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MOV10 RNA helicase is a potent inhibitor of retrotransposition in cells.

Goodier JL, Cheung LE, Kazazian HH - PLoS Genet. (2012)

Bottom Line: However, unlike MOV10, these other helicases do not strongly inhibit retrotransposition, an activity dependent upon intact helicase domains.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.

View Article: PubMed Central - PubMed

Affiliation: McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. jgoodier@jhmi.edu

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.

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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. [35]. (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.
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pgen-1002941-g001: 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. [35]. (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.

Mentions: We tagged the C-terminus of ORF1 in L1-RP (an L1 highly active in cell culture assays; [33]) with a tandem hemagglutinin (HA)-FLAG tag to create the construct pc-L1-1FH. Tagging ORF1 in this manner diminishes but maintains activity of L1-RP in an enhanced green fluorescent protein (EGFP)-reporter assay for cell culture retrotransposition [34] (Figure S2). All evidence indicates that pc-L1-1FH is capable of immunoprecipitating basal L1 RNP complexes from cell lysates. Following transfection in 293T cells and α-FLAG agarose purification, we detected in cytoplasmic immunoprecipates both ORF1 and ORF2 proteins (Figure 1A and 1B), L1 RNA (Figure 1C), and robust RT activity (Figure 1D) as determined by an in vitro PCR-based assay [35].


MOV10 RNA helicase is a potent inhibitor of retrotransposition in cells.

Goodier JL, Cheung LE, Kazazian HH - PLoS Genet. (2012)

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. [35]. (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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pgen-1002941-g001: 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. [35]. (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.
Mentions: We tagged the C-terminus of ORF1 in L1-RP (an L1 highly active in cell culture assays; [33]) with a tandem hemagglutinin (HA)-FLAG tag to create the construct pc-L1-1FH. Tagging ORF1 in this manner diminishes but maintains activity of L1-RP in an enhanced green fluorescent protein (EGFP)-reporter assay for cell culture retrotransposition [34] (Figure S2). All evidence indicates that pc-L1-1FH is capable of immunoprecipitating basal L1 RNP complexes from cell lysates. Following transfection in 293T cells and α-FLAG agarose purification, we detected in cytoplasmic immunoprecipates both ORF1 and ORF2 proteins (Figure 1A and 1B), L1 RNA (Figure 1C), and robust RT activity (Figure 1D) as determined by an in vitro PCR-based assay [35].

Bottom Line: However, unlike MOV10, these other helicases do not strongly inhibit retrotransposition, an activity dependent upon intact helicase domains.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.

View Article: PubMed Central - PubMed

Affiliation: McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. jgoodier@jhmi.edu

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.

Show MeSH
Related in: MedlinePlus