MOV10 RNA helicase is a potent inhibitor of retrotransposition in cells

Abstract

MOV10 protein, a putative RNA helicase and component of the RNA-induced silencing complex (RISC), inhibits retrovirus replication. We show that MOV10 also severely restricts human LINE1 (L1), Alu, and SVA retrotransposons. MOV10 associates with the L1 ribonucleoprotein particle, along with other RNA helicases including DDX5, DHX9, DDX17, DDX21, and DDX39A. However, unlike MOV10, these other helicases do not strongly inhibit retrotransposition, an activity dependent upon intact helicase domains. MOV10 association with retrotransposons is further supported by its colocalization with L1 ORF1 protein in stress granules, by cytoplasmic structures associated with RNA silencing, and by the ability of MOV10 to reduce endogenous and ectopic L1 expression. The majority of the human genome is repetitive DNA, most of which is the detritus of millions of years of accumulated retrotransposition. Retrotransposons remain active mutagens, and their insertion can disrupt gene function. Therefore, the host has evolved defense mechanisms to protect against retrotransposition, an arsenal we are only beginning to understand. With homologs in other vertebrates, insects, and plants, MOV10 may represent an ancient and innate form of immunity against both infective viruses and endogenous retroelements.

Figure 1. Construct pc-L1-1FH successfully immunoprecipitates basal L1 RNP complexes (ORF1p, ORF2p, and L1 RNA) from 293T cell lysates following α-FLAG purification.

Detection in purified immunoprecipitates of (A) FLAG-HA-tagged ORF1 protein, (B) ORF2 protein, (C) L1 RNA detected by RT-PCR, and (D) ORF2 reverse transcriptase activity, assayed as described by Kulpa et al. . (E–I) Endogenous and ectopically expressed MOV10 protein and L1 ORF1p associate in multiple cell lines. (E) Immunoprecipitation of V5-tagged MOV10 by FLAG-tagged pc-L1-1FH depends upon the presence of RNA (lanes 2 and 3). A double point mutation in ORF1 of pc-L1-1FH known to inhibit RNA-binding, prevents efficient co-IP of MOV10 protein (lane 4). Removing the FLAG-HA-tag from pc-L1-FH (pc-L1-RP) prevents IP of the L1 RNP and MOV10 protein on α-FLAG agarose (lane 5). (F) pc-L1-1FH-generated RNPs associate with endogenous MOV10 protein in 293T cells. (G) Transfected V5-tagged MOV10 and cold shock domain protein YBX1, but not fibrillarin (FBL) or empty vector, immunoprecipitate endogenous ORF1p from 2102Ep cells. (H) α-ORF1 (AH40.1) antibody co-IPs endogenous MOV10 protein from 2102Ep cells (lane 1). Lane 3: lysate of 293T cells transfected with MOV10-V5-His₆ WT as a marker for MOV10 protein. (I) Similarly, immunoprecipitation using α-MOV10 antibody yields endogenous ORF1p (lane 1). Lane 4: α-FLAG-tag IP from 293T cells transfected with pc-L1-1FH as a marker for ORF1p.

Figure 2. Ectopically expressed and endogenous MOV10 closely colocalizes with L1 ORF1p in multiple cell lines.

(A) EGFP-tagged ORF1p colocalizes with V5-tagged MOV10 in the cytoplasm and foci of 293T cells. (B) A mutation in the RRM RNA-binding domain of EGFP-ORF1p diminishes, but does not abolish, colocalization with MOV10 in cytoplasmic foci. (C) EGFP-ORF1p colocalizes with endogenous MOV10 in cytoplasmic granules of 2102Ep cells. (D) Endogenous ORF1p extensively colocalizes with RFP-tagged MOV10 in 2102Ep cells. (E) In 293T cells mouse mMOV10L is present in some, but not all ORF1p-marked cytoplasmic bodies, and significantly less often than MOV10. Scale bar: 10 µm.

Figure 3. Evidence from cell culture retrotransposition assays that MOV10 inhibits insertion of non-LTR retrotransposons.

(A) The reporter construct 99 PUR RPS EGFP was cotransfected in 293T cells with empty vector (pcDNA3) or constructs expressing V5-MOV10 or other epitope-tagged L1 RNP-associated helicases. Constructs are numbered and named at the bottom of Panel C. Five days later, percentages of EGFP-positive cells (ie. cells with a retrotransposition event) were determined by flow cytometry. Each construct pair was tested in quadruplicate (n = 4), and results are normalized to pcDNA3 empty vector control (#2). An L1 mutant, 99 PUR JM111 EGFP (JM111), was used as negative control for retrotransposition and FACs gating (#1). V5-DDX17 (isoform (iso)2, #7), is generated from an abbreviated transcript variant spanning only residues 549 to 729 of the C-terminal region of full-length DDX17 (#6). (B) Same as (A) in HeLa-HA cells. (C) To assess transfection efficiency and cell toxicity, helicase constructs were cotransfected with CEP-EGFP and fluorescent cells were assayed 4 days later. Results are from 293T cells. (D) Expression of MOV10 inhibits retrotransposition in a dose-dependent manner. Decreasing microgram amounts of MOV10-expessing plasmid, mixed with empty vector to normalize DNA concentrations, were cotransfected with the L1-reporter construct, 99 PUR RPS EGFP, and assayed for retrotransposition at 5 days. Western blot analysis of cytoplasmic lysates (below) shows that even very small amounts of exogenous MOV10 protein can inhibit retrotransposition. High-molecular weight aggregates of overexpressed MOV10 protein are also visible on overexposed gels (marked by *). (E) Loss of endogenous MOV10 expression increases retrotransposition. Stable Hela and 293T cell lines were established by infection with lentiviral particles expressing shRNAs against MOV10, GAPDH or empty vector, followed by selection with puromycin. These cell lines were then tested for retrotransposition competency of 99 PUR RPS EGFP. Results are normalized to empty vector control. Bottom panels: Western blot showing that shE1 and shE2 decrease endogenous MOV10 protein levels by about 90 percent in 293T cells, and loading control blot showing β-tubulin. (F) Overexpression of MOV10 decreases Alu and SVA retrotransposition in HeLa-HA cells. As described in Dewannieux et al. , a Ya5 Alu is cloned in a plasmid containing the 7SL pol III enhancer and neo^TET cassette interrupted by a Tetrahymena self-splicing intron. Upon transcription, the intron is spliced out. When this construct is co-expressed with L1 ORF2 alone (#3 and 4) or full-length L1-RP (#5 and 6), Alu RNAs are reverse transcribed along with the neo gene and integrated into the genome to confer neomycin resistance. Following 15 days of treatment with neomycin, resistant colonies were stained and counted. Either empty vector (#1, 3, 5, 7, and 9) or V5-MOV10 plasmid (#2, 4, 6, 8 and 10) was included in the reactions. Retrotransposition data for SVA.2 and L1-RP containing the mneoI antibiotic-selection cassette are also shown (#7–10). Colony counts are not normalized. To the right are representative T₇₅ flasks with Giemsa-stained Alu retrotransposition-positive colonies in the absence (left) or presence (right) of MOV10. (G) The SVA EGFP retrotransposition assay was performed in 293T cells with SVA.2 EGFP cotransfected with constructs expressing L1 ORF1 and ORF2 separately, in the presence or absence of MOV10. (H) Mutations in helicase motifs I–VI impede anti-retrotransposition activity of MOV10. Top panel: MOV10-V5-His₆ wild-type and mutant proteins were tested for effect on retrotransposition of 99 PUR RPS EGFP. Results are normalized to empty vector control (#2). Middle panel: mutant and wild-type MOV10-V5-His₆ proteins are expressed at similar levels in 293T cells. Bottom panel: mutant and wild-type MOV10-V5-HIS₆ proteins all efficiently co-IP with L1 RNPs expressed from pc-L1-1FH.

Figure 4. ATP-dependent RNA helicases other than MOV10 also associate with L1 RNPs expressed from pc-L1-1FH.

In α-FLAG immunoprecipitates, only association with myc-tagged full-length DDX17 (iso1) is resistant to RNase digestion.

Figure 5. MOV10 inhibits exogenous L1 RNP expression in cells.

(A–C) Analysis of 293T cell lysates showing that MOV10 expression has no discernable effect on total protein production as determined by (A) Coomassie blue staining, or (B) Western blot detection of constitutively expressed proteins. (C) However, MOV10 attenuates expression of ORF1p from pc-L1-1FH. Note, α-ORF1 AH40.1 only faintly detects endogenous ORF1 protein in this blot exposure. (D–G) Analysis of immunoprecipitate samples following IP of pc-L1-1FH. Levels of (D) ORF1p, (E) ORF2p, (F) ORF2p RT activity, and (G) L1 RNA are strongly diminished in the presence of MOV10. α-ORF2-N occasionally detects a slightly smaller than expected band in untransfected cells (labeled NS in (E)). It is not known if this band is non-specific or a truncated form of endogenous ORF2p. Lane names and numbers at the bottom refer to all panels A–G. (H) Analysis of 293T immunoprecipitate samples following α-FLAG IP of cotransfected pc-L1-1FH. Introducing mutations in helicase domains of MOV10 significantly abrogates its inhibition of ORF1p expression from pc-L1-1FH (compare also with panel D). (I) V5-MOV10 protein was expressed in 2102Ep cells, and endogenous ORF1 protein was detected in lysates with α-ORF1 AH40.1 antibody. Endogenous ORF1p levels decrease in the presence of MOV10 wild-type protein, but to lesser degree with MOV10 mutants (lower panel, lanes 4–6). ImageJ software (NIH) was used to quantitate band intensities, and their absolute readings are arrayed below the figure panels (H and I).

Figure 6. Polycomb group (PcG) multiprotein PRC1-like complex component Chromobox homolog 7 (CBX7) associates with the L1-RNP and inhibits retrotransposition.

(A) Lanes 1–3: CBX7 binds weakly with the L1 RNP in an RNA-dependent manner. Lanes 4–9: Co-immunoprecipitation of V5-tagged CBX7 by pc-L1-1FH is greatly enhanced by coexpression of tagged MOV10 proteins. (B) When overexpressed in the cell culture assay, both CBX7 and PRC1 component CBX8 significantly inhibit L1 retrotransposition, without obvious cell toxicity (C).