title: Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants.
abstract: Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5'-untranslated regions (5'-UTR) due to differential splicing. The 5'-UTR variant ACD' is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication.
authors.affiliation: Center for Host Defense, Inflammation and Lung Disease (CHILD) Research, Department of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania;
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Tsotakos
authors.affiliation: Center for Host Defense, Inflammation and Lung Disease (CHILD) Research, Department of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania;
authors.forename: Patricia
authors.initials: P
authors.lastname: Silveyra
authors.affiliation: Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania;
authors.forename: Zhenwu
authors.initials: Z
authors.lastname: Lin
authors.affiliation: Center for Host Defense, Inflammation and Lung Disease (CHILD) Research, Department of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania; Department of Public Health Sciences, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania; and.
authors.forename: Neal
authors.initials: N
authors.lastname: Thomas
authors.affiliation: Center for Host Defense, Inflammation and Lung Disease (CHILD) Research, Department of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania;
authors.forename: Mudit
authors.initials: M
authors.lastname: Vaid
authors.affiliation: Center for Host Defense, Inflammation and Lung Disease (CHILD) Research, Department of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania; Department of Obstetrics and Gynecology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania jfloros@psu.edu.
authors.forename: Joanna
authors.initials: J
authors.lastname: Floros
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects / Q000502:physiology*; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D018387:Codon, Initiator / Q000502:physiology*; D003907:Dexamethasone / Q000494:pharmacology; D004731:Endotoxins / Q000494:pharmacology; D005091:Exons / Q000502:physiology; D005260:Female; D006801:Humans; D008168:Lung / Q000378:metabolism*; D008297:Male; D008861:Microsomes / Q000378:metabolism; D009154:Mutation; D021382:Protein Sorting Signals / Q000502:physiology; D037662:Pulmonary Surfactant-Associated Protein A / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000893:Anti-Inflammatory Agents; D018387:Codon, Initiator; D004731:Endotoxins; D021382:Protein Sorting Signals; D037662:Pulmonary Surfactant-Associated Protein A; D012333:RNA, Messenger; C445842:SFTPA1 protein, human; D003907:Dexamethasone
keywords: ribosome binding site; scanning mechanism; translation regulation; uAUG