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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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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).
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pgen-1002941-g005: 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).

Mentions: Finally, we examined the effect of MOV10 protein on expression of L1s in cell culture. Ectopic expression of MOV10 was without obvious effect on global protein expression, as evidenced by Coomassie blue staining of cell lysates and Western blot detection of constitutively expressed proteins such as β-tubulin (Figure 5A and 5B). On the other hand, levels of ORF1p and ORF2p expressed from pc-L1-1FH were significantly reduced in both cell lysates and immunoprecipitates in the presence of transfected MOV10 (Figure 5C–5E). ORF2 RT activity was almost undetectable, and L1-RP RNA levels were diminished (Figure 5F and 5G). Introducing mutations in MOV10 helicase domains I, II, or III significantly restored expression of ectopic ORF1p as compared with inhibition by wild-type MOV10, consistent with the inability of these mutants to strongly restrict retrotransposition (Figure 5H, compare lane 1 with lanes 4–6; Figure 3H).


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

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

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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475670&req=5

pgen-1002941-g005: 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).
Mentions: Finally, we examined the effect of MOV10 protein on expression of L1s in cell culture. Ectopic expression of MOV10 was without obvious effect on global protein expression, as evidenced by Coomassie blue staining of cell lysates and Western blot detection of constitutively expressed proteins such as β-tubulin (Figure 5A and 5B). On the other hand, levels of ORF1p and ORF2p expressed from pc-L1-1FH were significantly reduced in both cell lysates and immunoprecipitates in the presence of transfected MOV10 (Figure 5C–5E). ORF2 RT activity was almost undetectable, and L1-RP RNA levels were diminished (Figure 5F and 5G). Introducing mutations in MOV10 helicase domains I, II, or III significantly restored expression of ectopic ORF1p as compared with inhibition by wild-type MOV10, consistent with the inability of these mutants to strongly restrict retrotransposition (Figure 5H, compare lane 1 with lanes 4–6; Figure 3H).

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