Worster B M, Yeoman M S, Benjamin P R
Sussex Centre for Neuroscience, School of Biological Sciences, University of Sussex, Brighton, East Sussex, UK.
Eur J Neurosci. 1998 Nov;10(11):3498-507. doi: 10.1046/j.1460-9568.1998.00361.x.
MALDI-ToF MS (matrix-assisted laser desorption/ionization time of flight mass spectrometry) has become a fast, reliable and sensitive technique for the identification of neuropeptides in biological tissues. Here, we applied this technique to identified neurons of the cardioregulatory network in the snail Lymnaea that express the FMRFamide gene. This enabled us to study the complex processing of the FMRFamide gene at the level of single identified neurons. In the CNS of Lymnaea, FMRFamide-like and additional peptides are encoded by a common, multiexon gene. Alternate mRNA splicing of the FMRFamide gene leads to the production of two different mRNAs. Type 1 mRNA (exon II) encodes for the tetrapeptides (FLRF/FMRFamide), whereas Type 2 (exons III-V) encodes for the heptapeptides (SDPFLRFamide/GDPFLRFamide). Previous in situ hybridization and immunocytochemical studies indicated that these two transcripts are expressed in the CNS neurons of Lymnaea in a differential and mutually exclusive manner. Two single identified neurons of the cardiorespiratory network, the Ehe neuron and the visceral white interneuron (VWI), were known to express the FMRFamide gene (Ehe, type 1 mRNA; VWI, type 2 mRNA). MALDI-ToF MS analysis of these neurons and other neurons expressing the FMRFamide gene confirmed the mutually exclusive expression of the distinct sets of peptides encoded on the two transcripts and revealed the pattern of post-translational processing of both protein precursors. From the gene sequence it was predicted that 16 final peptide products from the two precursor proteins could possibly exist. We showed that most of these peptides were indeed present in the identified neurons (13) while others were not (three), suggesting that not all of the potential cleavage sites within the two precursors are utilized. In this way, the neuronal expression of the full range of the peptide products resulting from alternative mRNA splicing was revealed for the first time.
基质辅助激光解吸/电离飞行时间质谱（MALDI-ToF MS）已成为一种用于鉴定生物组织中神经肽的快速、可靠且灵敏的技术。在此，我们应用该技术鉴定了田螺中表达FMRF酰胺基因的心脏调节网络神经元。这使我们能够在单个已鉴定神经元水平上研究FMRF酰胺基因的复杂加工过程。在田螺的中枢神经系统中，FMRF酰胺样肽和其他肽由一个常见的多外显子基因编码。FMRF酰胺基因的可变mRNA剪接导致产生两种不同的mRNA。1型mRNA（外显子II）编码四肽（FLRF/FMRF酰胺），而2型（外显子III-V）编码七肽（SDPFLRF酰胺/GDPFLRF酰胺）。先前的原位杂交和免疫细胞化学研究表明，这两种转录本在田螺的中枢神经系统神经元中以差异且相互排斥的方式表达。已知心脏呼吸网络中的两个单个已鉴定神经元，即Ehe神经元和内脏白色中间神经元（VWI），表达FMRF酰胺基因（Ehe，1型mRNA；VWI，2型mRNA）。对这些神经元以及其他表达FMRF酰胺基因的神经元进行的MALDI-ToF MS分析证实了两种转录本上编码的不同肽组的相互排斥表达，并揭示了两种蛋白质前体的翻译后加工模式。从基因序列预测，两种前体蛋白可能产生16种最终肽产物。我们表明，这些肽中的大多数确实存在于已鉴定的神经元中（13种），而其他的则不存在（3种），这表明并非两种前体中的所有潜在切割位点都被利用。通过这种方式，首次揭示了由可变mRNA剪接产生的全部肽产物的神经元表达情况。
