Alternative splicing in single cells dissected from complex tissues: separate expression of prepro-tachykinin A mRNA splice variants in sensory neurones.
Tachykinins play an important role in peripheral inflammatory diseases and disorders of the CNS. Most members of the tachykinin family are generated by alternative post-transcriptional splicing of the prepro-tachykinin (PPT) A gene. Here, we examined the simultaneous expression of PPT-A splice variants in individual neurones of the nodose ganglion. In extracts of ganglia, the expression of the four PPT-A mRNA splice variants and their four encoded peptides was shown by RT-PCR and combined HPLC and radioimmunoassay respectively. In order to examine prepro-tachykinin A expression in individual cells, single neurones were isolated from the ganglia using laser-assisted microdissection and processed for RT-PCR. Some 31.9% of the neurones investigated expressed a specific PPT-A transcript. Each individual neurone was found to express only a single splice variant. This is the first study to analyse the differential expression of PPT-A splice variants at the single-cell level. In view of the large number of alternatively spliced genes in the human genome and the resulting profound physiological effects, including several diseases, the technique described here is useful for isolating cells without possible confounding effects of dissociated neuronal cultures. For PPT-A, the results indicate that alternative post-transcriptional splicing determines the tachykinergic phenotype and may therefore have important functional implications.
Springer
Jochen
J
Department of Anatomy and Cell Biology, Justus-Liebig-University, Giessen, Germany. jochen.springer@charite.de
McGregor
Gerard P
GP
Fink
Ludger
L
Fischer
Axel
A
D017398:Alternative Splicing; D000818:Animals; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D005260:Female; D007834:Lasers; D008297:Male; D051379:Mice; D009475:Neurons, Afferent / Q000737:chemistry / Q000378:metabolism*; D009620:Nodose Ganglion / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis*; D011863:Radioimmunoassay; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D015320:Tachykinins / Q000235:genetics*
D011498:Protein Precursors; D012333:RNA, Messenger; D015320:Tachykinins; C044206:preprotachykinin