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miR-128 represses L1 retrotransposition by binding directly to L1 RNA

Nature Structural & Molecular Biology volume 22, pages 824–831 (2015)Cite this article

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Abstract

Long interspersed element 1 (LINE-1 or L1) retrotransposons compose 17% of the human genome. Active L1 elements are capable of replicative transposition (mobilization) and can act as drivers of genetic diversity. However, this mobilization is mutagenic and may be detrimental to the host, and therefore it is under strict control. Somatic cells usually silence L1 activity by DNA methylation of the L1 promoter. In hypomethylated cells, such as cancer cells and induced pluripotent stem cells (iPSCs), a window of opportunity for L1 reactivation emerges, and with it comes an increased risk of genomic instability and tumorigenesis. Here we show that miR-128 represses new retrotransposition events in human cancer cells and iPSCs by binding directly to L1 RNA. Thus, we have identified and characterized a new function of microRNAs: mediating genomic stability by suppressing the mobility of endogenous retrotransposons.

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Figure 1: L1 activity is derepressed in hESCs, iPSCs and teratoma cells.
Figure 2: Identification of miRs that regulate L1 retrotransposition.
Figure 3: miR-128 represses L1 activity, whereas miR-128 neutralization derepresses L1 retrotransposition in various cell lines.
Figure 4: miR-128 represses L1 activity, whereas miR-128 neutralization derepresses L1 retrotransposition in iPSCs.
Figure 5: miR-128 represses L1 activity by binding directly to ORF2 RNA.
Figure 6: miR-128 binds directly to L1 ORF2 RNA in cells.

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Acknowledgements

We thank J. Moran (University of Michigan Medical School) and M. An (Washington State University) for generously sharing pJM101/L1RP and pWA355 plasmids, M. Waterman (University of California, Irvine) for sharing the CCICs and H. Fan and A. James (University of California, Irvine) for comments and critical reading of the manuscript. This work was supported by the University of California Cancer Research Coordinating Committee 55205 (I.M.P.), American Cancer Society Institutional Research Grant 98-279-08 (I.M.P.), a University of California Irvine Institute for Memory Impairments and Neurological Disorders grant (I.M.P.), US National Institutes of Health T32 NS082174-02 (K.J.S.) and California Institute of Regenerative Medicine TG2-01152 (A.I.)

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Author notes

  1. Matthias Hamdorf, Adam Idica and Dimitrios G Zisoulis: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology and Biochemistry, Francisco J. Ayala School of Biological Sciences, University of California, Irvine, Irvine, California, USA

    Matthias Hamdorf, Adam Idica, Lindsay Gamelin, Charles Martin, Katie J Sanders & Irene M Pedersen

  2. Regulus Therapeutics, San Diego, California, USA

    Dimitrios G Zisoulis

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  1. Matthias Hamdorf
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Contributions

I.M.P. originally developed the concept, and M.H. further elaborated on it and designed the experiments together with I.M.P. and D.G.Z.; M.H., A.I., D.G.Z. and L.G. performed experiments and analyzed the data. C.M. established the RNA stem cell–reprogramming approach and carried out reprogramming of skin fibroblasts and characterization of iPSCs with A.I. and K.J.S.; I.M.P. wrote the paper with input from D.G.Z, M.H. and A.I., who contributed equally to this work.

Corresponding author

Correspondence to Irene M Pedersen.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Endogenous miR-128 expression levels in different cell types.

miR-128 expression levels were determined in human foreskin fibroblasts (hBJ), HEK293T (293T), HeLa, H23, Tera-1 (Tera), colon cancer initiating cells (CCIC), IMR90, RNA-iPSC-1, RNA-iPSC-3, RNA-iPSC-7 by miR-128 specific RT and miR-specific qPCR analysis, which were normalized to RNU5A expression levels.

Supplementary Figure 2 Effect of miR-128 on L1 RNA levels in HeLa, Tera (ORF2 and full length), H23 and HEK293T cells, and GAPDH levels in RIP HeLa cell lysate.

(A) Relative RNA levels of L1 ORF2 RNA in HeLa cells transiently transfected with miR mimics or inhibitors and L1-Neo-reporter plasmid (anti-miR-128, n=3 independent biological replicates, or miR-128 n=2 independent biological replicates, range of values shown). (B) Relative ORF2 RNA levels in Tera-1 cells transiently transfected with miR mimics or inhibitors (n=2 independent biological replicates, range of values shown). (C) Relative full-length L1 RNA (5’UTR, ORF1 and ORF2) of stably transduced HeLa cells (n=4 technical replicates). (D) Non-small cell lung cancer cells (H23, n=3 independent biological replicates) and HEK293T (293T, n=3 independent biological replicates) cells were transfected with miR-128, anti-miR-128 or miR-control mimics. After 48 hours cells were lysed and RNA was used to determine expression levels of ORF2 using specific L1 qPCR analysis, normalized to beta-2-microglobulin expression levels. H23 and 293Ts show the same effect of ORF2 RNA regulation as we observed for HeLa, Tera, and stem cells. Overexpression of miR-128 leads to a downregulation of ORF2 and knock down of miR-128 by anti-miR-128 leads to higher levels of ORF2. (E) Relative mRNA levels of GAPDH in HeLa cells lentivirally transduced with miR constructs and transfected with L1 wild-type plasmid (miR-128 and anti-miR-128) or mutant L1 plasmid (miR-128 binding site mutated transfected into stably transduced with miR-128) normalized to GUSB (n=3, independent biological replicates). The next panel is relative IP fraction of GAPDH transcript levels associated with Argonaute complexes in stably transduced miR-128 and anti-miR-128 cells (both transfected with L1 wild-type plasmid), from the same lysates as the previous panel, normalized to let-7 levels. The next panel is relative IP fraction of GAPDH transcript levels associated with Argonaute complexes in stably transduced miR-128 cells transfected with L1 wild-type or L1 binding site mutated from the same lysates as the first panel, normalized to let-7 levels. Throughout the figure, error bars represent SEM for n≥3 and range of values for n=2. p-values from two-tailed Student’s t-tests: *:p<0.05, **:p <0.01, ***:p <0.001, ****:p <0.0001.

Supplementary Figure 3 Transfection controls in HeLa cells.

HeLa cells were transduced (HeLa stable) with lentiviral miR-128, anti-miR-128 or control-miRs, then transfected with WT L1 neomycin-reporter plasmid (WT) or mutant L1 neomycin-reporter plasmid (mutant), or transiently transfected with miR-128, anti-miR-128 or control mimics (HeLa transient). miR-128 expression levels were determined by miR-128-specific RT and miR-specific qPCR, which were normalized to RNU5A expression levels. We observed strong overexpression of miR-128 in cells transduced with the miR-128 overexpression virus and downregulation in cells transduced with virus expressing a hairpin inhibitor anti-miR-128 (HeLa stable). Similar results were observed when we transfected HeLa cells with miR-128 mimics and hairpin inhibitor anti-miR-128 (HeLa transient). When we compare the level of miR-128 overexpression for the experiment where we used the WT and the mutant L1 reporter level (mutant), we observed equal overexpression. As anti-miR introductions can work as a sponge, and thus anti-miR treatment can be underestimated when measuring miR levels, we also measure the effect of miR-128 and anti-miR-128 on a published target (Bmi1) in HeLa cells. Mean of technical triplicates are shown +/- SD. For the secondary target, Bmi1, we found that miR-128 overexpression leads to a downregulation of Bmi1 in the stable cell lines as well as in the transient transfected cells. Downregulation of miR-128 results in an enhanced Bmi1 expression. These data correspond to the detected miR-128 levels.

Supplementary Figure 4 L1 reporter plasmid and transfection control in HeLa cells.

Partial map and sequence of the used L1 ORF-2 from the pJM101/L1RP reporter. The miR-128 seed sequence is indicated in red. The start ATG of ORF-2 is indicated in blue. We also verified similar plasmid levels in miR-modulated HeLa cells, by determining level of plasmid backbone (hygromycin constitutively expressed from backbone of plasmid, amplification site is indicated by red lines on map) expression, when normalized to beta-2-microglobulin expression levels. Stable miR lines and transient miR lines show that the hygromycin expression levels of all treatments are similar reflecting an equal transfection of the plasmid into all cells. HeLa plasmid levels (pJM101) control for Colony Formation Assay verified that miR control modulation itself did not affect plasmid transfection levels (neo resistant colonies) as compared to cells not treated with miR controls. HeLa (mutant) plasmid levels (Fig 4). Plasmid levels were verified by DNA extraction of transfected cells and qPCR with backbone-specific primers then normalized to genomic DNA (PBGD). For the control and the mutant plasmid, we observed equal transfection efficiency in control and miR-128 and anti-miR-128 cells

Supplementary Figure 5 Transfection controls in iPSCs.

Efficient miR transduction of iPS cells, were determined by GFP that is co-expressed with the miR (scale bar=400 µm). Next we determined miR-128 expression levels in iPSC lines transiently or stably (IMR90 and RNA-iPSC-1) transfected/transduced with miR-128, anti-miR-128 and miR-controls, by performing specific RT and q-PCR analysis, normalized to RNU5A expression levels, or by analyzing expression levels of a secondary miR-128 target (Bmi1). Finally similar plasmid levels between miR modulated iPSC lines were verified by transfection of a plasmid encoding a constitutively active neomycin-resistance gene in the colony formation assay. For the transient transfected stem cells, we found a strong upregulated miR-128 level when we introduced the miR-128 mimic (upper middle panel) and a downregulation when we transfected the hairpin anti-miR-128 inhibitor. For the stable cell lines we found a strong upregulation in the stable overexpression cell line (lower middle panel) but for the downregulation with the anti-miR-128 we didn´t observe a strong effect. As we know that anti-miR introductions can work as a sponge and not always leads directly to miR degradation, we tested Bmi as a secondary target and there is an enhanced mRNA level of Bmi in the anti-miR-128 cells that leads us to the conclusion that the downregulation of miR-128 worked in the stable cells.

Supplementary Figure 6 Conservation of miR-128–binding site and mutated miR-128–binding site on L1 mRNA.

(A) Schematic of miR-128 binding site in ORF2 of L1 mRNA. Mutant plasmid (mutated seed) encodes full length L1 mRNA but with a silent mutation in the seed sequence binding site for miR-128, thus encoding the functional protein, but not serving as a perfect miR-128 target mRNA (shown in red). Table represents schematic of experimental set-up. Expected miR-128 expression levels are shown and L1 including miR-128 binding site expression levels are shown, box on plasmid symbolizes the mutated miR-128 binding site. (B) DNA sequence alignment of homo sapiens (NCBI:M80343), chimpanzee (NCBI:KF661301), mouse (L1Base:ID1048) and rat (L1Base:ID358) as well as hsa-miR-128 (converted to the corresponding DNA sequence. Blue indicates homology between n>2 and red represents homology between all four species. NCBI (http://www.ncbi.nlm.nih.gov/pubmed/); L1Base (http://line1.bioapps.biozentrum.uni-wuerzburg.de/l1base.php).

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Hamdorf, M., Idica, A., Zisoulis, D. et al. miR-128 represses L1 retrotransposition by binding directly to L1 RNA. Nat Struct Mol Biol 22, 824–831 (2015). https://doi.org/10.1038/nsmb.3090

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