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IFNL3 mRNA Structure Is Remodeled by a Functional Non-Coding Polymorphism Associated With Hepatitis C Virus Clearance

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IFNL3 mRNA Structure Is Remodeled by a Functional Non-Coding Polymorphism Associated With Hepatitis C Virus Clearance

Yi-Fan Lu et al. Sci Rep.

Abstract

Polymorphisms near the interferon lambda 3 (IFNL3) gene strongly predict clearance of hepatitis C virus (HCV) infection. We analyzed a variant (rs4803217 G/T) located within the IFNL3 mRNA 3' untranslated region (UTR); the G allele (protective allele) is associated with elevated therapeutic HCV clearance. We show that the IFNL3 3' UTR represses mRNA translation and the rs4803217 allele modulates the extent of translational regulation. We analyzed the structures of IFNL3 variant mRNAs at nucleotide resolution by SHAPE-MaP. The rs4803217 G allele mRNA forms well-defined 3' UTR structure while the T allele mRNA is more dynamic. The observed differences between alleles are among the largest possible RNA structural alterations that can be induced by a single nucleotide change and transform the UTR from a single well-defined conformation to one with multiple dynamic interconverting structures. These data illustrate that non-coding genetic variants can have significant functional effects by impacting RNA structure.

Figures

Figure 1
Figure 1. Variant IFNL3 3′ UTRs differentially inhibit reporter gene expression.
(A) IFNL genes and genetic variants. (B) The IFNL3 mRNA 3′ UTR sequence. The three AREs (1–3) are indicated in red and the rs4803217 SNP is bracketed. (C) IFNL3 reporter constructs. Transcription of each reporter mRNA is directed by the constitutive CMV promoter. (D) Relative levels of each RLuc reporter mRNA. RNA levels were normalized to GAPDH; asterisk indicates that differences are significant at the p < 0.05 level. (E) RLuc protein levels. Protein levels were measured by luciferase assay and normalized to total protein. Triple asterisks indicate that differences are significant at the p < 0.001 level. All data are shown as mean values ± s.d.
Figure 2
Figure 2. Polysome profiles of IFNL3 reporter mRNAs.
(A) Top: Representative ribosome profiles. Locations of the 40S subunit, 80S monosome, and polysome peaks are indicated. Profiles were obtained using a sucrose density gradient, monitored by absorbance trace (254 nm). Bottom: Analysis of extracted total RNA samples in each gradient fraction for each IFNL3 reporter cell line. Ribosomal RNA species are indicated at right. (B) Relative levels of RLuc reporter mRNA normalized to GAPDH mRNA for each IFNL3 cell line as a function of ribosome gradient fraction. The relative levels (%) of each mRNA in each gradient fraction are indicated. (C) Areas underneath the lines in Figures 2B and S3 were quantified for both polysome fractions (fractions 7–12) and non-polysome fractions (fractions 1–6). The plot shows the differences in area between the G and T alleles (G minus T). Unpaired t-test (two-tailed) was used to calculate the P value from the three independent experiments (p = 0.01). Error bar represents the SEM.
Figure 3
Figure 3. SNP-induced structural changes in the IFNL3 3′ UTR.
(A) SHAPE reactivity profiles. Low median SHAPE reactivities correspond to highly structured regions in the RNA. Reactivities are shown as the median over 5-nt windows. “Nucleotide position” values indicate nt locations in the context of the total SHAPE data (B) Shannon entropies (medians over 5-nt windows). Peaks indicate regions with high Shannon entropies and that likely adopt multiple conformations. (C) Correlation of median SHAPE reactivities between the G and T alleles. R2 was calculated over 100-nt windows. (D) SHAPE-directed RNA secondary structure models for the 3′ UTR. Base pairs are shown as arcs. Arcs are colored by pairing probability.
Figure 4
Figure 4. Secondary structure models for the IFNL3 3′ UTR.
(A) SHAPE-directed RNA secondary structure models of the IFNL3 3′ UTR G (top) and T (bottom) alleles. rs4803217 site is shown for each structure. Nucleotides are colored by SHAPE reactivity. Highly probable (>80%) helices for each allele are shown. Locations of AU-rich elements are indicated on each allele structure. (B) Calculated free energy change increments (ΔΔG) for all possible nucleotides changes in the IFNL3 3′ UTR (left) or complete intact mRNA (right). Batch calculation was performed by mfold 3.0.
Figure 5
Figure 5. Site-directed mutagenesis of the IFNL3 3′ UTR.
(A) The rs4803217 G and T allele reporter constructs were compared to three mutants in stable HeLa cell lines. (B) Normalized relative light units (RLU) for each reporter construct is shown above. Data are shown as mean values ± s.d. and folding free energies for the full 3′ UTR sequence are shown.

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