Cap-independent translation of human SP-A 5'-UTR variants: a double-loop structure and cis-element contribution.
Human surfactant protein A (hSP-A), a molecule of innate immunity and surfactant-related functions, consists of two functional genes, SP-A1 and SP-A2. SP-A expression is regulated by several factors including environmental stressors. SP-A1 and SP-A2 5'-untranslated region (5'-UTR) splice variants have a differential impact on translation efficiency and mRNA stability. To study whether these variants mediate internal ribosome entry site (IRES) activity (i.e., cap-independent translation), we performed transient transfection experiments in H441 cells with constructs containing one SP-A1 (A'D', AB'D', or A'CD') or SP-A2 (ABD) 5'-UTR splice variant between the Renilla and firefly luciferase genes of a bicistronic reporter vector. We found that 1) variants A'D', ABD, and AB'D' exhibit significantly higher IRES activities than negative control (no SP-A 5'-UTR) and A'CD' has no activity; the order of highest IRES activity was ABD > A'D' > AB'D; 2) IRES activity of ABD significantly increased in response to diesel particulate matter (20 microg/ml) but not in response to ozone (1 ppm for 1 h); 3) deletion mutants of ABD revealed regulatory elements associated with IRES activity; one at the end of exon A attenuated activity, whereas a region containing a short adenosine-rich motif in the second half of exon B and the start of exon D enhanced activity; 4) elimination of a predicted double-loop structure or increase in free energy significantly reduced IRES activity; 5) elimination of one or both double-loop structures in A'D' did not affect cap-dependent translation activity. Thus several factors, including cis-elements and secondary structure type and stability, are required for hSP-A 5'-UTR variant-mediated cap-independent translation.
Wang
Guirong
G
Department of Pediatrics, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Guo
Xiaoxuan
X
Silveyra
Patricia
P
Kimball
Scot R
SR
Floros
Joanna
J
D020121:5' Untranslated Regions / Q000235:genetics*; D017398:Alternative Splicing / Q000187:drug effects / Q000235:genetics; D001483:Base Sequence; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010126:Ozone / Q000494:pharmacology; D052638:Particulate Matter / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects; D037662:Pulmonary Surfactant-Associated Protein A / Q000235:genetics*; D012315:RNA Caps / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017384:Sequence Deletion
D020121:5' Untranslated Regions; D052638:Particulate Matter; D037662:Pulmonary Surfactant-Associated Protein A; D012315:RNA Caps; D010126:Ozone