Synthetic oligonucleotides were used to screen a rat striatal cDNA library for sequences corresponding to the tachykinin peptides substance P and neurokinin A. The cDNA library was constructed from RNA isolated from the rostral portion of the rat corpus striatum, the site of striatonigral cell bodies. Two types of cDNAs were isolated and dermed by restriction enzyme analysis and DNA sequencing to encode both substance P and neurokinin A. The two predicted preprotachykinin protein precursors (130 and 115 amino acids in length) differ from each other by a pentadecapeptide sequence between the two tachykinin sequences, and both precursors possess appropriate processing signals for substance P and neurokinin A production. The presence of a third preprotachykinin mRNA of minor abundance in rat striatum was established by S1 nuclease protection experiments. This mRNA encodes a preprotachykinin of 112 amino acids containing substance P but not neurokinin A. These three mRNAs are derived from one rat gene as a result of differential RNA processing; thus, this RNA processing pattern further increases the diversity of products that can be generated from the preprotachykinin gene.Of all the naturally occurring neuropeptides, the undecapeptide substance P (SP) is perhaps the peptide best documented as a neurotransmitter and/or neuromodulator substance in both central and peripheral nervous systems (1, 2). SP belongs to a family of structurally related peptides, the tachykinins. Peptides in this family include neurokinin A (NKA, also known as substance K) and neurokinin B (see ref.3 for discussion); they all exhibit similar biological activities-including contraction of smooth muscle and hypotension-and share a common COOH-terminal sequence, PheXaa-Gly-Leu-Met-NH2 but possess distinct NH2-terminal sequences that convey receptor specificities.Little is known about the biosynthesis of the tachykinins and the regulation ofpreprotachykinin (PPT) gene expression (4). Nawa et al. (5) have recently cloned cDNAs that correspond to two types of bovine PPT mRNAs (a and /3) that are derived from one gene (5, 6). Because most information about the localization and functions of the tachykinins and their receptors has involved the rat nervous system, it is the system of choice for an analysis of tachykinin gene expression. It is important to identify the primary structure of rat PPT mRNAs, the precursor proteins encoded by these mRNAs, and the specific gene(s) encoding the PPT mRNAs as well as to understand its regulation. The development of a specific and sensitive assay for individual PPT mRNAs is also necessary. We report here progress made toward these goals, describing three rat PPH mRNAs that encode SP and NKA derived from a single gene; differential RNA processing yields the structural differences of these peptide precursors. A preliminary account of this work has appeared (7). (Holtzmann, Madison, WI) was used for RNA isolation by the guanidine isothiocyanate method (9). Poly(A)+ RNA was isolated using oligo(dT)-cellulos...
The rat preprotachykinin (PPT) gene encoding the neuropeptides substance P (SP), neurokinin A (NKA), neuropeptide K (NPK), and neuropeptide gamma was isolated from a lambda Charon 4A genomic library. Two overlapping clones contained all of the exons present in beta-PPT, including some 7 and 9 kb 5' and 3' flanking sequence, respectively. The presence of 1 major and 2 minor transcription initiation sites was determined from primer extension and nuclease protection experiments. Analysis of the nucleotide sequence homology between the rat and bovine revealed the presence of highly conserved regions throughout the entire coding region and within the 5' flanking sequences. Primer extension and nuclease protection experiments demonstrated that the primary transcript is differentially spliced primarily into gamma- and beta-PPT mRNA in all tissues examined in the adult rat where the gene is expressed. beta-PPT mRNA contains all of the exons, whereas gamma-PPT mRNA lacks exon 4, which encodes part of the N-terminus of NPK. The alpha-PPT mRNA, which lacks exon 6 (the sequence encoding NKA and processing sites), comprises about 1% of the total PPT mRNA. An RNA secondary structure model is proposed to account for these specific exon exclusion events in the RNA splicing process. These results are discussed with regard to the mechanisms regulating SP gene expression and the functional significance of differential RNA splicing in the rat.
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