Susan E. Saunders

Chapter 9 Alternative mRNA splicing in the nervous system

Publisher Summary The product of transcription, termed the precursor messenger RNA (pre-mRNA), heterogenous nuclear RNA (hnRNA) or the primary transcript, undergoes a series of nuclear processing events before a mature messenger RNA (mRNA) is produced. This chapter describes the factors that regulate the synthesis of mRNA, particularly the splicing of neuropeptide precursor mRNAs. Eukaryotic gene expression requires the activities of complex biochemical machinery to transcribe, process, and transport mature mRNA before it can be translated to yield functional products. The functional consequence of most cases of alternative splicing is the production of protein isoforms sharing extensive regions of identity and varying only in specific domains. While the splicing strategy of the Drosophila gene appears simple, it may use the mechanisms of posttranslational processing to generate diversity from its transcriptional unit. The data show that the Lymnaea gene is similarly expressed in a wide variety of cell types. However, unlike the Drosophila central nervous system (CNS), which because of its small size has not been extensively studied in terms of neuropeptide physiology, the Lymnaea CNS proves to be an ideal model system in which to perform the studies of peptidergic and nonpeptidergic neural systems.

Multimeric CREB-binding sites in the promoter regions of a family of G-protein-coupled receptors related to the vertebrate galanin and nociceptin/orphanin-FQ receptor families

Four related genes encoding a family of G‐protein‐coupled receptors (GPCRs) have been isolated from the mollusc Lymnaea stagnalis. The coding regions of this family of receptors share 97–99% sequence similarity at both the protein and nucleotide level, and they also share high sequence identity with vertebrate galanin and orphanin‐FQ/nociceptin GPCR families. Analysis of the promoter regions reveals shared domains, some of which encode highly conserved repeating units. One 27‐bp repeating unit, which encodes a c‐AMP response element (CRE) and binds CREB protein, is repeated 14 times in one promoter. In situ hybridization showed expression of these receptors in identified neurons of several behaviourly important networks including those involved in feeding and ion and water regulation. These Lymnaea receptors are likely to represent members of a novel family of invertebrate neuropeptide receptors extensively regulated in response to intracellular signalling cascades.

5 – Neuropeptide FMRFamide Gene Expression

This chapter describes a variety of techniques to isolate (starting from the amino acid sequence only) and to analyze FMRFamide (Phe-Met-Arg-Phe- NH2) gene expression.* These include the polymerase chain reaction (PCR), DNA sequencing, in situ hybridization, and immunocytochemistry. The central nervous system of the pond snail Lymnaea was shown to contain two classes of peptide FMRFamide and GDP/SDPFLRFamide (1). These peptides are important physiologically in regulating heart rate in the animal and are also involved in other physiological systems (2). We describe here a variety of techniques used to determine whether the different classes of peptide are encoded by the same gene, how they are expressed at the RNA level, and whether the peptide products encoded by the genes are translated and expressed.