Human SP-A1 (SFTPA1) variant-specific 3' UTRs and poly(A) tail differentially affect the in vitro translation of a reporter gene.
Human surfactant protein A (SP-A) is encoded by two functional genes (SFTPA1, SFTPA2) with a high degree of sequence identity. Sequence differences among these genes and their genetic variants have been observed at the 5' and 3' untranslated regions (UTRs). In this work, we studied the impact on translation of the SFTPA1 (hSP-A1) and SFTPA2 (hSP-A2) gene 5' UTR splice variants and 3' UTR sequence variants, in the presence or absence of poly(A) tail. We generated constructs containing the luciferase reporter gene flanked upstream by one of the hSP-A 5' UTR splice variants and/or downstream by one hSP-A 3' UTR sequence variant. mRNA transcripts were prepared by in vitro transcription and used for either in vitro translation with a rabbit reticulocyte lysate or transient transfection of the lung adenocarcinoma cell line NCI-H441. The luciferase activity results indicate that hSP-A 5' UTR and 3' UTR together have an additive effect on translation. In this context, the hSP-A1 6A(3) and 6A(4) 3' UTR variants exhibited higher translation efficiency than the 6A(2) variant (P <0.05), whereas no significant difference was observed between the two hSP-A2 3' UTRs studied (1A(0), 1A(3)). Further sequence analysis revealed that a deletion of an 11-nucleotide (nt) element in both the 6A(3) and 6A(4) 3' UTR variants changes the predicted secondary structure stability and the number of putative miRNA binding sites. Removal of this 11-nt element in the 6A(2) 3' UTR resulted in increased translation, and the opposite effect was observed when the 11-nt element was cloned in a guest 3' UTR (6A(3), 6A(4)). These results indicate that sequence differences among hSP-A gene variants may account for differential regulation at the translational level.
Silveyra
Patricia
P
Penn State Center for Host Defense, Inflammation, and Lung Disease Research, Department of Pediatrics, Pennsylvania State University, Hershey, USA.
Wang
Guirong
G
Floros
Joanna
J
D020413:3' Untranslated Regions / Q000235:genetics*; D020121:5' Untranslated Regions / Q000235:genetics*; D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D000818:Animals; D001483:Base Sequence; D014644:Genetic Variation; D006801:Humans; D008156:Luciferases / Q000378:metabolism*; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D035683:MicroRNAs / Q000502:physiology; D008969:Molecular Sequence Data; D011061:Poly A / Q000235:genetics*; D014176:Protein Biosynthesis; D037662:Pulmonary Surfactant-Associated Protein A / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D035683:MicroRNAs; D037662:Pulmonary Surfactant-Associated Protein A; C445842:SFTPA1 protein, human; C445843:SFTPA2 protein, human; D011061:Poly A; D008156:Luciferases