Human SP-A1 (SFTPA1) variant-specific 3' UTRs and poly(A) tail differentially affect the in vitro translation of a reporter gene

Abstract

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.

Fig. 1.

A: diagram of the human surfactant protein A (SP-A) gene locus. Two human SP-A (hSP-A) genes (SP-A1, SP-A2) of ∼5 kb and a pseudogene (P) are located in the long arm of chromosome 10 in opposite transcriptional orientation. B: schematic representation of the hSP-A1 and hSP-A2 gene structure. The 5′ untranslated region (UTR) contains 4 exons (A–D) of variable size: A (44 nt), A′ (39 nt), A″ (34 nt), B (30 nt), B′ (70 nt), C (60 nt), C′ (63 nt), D (26 nt), and D′ (23 nt). Exons A, A′, and A″ are represented by A*. The coding region also contains 4 exons: I (171 nt), II (117 nt), III (75 nt), and IV (125 nt), that is continued by the hSP-A 3′ UTR (1.3 kb). C: alternative splicing patterns found at the 5′ UTR for the hSP-A1 and hSP-A2 mRNA variants.

Fig. 2.

A depicts the basic construct used to generate mRNA with the hSP-A 5′ UTR gene variants (hSP-A1: AD′; hSP-A2: ABD, ABD′; con: no 5′UTR) and/or 3′ UTR variants (hSP-A1: 6A², 6A³, 6A⁴; hSP-A2: 1A⁰, 1A³; con: no 3′ UTR). The previously generated rpcDNA3/5′-UTR/Luc vector was modified as described in materials and methods. A fragment of ∼1.3 kb (3′ UTR) containing an SpeI site for linearization was inserted into the XhoI site of the vector downstream of the luciferase stop codon. B shows the experimental constructs (n = 12) used for in vitro transcription. The start (ATG) and termination (TAA) codons are shown. At the 5′ end of luciferase, the hSP-A2 constructs contain either the ABD or ABD′ variants, whereas the hSP-A1 contains the AD′ variant. These 5′ UTRs are the predominant splice variants previously identified for each gene. The control constructs lack the hSP-A 5′ UTR upstream luciferase. All constructs contain a variant-specific 3′ UTR as well as XhoI restriction site to obtain transcripts without 3′ UTR. C depicts a 0.8% agarose denaturing RNA gel with different size transcripts in the presence and absence of poly(A).

Fig. 3.

Comparison of the translational efficiency measured as Firefly/Renilla luciferase activity ratio among hSP-A1 and hSP-A2 5′ UTR and/or 3′ UTR variants, in the presence or absence of poly(A) tail. Capped mRNAs containing hSP-A 5′ UTR splice variants (hSP-A1: AD′; hSP-A2: ABD, ABD′) and/or hSP-A 3′ UTR sequence variants (hSP-A1: 6A², 6A³; 6A⁴; hSP-A2: 1A⁰, 1A³) flanking the luciferase reporter gene (Luc) were generated by in vitro transcription using the mMessage mMachine Ultra kit (Ambion) and translated in vitro with the rabbit reticulocyte lysate system (RRL), in the presence of a Renilla luciferase control transcript, as described in materials and methods. Results are shown as means ± SE (n = 9–15). Comparisons with P < 0.05 were considered significant, and are marked with superscripts (a–e), as follows. hSP-A 5′ UTR modulated translation with different efficiencies (ABD = ABD′ > AD′ > no 5′ UTR, P < 0.05) in the absence of 3′ UTR in poly(A)⁻ (a) and poly(A)⁺ (b) mRNA; c: all the hSP-A 3′ UTR variants increased translation over control [no 5′ UTR, no 3′ UTR, and poly(A)⁻], but no differences were observed among variants in the presence or absence of poly(A); d: for hSP-A1 and hSP-A2, an increased translation over control [no 5′ UTR, no 3′ UTR, and poly(A)⁻] was observed in the presence of both 5′ UTR and 3′ UTR, but no differences were observed among variants; e: the poly(A) tail increased the efficiency of translation in mRNAs without 3′ UTR.

Fig. 4.

Relative Luciferase activity over time in NCI-H441 cells transfected with 2 experimental mRNAs. The Firefly to Renilla Luc activity ratio was calculated after transfection of cells with 1.5 μg of mRNA using the TransIT-mRNA transfection kit (MirusBio) for short (30–240 min) or long (0.5–24 h; inset) periods of time. The 90-min time point (arrow) was chosen for further experiments.

Fig. 5.

Human SP-A 5′ UTR and 3′ UTR variants differentially regulate the translation of the reporter gene. Translational efficiency was measured in the lung cell line NCI-H441, as the ratio of Firefly and Renilla luciferase activities, after 90 min of transfection with mRNA transcripts containing hSP-A 5′ UTR variants (A), hSP-A 3′ UTR variants (B), or the combination of both hSP-A1 or hSP-A2 5′ UTR and 3′ UTR variants (C), in the presence or absence of poly(A) tail. NCI-H441 cells were transiently transfected with the experimental (Firefly luciferase) and the control (Renilla luciferase) mRNA using the TransIT-mRNA transfection kit (MirusBio), and activity was determined as described in materials and methods. Results are shown as means ± SE (n = 9–15). a–k: statistically significant (P < 0.05) differences are marked by different symbols, as indicated in each panel. A: in the absence of 3′ UTR: a, poly(A)⁺ RNA displayed higher activity than poly(A)⁻ RNA (P < 0.05); b and c, a differential efficiency of translation was observed among 5′ UTR variants (ABD = ABD′ > AD′ > con) (P < 0.05). B: the 3′ UTR increased the efficiency of translation in all variants in absence of 5′ UTR. C: in transcripts containing both 5′ UTR and 3′ UTR, the efficiency of translation (d–k) was significantly higher (P < 0.05) for the hSP-A1 6A³ and 6A⁴ variants than the 6A² and all hSP-A2 variants. To enable a better comparison of the data, C shows some of the data already shown in A and B (transcripts without 3′ UTR or 5′ UTR, respectively).

Fig. 6.

A deletion in the 3′ UTR alters the predicted mRNA stability and secondary structure of the hSP-A variants. The RNAfold online software was used to obtain the secondary structures for the hSP-A 3′ UTR mRNA sequences used in this study. A comparison among the minimum free energy secondary structures of the 6A², 6A³, and 6A⁴ 3′ UTR variants is shown (right). The specific region containing the 11-nt deletion-insertion element is amplified and shown (left) (nt 383–500 of the mRNA 3′ UTR from the luciferase stop codon). The minimum free energy (dG) was calculated for all the structures. The colors indicate the propensity of individual nucleotides to participate in base pairs and whether or not a predicted base pair is well determined. The scale ranges from red (highest probability) to blue-violet (lower probability). The 6A³ and 6A⁴ 3′ UTR variants (lacking the 11-nt element) revealed a more stable secondary structure, with lower dG and higher base pair probabilities, and with lower positional entropy (not shown).

Fig. 7.

The 11-nt element of the hSP-A 3′ UTR revealed variant-specific predicted miRNA binding sites. Top illustrates the sequence alignment of the 3′ UTR 11-nt insertion-deletion element adjacent region (nt 387–436 of the 3′ UTR). At bottom, a graphic representation of the putative human miRNA interacting sites identified by the RNAreg and PITA software (described in materials and methods). Seven human microRNAs (hsa-mir) were predicted to interact with this region of the hSP-A2 (1A⁰ and 1A³) and the hSP-A1 6A² 3′ UTR variants, but not with that of the hSP-A1 6A³ and 6A⁴ variants.

Fig. 8.

miRNA expression in NCI-H441 cells and RRL small RNA fractions. A: PAGE analysis of the small RNA fractions purified by the mirVANA miRNA Isolation Kit (Ambion) from NCI-H441 cells (lane 1) and RRL (lane 2). B: the presence of the human microRNAs hsa-mir-183, hsa-mir-194, hsa-mir-449b, hsa-mir-612, hsa-mir-654-5p, hsa-mir-767-3p, and hsa-mir-885-3p, was analyzed by the All-in-One miRNA qRT-PCR detection kit (GeneCopoeia) with specific primers, as described in materials and methods. PCR product bands were detected for hsa-mir-183, 449b, 654-5p, and 885-3p in the NCI-H441 cell small RNA. A low intensity band was also detected for hsa-mir-612 in the human lung cell extracts. No amplification was detected in the RRL samples or the negative control where reverse transcriptase (RT−) was omitted.

Fig. 9.

Effect of the 11-nt element on translation in NCI-H441 cells. Translation efficiency after 90 min of transfection with mRNAs in which the 11-nt element (CTGCCTGCCCA) was: 1) cloned (ins) in the 3′ UTR of the 6A³ and 6A⁴ variants or 2) removed (del) from the 6A² variant. Relative luciferase activities (Firefly to Renilla) are shown as means ± SE (n = 5–7). The removal of the 11-nt element in the 6A² variant significantly increased translation of the transcripts (*P < 0.05), whereas the insertion of the element in the 3′ UTR of the 6A³ and 6A⁴ variants decreased translation (**P < 0.05) in the absence (left) or presence (right) of poly(A) tail. The poly(A) tail exhibited a positive effect on translation not only with the wild-type transcripts but also with mutant transcripts.