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Activity of cap‐independent translation depends on a double‐loop structure in human SP‐A 5'‐UTR variants
Author(s) -
Wang Guirong,
Guo Xiaoxuan,
Kimball Scot R.,
Floros Joanna
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.785.1
Subject(s) - internal ribosome entry site , untranslated region , five prime untranslated region , translation (biology) , microbiology and biotechnology , biology , exon , messenger rna , eukaryotic translation , enhancer , gene , genetics , gene expression
Human surfactant protein A (SP‐A), a molecule of innate immunity and surfactant‐related functions, consists of two functional genes, SP‐A1 and SP‐A2. Several SP‐A 5′UTR splice variants have been identified with differences in translation efficiency and mRNA stability. To study whether these 5′UTR variants are subject to internal ribosome entry site (IRES) activity (i.e. Cap‐independent translation activity), we generated several constructs using a bicistronic reporter vector, in which each contained one SP‐A 5′UTR variant (A′D′, ABD, AB′D′, or A′CD′) between the Renilla and firefly luciferase genes. The results indicated that variants A′D′, ABD, AB′D′ exhibit significantly higher IRES activities than the negative control (plasmid without 5′UTR insert), but the A′CD′ had no IRES activity. Deletion mutation analysis of the ABD variant revealed that at least two cis‐acting regulatory elements associate with IRES activity. An element at the end of exon A attenuated activity whereas elements in the second half of exon B and the start of exon D enhanced activity. Further analysis by predicted mRNA secondary structure and site‐directed mutation, revealed that the activity of the enhancer element depends on a double‐loop structure. Similarly, elimination of a double‐loop structure in the A′D′ variant resulted in a decrease in IRES activity, but with no impact on cap‐dependent translation activity. Supported by NIH HL‐34788