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This Article Full Text Full Text (PDF) All Versions of this Article: 296/4/L635    most recent 90508.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wang, G. Articles by Floros, J. Search for Related Content PubMed PubMed Citation Articles by Wang, G. Articles by Floros, J.

Cap-independent translation of human SP-A 5'-UTR variants: a double-loop structure and cis-element contribution

¹Penn State Center for Host Defense, Inflammation, and Lung Disease (CHILD) Research, Department of Pediatrics, and Departments of ²Cellular and Molecular Physiology and ³Obstetrics and Gynecology, Penn State Hershey College of Medicine, Pennsylvania State University, Hershey, Pennsylvania

Submitted 29 September 2008 ; accepted in final form 27 January 2009

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 µg/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.

circular RNA; internal ribosome entry site; H441 cells; diesel particulate matter; secondary structure of mRNA; surfactant protein A; 5'-untranslated region