title: Nicotinic acetylcholine receptor subunits α4 and α5 associated with smoking behaviour and lung cancer are regulated by upstream open reading frames.
abstract: Nicotinic acetylcholine receptor subunits (nAChR) are associated with different aspects of smoking behaviour as well as with smoking related disorders. Several of these subunits have been found to be upregulated in smokers or differentially expressed in lung tumor cells. The mechanisms behind these observations are not known but assumed to be mainly post-transcriptional. Many post-transcriptional mechanisms are initiated by functionally relevant sequence motifs within untranslated gene regions, such as upstream open reading frames (uORFs). We performed a systematic search in all smoking-associated neuronal nAChR subunits and identified functionally relevant uORFs in CHRNA4 and CHRNA5. Luciferase experiments showed that these uORFs are able to significantly decrease protein expression. Our quantitative real-time PCR (qPCR) results strongly suggest that the observed effects originate at the translation rather than at the transcription level. Interestingly, the CHRNA4 uORF was only functionally relevant when expressed in the shorter isoform of this gene. Therefore, the data presented in this study strongly points towards an important role of uORFs within the 5'UTR of CHRNA4-isoform 1 and CHRNA5 as regulators of protein translation. Moreover, the shared uORF of CHRNA4-isoform 1/isoform 2 represents the first example of a sequence context-dependent uORF.
authors.affiliation: Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University Munich, Munich, Germany. Marlene.Eggert@med.uni-muenchen.de
authors.forename: Marlene
authors.initials: M
authors.lastname: Eggert
authors.forename: Eric
authors.initials: E
authors.lastname: Aichinger
authors.forename: Michael W
authors.initials: MW
authors.lastname: Pfaffl
authors.forename: Ortrud K
authors.initials: OK
authors.lastname: Steinlein
authors.forename: Martina
authors.initials: M
authors.lastname: Pfob
mesh_terms: D020121:5' Untranslated Regions; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D011978:Receptors, Nicotinic / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D012907:Smoking / Q000235:genetics / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D008156:Luciferases; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D011978:Receptors, Nicotinic; C447158:CHRNA5 protein, human