encoding the vasopressin (VP) and oxytocin (OT) precursors are expressed in magnocellular neurons of the hypothalamo-neurohypophyseal system. The neuropeptides have a dual function: (1) they are secreted from the nerve terminals into the systemic circulation to act as hormones on various peripheral target organs; and (2) VP and OT are also released from the dendrites into the central nervous system where they presumably play a role as either neurotransmitters or as modulators of the classical transmitters. Substantial amounts of VP and OT mRNAs are sorted to both axons and dendrites. Since the latter are equipped with components of the translation machinery, the peptide hormone precursors are likely to be locally synthesized in dendrites of magnocellular neurons. Evidence for axonal precursor synthesis, on the other hand, has not been obtained. Subcellular mRNA localization is a complex pathway. It is determined by sequences (cis-acting elements) within the RNA and proteins (trans-acting factors) which interact with these elements in order to guide the molecules to their ultimate destination. We have investigated the mechanisms involved in mRNA targeting in neurons by using VP mRNA as a model system. Recombinant eukaryotic expression vectors harboring the VP cDNA have been microinjected into the cell nuclei of cultured superior cervical ganglion (SCG) neurons. The subcellular distribution of the vector-expressed mRNAs was determined by non-radioactive in situ hybridization techniques. This revealed transport of VP mRNA to the dendrites, but not to the axonal compartment of SCG neurons. A complex dendritic localizer sequence (DLS) that spans part of the coding region as well as the 3'-untranslated region was identified by microinjecting constructs encoding partial sequences of the VP mRNA. In order to characterize trans-acting factors interacting with this element, protein/RNA binding experiments with radiolabeled in vitro synthesized VP RNA probes and proteins extracted from rat brain have been carried out. A protein specifically interacts with the DLS of the VP mRNA but not with sequences that obviously lack a role in subcellular RNA transport. Biochemical purification revealed that this protein is the multifunctional poly(A)-binding protein (PABP). It is well known for its ability to bind with high affinity to poly(A) tails of mRNAs, prerequisite for mRNA stabilization and stimulation of translational initiation, respectively. With lower affinities, PABP can also associate with non-poly(A) sequences. The physiological consequences of these PABP/RNA interactions include functions such as translational silencing. The translational state of mRNAs subject to dendritic sorting is most likely influenced by external stimuli. Consequently, PABP could represent one of several components necessary to regulate local synthesis of the VP precursor and possibly of other proteins.
编码抗利尿激素（VP）和催产素（OT）前体的基因在下丘脑 - 神经垂体系统的大细胞神经元中表达。这些神经肽具有双重功能：（1）它们从神经末梢分泌到体循环中，作为激素作用于各种外周靶器官；（2）VP和OT也从树突释放到中枢神经系统中，在那里它们可能作为神经递质或经典递质的调节剂发挥作用。大量的VP和OT mRNA被分选到轴突和树突中。由于后者配备了翻译机制的组件，肽激素前体可能在大细胞神经元的树突中局部合成。另一方面，尚未获得轴突前体合成的证据。亚细胞mRNA定位是一个复杂的过程。它由RNA中的序列（顺式作用元件）和与这些元件相互作用以引导分子到达其最终目的地的蛋白质（反式作用因子）决定。我们以VP mRNA为模型系统研究了神经元中mRNA靶向所涉及的机制。携带VP cDNA的重组真核表达载体已被显微注射到培养的颈上神经节（SCG）神经元的细胞核中。通过非放射性原位杂交技术确定载体表达的mRNA的亚细胞分布。这揭示了VP mRNA向SCG神经元树突的转运，但未向轴突部分转运。通过显微注射编码VP mRNA部分序列的构建体，鉴定了一个跨越部分编码区以及3' - 非翻译区的复杂树突定位序列（DLS）。为了表征与该元件相互作用的反式作用因子，已经进行了用放射性标记的体外合成VP RNA探针和从大鼠脑中提取的蛋白质进行的蛋白质/RNA结合实验。一种蛋白质特异性地与VP mRNA的DLS相互作用，但不与明显在亚细胞RNA转运中不起作用的序列相互作用。生化纯化表明该蛋白质是多功能聚（A）结合蛋白（PABP）。众所周知，它能够以高亲和力结合mRNA的聚（A）尾巴，分别是mRNA稳定化和刺激翻译起始的前提条件。以较低的亲和力，PABP也可以与非聚（A）序列结合。这些PABP/RNA相互作用的生理后果包括翻译沉默等功能。经历树突分选的mRNA的翻译状态很可能受到外部刺激的影响。因此，PABP可能是调节VP前体以及可能其他蛋白质局部合成所需的几个组件之一。
