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

Patricia Silveyra, Guirong Wang, Joanna Floros

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15 Citations (Scopus)

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 6A3 and 6A4 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 (1A0, 1A 3). Further sequence analysis revealed that a deletion of an 11-nucleotide (nt) element in both the 6A3 and 6A4 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 6A2 3′ UTR resulted in increased translation, and the opposite effect was observed when the 11-nt element was cloned in a guest 3′ UTR (6A3, 6A4). These results indicate that sequence differences among hSP-A gene variants may account for differential regulation at the translational level.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume299
Issue number4
DOIs
StatePublished - Oct 1 2010

Fingerprint

3' Untranslated Regions
Reporter Genes
Messenger RNA
5' Untranslated Regions
Nucleotides
Luciferases
Genes
Pulmonary Surfactant-Associated Protein A
In Vitro Techniques
Reticulocytes
MicroRNAs
Transfection
Sequence Analysis
Binding Sites
Rabbits
Cell Line

All Science Journal Classification (ASJC) codes

  • Physiology
  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology

Cite this

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title = "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 6A3 and 6A4 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 (1A0, 1A 3). Further sequence analysis revealed that a deletion of an 11-nucleotide (nt) element in both the 6A3 and 6A4 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 6A2 3′ UTR resulted in increased translation, and the opposite effect was observed when the 11-nt element was cloned in a guest 3′ UTR (6A3, 6A4). These results indicate that sequence differences among hSP-A gene variants may account for differential regulation at the translational level.",
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N2 - 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 6A3 and 6A4 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 (1A0, 1A 3). Further sequence analysis revealed that a deletion of an 11-nucleotide (nt) element in both the 6A3 and 6A4 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 6A2 3′ UTR resulted in increased translation, and the opposite effect was observed when the 11-nt element was cloned in a guest 3′ UTR (6A3, 6A4). These results indicate that sequence differences among hSP-A gene variants may account for differential regulation at the translational level.

AB - 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 6A3 and 6A4 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 (1A0, 1A 3). Further sequence analysis revealed that a deletion of an 11-nucleotide (nt) element in both the 6A3 and 6A4 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 6A2 3′ UTR resulted in increased translation, and the opposite effect was observed when the 11-nt element was cloned in a guest 3′ UTR (6A3, 6A4). These results indicate that sequence differences among hSP-A gene variants may account for differential regulation at the translational level.

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