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.

Non-coding functions of alternative pre-mRNA splicing in development

“…Surfactant protein A (Sp-A) is a surface-coating protein involved in immune response and normal functioning of the lung [38] . The human SP-A locus comprises two functional genes called SP-A1 and SP-A2 and both of them can generate a number of 5′UTR variants through combinatorial utilization of four alternative exons [38] , [39] , [40] . At least one of these mRNA species, the Sp-A1 ACD’ isoform, contains an uORF that has been shown to dampen protein production [40] .…”

Human Surfactant Protein SP-A1 and SP-A2 Variants Differentially Affect the Alveolar Microenvironment, Surfactant Structure, Regulation and Function of the Alveolar Macrophage, and Animal and Human Survival Under Various Conditions

“…A number of variants have been identified and characterized for each functional gene that may differentially affect regulation and/or function of alveolar cells, or the structure of surfactant films. These include variants resulting from alternative splicing events and variable translation initiation sites in the 5'-UTR (28)(29)(30), as well as isoforms with different signal peptide cleavage sites (23). Some of these have been shown to have an impact on translation efficiency and mRNA stability (31,32).…”

Differential Sex-Dependent Regulation of the Alveolar Macrophage miRNome of SP-A2 and co-ex (SP-A1/SP-A2) and Sex Differences Attenuation after 18 h of Ozone Exposure

“…Previous studies have demonstrated differences between SP-A1 and SP-A2 that include both qualitative (i.e., functional, biochemical, and/or structure) [ 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 ] and quantitative (regulatory) changes [ 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 ]. A recent study [ 60 ] examined SP-A genotype-dependent alterations in the bronchoalveolar lavage (BAL) proteome, and explored the effects of sex, infection, and ozone-induced oxidative stress on these changes.…”