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Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames

Nature Biotechnology volume 34, pages 875–880 (2016)Cite this article

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Abstract

Increasing the levels of therapeutic proteins in vivo remains challenging. Antisense oligonucleotides (ASOs) are often used to downregulate gene expression1 or to modify RNA splicing2,3, but antisense technology has not previously been used to directly increase the production of selected proteins. Here we used a class of modified ASOs that bind to mRNA sequences in upstream open reading frames (uORFs) to specifically increase the amounts of protein translated from a downstream primary ORF (pORF). Using ASO treatment, we increased the amount of proteins expressed from four genes by 30–150% in a dose-dependent manner in both human and mouse cells. Notably, systemic treatment of mice with ASO resulted in an ∼80% protein increase of LRPPRC. The ASO-mediated increase in protein expression was sequence-specific, occurred at the level of translation and was dependent on helicase activity. We also found that the type of RNA modification and the position of modified nucleotides in ASOs affected translation of a pORF. ASOs are a useful class of therapeutic agents with broad utility.

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Figure 1: Antisense ASOs targeting the uAUG region specifically increased protein production from the pORF.
Figure 2: ASO modification and length effects on RNase H1 protein level.
Figure 3: uORF ASOs increase translation in a helicase-dependent manner.
Figure 4: uORF ASOs can increase protein levels of different genes in different species or in mice.

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Acknowledgements

This work was supported by Ionis Pharmaceuticals internal funding. We thank M. Ramesh for technique assistance; H. Murray and C.L. De Hoyos for help in the animal study; S. Damle for microarray data processing and deposition; T. Reigle for help in figure preparation; and F. Bennett, E. Swayze, W. Lima and S. Wang for discussions.

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Authors and Affiliations

  1. Department of Core Antisense Research, Ionis Pharmaceuticals, Inc., Carlsbad, California, USA

    Xue-hai Liang, Wen Shen, Hong Sun, Timothy A Vickers & Stanley T Crooke

  2. Department of Medicinal Chemistry, Ionis Pharmaceuticals, Inc., Carlsbad, California, USA

    Michael T Migawa

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  1. Xue-hai Liang
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Contributions

X.L., W.S. and S.T.C. designed the research; X.L., W.S., H.S. and T.A.V. performed the experiments; and all authors analyzed the data. M.T.M. provided ASOs and measured Tm. X.L. and S.T.C. wrote the manuscript.

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Correspondence to Xue-hai Liang.

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Competing interests

All authors are employees of Ionis Pharmaceuticals. Patents related to this study have been applied for (S.T.C., X.L., and W.S., patent application number: PCT/US2015/060938).

Integrated supplementary information

Supplementary Figure 1 ASOs targeting RNase H1 uORF can specifically increase RNase H1 protein levels and activity.

(a) qRT-PCR analyses for RNase H1 mRNA levels in HeLa cells transfected for 24 hr with different uORF ASOs, as used in Figure 1b. (b) qRT-PCR analyses for U16 RNA levels in HeLa cells treated for 24 hr with uORF ASO, followed by transfection of gapmer ASO462026 targeting U16 RNA for 4 hr. (c) qRT-PCR analyses for NCL1 mRNA levels in HeLa cells treated for 24 hr with uORF ASO, followed by transfection of gapmer ASO110074 targeting NCL1 mRNA for 4 hr, as in panel (b). The error bars for qRT-PCR panels are standard deviation of three independent experiments. (d) Western analysis of RNase H1 protein in HEK293 cells treated with ASO761909. The relative ratios of RNase H1 normalized to loading control protein Ku70 are listed below the lanes.

Supplementary Figure 2 The increase of RNase H1 protein is specific to the uORF ASO.

(a) qRT-PCR analyses for RNaseH1 mRNA levels in HeLa cells treated with or without uORF ASO761909 and complementary ASO761929, as used in Figure 1f. (b) Equal amount of S35-methionine labeled HeLa cell lysates treated with ASO761909 was analyzed by SDS-PAGE, and visualized by autoradiography. (c) Western analyses for the levels of eIF2a and phosphorylated eIF2a (Ser51) proteins in HeLa cells treated or not treated with 25 nM uORF ASOs for 16 hr. (d) Predicted off-target genes with imperfect base-pairing with ASO761909, as searched using Blast. (e) qRT-PCR results for the level of mRNAs in control cells or cells treated with 25 nM ASO761909 for 16 hr. Error bars indicate standard deviations of three independent experiments. (f) Western analysis for RNase H1, SPPL2B, and FGFR1 proteins in HeLa cells treated for different times with the uORF ASO. Ku70 served as a loading control.

Supplementary Figure 3 Effects of chemical modification on uORF ASO activity.

(a) Western for RNase H1 protein in HEK293 cells treated with uORF ASO for different times. (b) and (c) RNaseH1 protein levels in HEK293 cells tend to reduce by 16-mer and 20-mer PO/MOE ASOs, respectively, as determined by western analyses. The relative RNase H1 protein levels are listed below the lanes. (d) qRT-PCR analyses of RNase H1 mRNA levels in cells transfected with PO/MOE ASOs, as in panels (b) and (c). The error bars are standard deviations of three independent experiments.

Supplementary Figure 4 The RNase H1 uORF ASO did not act at mRNA level.

(a) qRT-PCR analysis for pre-mRNA levels of RNase H1 in cells treated with 25 nM ASOs for 16 hr. (b) The RNase H1 uORF ASO did not affect pre-mRNA splicing of RNase H1. RNase H1 mRNA in different test cells was detected by reverse-transcription-PCR, using primers specific to exon-exon junctions, followed by separation of PCR products in a 2% Agarose gel. PTEN mRNA was detected and served as a control for loading. The arrows indicate the specific PCR products. (c) qRT-PCR analyses for different RNAs in cytoplasmic and nuclear fractions prepared from cells treated with or without uORF ASO761909. Drosha and Malat1 RNAs served as control for cytoplasmic and nuclear RNAs, respectively. The error bars are standard deviations of three independent experiments. (d) Northern hybridization of RNase H1 mRNA after cleavage using specific oligonucleotides. The positions of oligonucleotides in RNase H1 3’ UTR region are depicted in upper panel. The arrows indicate the full length of the 3’ fragment after cleavage using different oligonucleotides. XL564 was used as probe for hybridization.

Supplementary Figure 5 RNase H1 mRNA shifted towards polysomes by uORF ASO treatment.

RNA was prepared from different fractions and subjected to qRT-PCR analysis, using primer probe sets specific to 28S rRNA (upper panel), RNase H1 mRNA (middle panel), and PTEN mRNA (lower panel), respectively. The percentages of each fraction are plotted. The error bars are standard deviation of 3 experiments. The 80S mono-ribosome region (Fractions 9-12) and polysome regions (fractions 15-25) are indicated by blue and red lines, respectively. The increased levels of RNase H1 mRNA in polysomes are marked with an arrow.

Supplementary Figure 6 RNase H1 uORF ASO can increase translation in a dual luciferase reporter assay.

(a) Schematic representation of the dual luciferase reporter system. The expression directions are indicated by arrows. (b) A uAUG→uUUG single nucleotide mutation. (c) A 7-nt mutation of the RNase H1 mRNA sequence and compensatory mutation of an ASO. The uAUG region is highlighted, and the mutated nucleotides are in red. (d) Luciferase activity assay for the effect of uAUG→uUUG mutation and ASO-binding site 7-nt mutations. The reporter constructs were transfected into HEK293 cells for 24 hr, and luciferase activity was measured. (e) uORF ASO761909 had no significant effect on luciferase activity for the uUUG reporter. The uUUG mutant reporter was transfected into HEK293 cells for 24 hr, followed by ASO treatment for an additional 16 hr. All the Luciferase experiments were repeated more than five times and representative results are shown. The error bars are standard deviation of three independent experiments. (f) Schematic depiction of the reporters with stepwise mutations (in red) and the ASO761909 base-pair patterns. The RNA mismatch nts with the ASO are underlined. (g) Luciferase activity assay for the effects of stepwise mutations in the reporters. The plasmids PXL59 (WT), PXL63 (M1), PXL64 (M2), PXL65 (M3), or PXL66 (M4) were transfected into HEK293 cells for 24 hr and ASO761909 was transfected at 40 nM for an additional 15 hr. The luciferase activity relative to non-ASO transfected control cells expressing the same reporter was plotted. The experiment with multiple duplicates was repeated five times and representative results are shown. The error bars are standard deviations of four independent experiments. (h) Schematic depiction of the base-pairing patterns between the reporters with stepwise mutations (in red) and ASO XL695. The blue letters in the ASO are compensatory mutations relative to ASO761909. The RNA mismatch nts with the ASO are underlined. (i) Luciferase activity assay for the reporters, as described in panel g, but treated with 40 nM ASO XL695 for 15 hr. The luciferase activity relative to non-ASO transfected control cells expressing the same reporter was plotted. The experiment with multiple duplicates was repeated five times and representative results are shown. The error bars represent standard deviation of four independent experiments. P-values were calculated based on unpaired t-test. *: P<0.05; **: P<0.01; ***: P<0.001; ****: P<0.0001. NS, not significant.

Supplementary Figure 7 uORF ASO effect on other targeted proteins.

(a) The uORF characteristics of four mRNAs of selected human and mouse genes. nt, nucleotide. aa, amino acid. (b) The 5’ UTR sequence of human SFXN3 mRNA. The uORF ASO position and the uORF sequence are shown. (c) qRT-PCR analyses of human SFXN3 mRNA in cells transfected with the uORF ASO. (d) An ASO with 2-nt mismatches (underlined) at the uAUG region significantly reduce the upregulation activity, as determined by western. Tubulin served as a loading control. The relative levels of SFXN3 are listed below the lanes.

Supplementary Figure 8 Mouse MRPL11 mRNA contains an uORF.

(a) The 5’ UTR sequence of mouse MRPL11 mRNA. The uORF ASO position is shown. (b) The mRNA levels of mouse MRPL11 in cells transfected with the uORF ASO were determined by qRT-PCR. The error bars are standard deviations of three independent experiments. (c) Luciferase activity assay for cells transiently transfected with a dual luciferase reporter construct, followed by treatment with different ASOs. The error bars are standard deviation of four independent experiments.

Supplementary Figure 9 Mouse LRPPRC protein was increased by uORF ASOs in vitro and in vivo.

(a) The 5’ UTR sequence of mouse LRPPRC mRNA. The position of the uORF ASO is shown. (b) Western analyses for LRPRPC protein levels in MHT cells treated for 10 hr with an 18-mer PO/Me ASO. ANXA2 served as a loading control. The relative LRPPRC protein levels are listed below the lanes. (c) qRT-PCR analyses for LRPPRC mRNA levels in MHT cells treated with uORF ASO761930. The error bars are standard deviations of three independent experiments. (d) Western analyses for LRPPRC protein in mouse liver homogenates treated with a control ASO or saline (N=3). hnRNP K served as a loading control. (e) Western analyses of LRPPRC in liver homogenates from mice (N=3) administrated twice with different doses of the uORF ASO. hnRNP K served as a loading control.

Supplementary Figure 10 Full length blot images presented in Figure 1.

The protein bands shown in Figure 1 are indicated by arrows.

Supplementary Figure 11 Full length blot images presented in Figure 2.

The protein bands shown in Figure 2 are indicated by arrows. The X in panel b indicates a duplicate sample that was not included in main figure.

Supplementary Figure 12 Full length blot images presented in Figure 3.

The protein bands shown in Figure 3 are indicated by arrows. The X in panel b indicates a positive control sample (CHX, cycloheximide treatment) that was not included in main figure.

Supplementary Figure 13 Full length blot images presented in Figure 4.

The protein bands shown in Figure 4 are indicated by arrows. The X in different panels indicates either different controls or at very high ASO doses that was not included in main figure, to increase conciseness. In panel c, the control sample used in main figure has higher level than the excluded control sample, thus the levels of increase for ASO treated samples are more conservative. In panel d, the untreated control was excluded since the cells in this sample were not mock transfected. In panel f, the LRPPRC band was confirmed using a different antibody.

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Liang, Xh., Shen, W., Sun, H. et al. Translation efficiency of mRNAs is increased by antisense oligonucleotides targeting upstream open reading frames. Nat Biotechnol 34, 875–880 (2016). https://doi.org/10.1038/nbt.3589

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