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Mapping the LINE1 ORF1 protein interactome reveals associated inhibitors of human retrotransposition.

Goodier JL, Cheung LE, Kazazian HH - Nucleic Acids Res. (2013)

Bottom Line: These elements have significant effects on gene organization and expression.By co-immunoprecipitation of tagged L1 constructs and mass spectrometry, we identified proteins associated with the L1 ORF1 protein and its ribonucleoprotein.We also assayed the effects of these proteins on cell culture retrotransposition and found strong inhibiting proteins, including some that control HIV and other retroviruses.

View Article: PubMed Central - PubMed

Affiliation: McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University School of Medicine.

ABSTRACT
LINE1s occupy 17% of the human genome and are its only active autonomous mobile DNA. L1s are also responsible for genomic insertion of processed pseudogenes and >1 million non-autonomous retrotransposons (Alus and SVAs). These elements have significant effects on gene organization and expression. Despite the importance of retrotransposons for genome evolution, much about their biology remains unknown, including cellular factors involved in the complex processes of retrotransposition and forming and transporting L1 ribonucleoprotein particles. By co-immunoprecipitation of tagged L1 constructs and mass spectrometry, we identified proteins associated with the L1 ORF1 protein and its ribonucleoprotein. These include RNA transport proteins, gene expression regulators, post-translational modifiers, helicases and splicing factors. Many cellular proteins co-localize with L1 ORF1 protein in cytoplasmic granules. We also assayed the effects of these proteins on cell culture retrotransposition and found strong inhibiting proteins, including some that control HIV and other retroviruses. These data suggest candidate cofactors that interact with the L1 to modulate its activity and increase our understanding of the means by which the cell coexists with these genomic 'parasites'.

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Related in: MedlinePlus

Ectopically expressed and endogenous proteins associate with L1 complexes in multiple cell lines. (A) V5-, 6xMyc- or GFP-tagged proteins exogenously expressed in 293T cells specifically co-immunoprecipitate with pc-L1-1FH, but not empty vector (pcDNA6 myc/his B) [IP: α-FLAG affinity gel, western blotting (WB): α-V5, α-Myc or α-GFP]. IP reactions were in the presence or absence of 15 μg/ml RNase (lanes 3–5). Lysate input samples are also shown (lanes 1 and 2). Several protein panels are reproduced from Goodier et al. (29). GFP-mIGF2BP1 is derived from mouse.The bottom-most panel is representative of tagged ORF1p in the input and IP fractions and confirms that RNase treatment does not affect ORF1p immunoprecipitation on α-FLAG agarose. Molecular weights shown include the epitope tag. The protein standard is See Blue Plus 2 (Invitrogen). (B) Co-IP of endogenous ORF1p from 2102Ep cells by selected V5-tagged proteins (IP: α-V5/IgG affinity gel). Upper rows: detection of ORF1p (WB: α-ORF1 AH40). Asterisk indicates proteins that strongly co-IP endogenous ORF1p. ‘o’ marks proteins that clearly associate with ORF1p on gel overexposure. Lower rows confirm successful IP of the test proteins (WB: α-V5). Exposure times are not necessarily the same for each lane. Input lysate fractions are shown in Supplementary Figure S1. (C) Co-IP of selected endogenous proteins by pc-L1-1FH from 293T cells (IP: α-FLAG affinity gel, WB: various antibodies). The antibody name is followed by the expected protein molecular weight. NCL has an expected weight of 77 kDa, but observed molecular weight of ∼100 kDa. As previously reported (27), an antibody against DDX39B [UAP56; (50)] detects a dominant band of 55 kDa in cytoplasmic lysates, and a smaller isoform of 49 kDa (the expected size for DDX39B) that co-IPs with tagged ORF1p. In total, 12 antibodies were tested; α-PCBP2 (Abnova), α-FBL (Santa Cruz) and α-TOP1 (Spring) failed to detect their endogenous targets in pc-L1-1FH immunoprecipitates. The bottom-most panel shows efficient immunoprecipitation of tagged ORF1p detected by α-FLAG antibody.
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gkt512-F3: Ectopically expressed and endogenous proteins associate with L1 complexes in multiple cell lines. (A) V5-, 6xMyc- or GFP-tagged proteins exogenously expressed in 293T cells specifically co-immunoprecipitate with pc-L1-1FH, but not empty vector (pcDNA6 myc/his B) [IP: α-FLAG affinity gel, western blotting (WB): α-V5, α-Myc or α-GFP]. IP reactions were in the presence or absence of 15 μg/ml RNase (lanes 3–5). Lysate input samples are also shown (lanes 1 and 2). Several protein panels are reproduced from Goodier et al. (29). GFP-mIGF2BP1 is derived from mouse.The bottom-most panel is representative of tagged ORF1p in the input and IP fractions and confirms that RNase treatment does not affect ORF1p immunoprecipitation on α-FLAG agarose. Molecular weights shown include the epitope tag. The protein standard is See Blue Plus 2 (Invitrogen). (B) Co-IP of endogenous ORF1p from 2102Ep cells by selected V5-tagged proteins (IP: α-V5/IgG affinity gel). Upper rows: detection of ORF1p (WB: α-ORF1 AH40). Asterisk indicates proteins that strongly co-IP endogenous ORF1p. ‘o’ marks proteins that clearly associate with ORF1p on gel overexposure. Lower rows confirm successful IP of the test proteins (WB: α-V5). Exposure times are not necessarily the same for each lane. Input lysate fractions are shown in Supplementary Figure S1. (C) Co-IP of selected endogenous proteins by pc-L1-1FH from 293T cells (IP: α-FLAG affinity gel, WB: various antibodies). The antibody name is followed by the expected protein molecular weight. NCL has an expected weight of 77 kDa, but observed molecular weight of ∼100 kDa. As previously reported (27), an antibody against DDX39B [UAP56; (50)] detects a dominant band of 55 kDa in cytoplasmic lysates, and a smaller isoform of 49 kDa (the expected size for DDX39B) that co-IPs with tagged ORF1p. In total, 12 antibodies were tested; α-PCBP2 (Abnova), α-FBL (Santa Cruz) and α-TOP1 (Spring) failed to detect their endogenous targets in pc-L1-1FH immunoprecipitates. The bottom-most panel shows efficient immunoprecipitation of tagged ORF1p detected by α-FLAG antibody.

Mentions: For each co-immunoprecipitation of Figure 3A, extracts from 3.5 × 106 293T cells transfected with L1 and test protein constructs in T75 flasks were prepared in 370 μl of buffer A supplemented as aforementioned. Lysates containing test proteins of predominantly nuclear localization were sonicated. Reactions transfected with pc-L1-1FH were immunoprecipitated with 30 μl of anti-FLAG M2 affinity gel and eluted as aforementioned. RNase inhibitors were omitted from samples treated with 15 μg/ml DNase-free RNase (Roche). To detect their interaction with endogenous ORF1p, V5-tagged proteins were transfected in 7 × 106 2102Ep embryonal carcinoma cells (Figure 3B), immunoprecipitated on Protein G Agarose/Salmon Sperm DNA (Upstate) and eluted in SDS loading buffer with heating at 95°C, followed by addition of β-mercaptoethanol.


Mapping the LINE1 ORF1 protein interactome reveals associated inhibitors of human retrotransposition.

Goodier JL, Cheung LE, Kazazian HH - Nucleic Acids Res. (2013)

Ectopically expressed and endogenous proteins associate with L1 complexes in multiple cell lines. (A) V5-, 6xMyc- or GFP-tagged proteins exogenously expressed in 293T cells specifically co-immunoprecipitate with pc-L1-1FH, but not empty vector (pcDNA6 myc/his B) [IP: α-FLAG affinity gel, western blotting (WB): α-V5, α-Myc or α-GFP]. IP reactions were in the presence or absence of 15 μg/ml RNase (lanes 3–5). Lysate input samples are also shown (lanes 1 and 2). Several protein panels are reproduced from Goodier et al. (29). GFP-mIGF2BP1 is derived from mouse.The bottom-most panel is representative of tagged ORF1p in the input and IP fractions and confirms that RNase treatment does not affect ORF1p immunoprecipitation on α-FLAG agarose. Molecular weights shown include the epitope tag. The protein standard is See Blue Plus 2 (Invitrogen). (B) Co-IP of endogenous ORF1p from 2102Ep cells by selected V5-tagged proteins (IP: α-V5/IgG affinity gel). Upper rows: detection of ORF1p (WB: α-ORF1 AH40). Asterisk indicates proteins that strongly co-IP endogenous ORF1p. ‘o’ marks proteins that clearly associate with ORF1p on gel overexposure. Lower rows confirm successful IP of the test proteins (WB: α-V5). Exposure times are not necessarily the same for each lane. Input lysate fractions are shown in Supplementary Figure S1. (C) Co-IP of selected endogenous proteins by pc-L1-1FH from 293T cells (IP: α-FLAG affinity gel, WB: various antibodies). The antibody name is followed by the expected protein molecular weight. NCL has an expected weight of 77 kDa, but observed molecular weight of ∼100 kDa. As previously reported (27), an antibody against DDX39B [UAP56; (50)] detects a dominant band of 55 kDa in cytoplasmic lysates, and a smaller isoform of 49 kDa (the expected size for DDX39B) that co-IPs with tagged ORF1p. In total, 12 antibodies were tested; α-PCBP2 (Abnova), α-FBL (Santa Cruz) and α-TOP1 (Spring) failed to detect their endogenous targets in pc-L1-1FH immunoprecipitates. The bottom-most panel shows efficient immunoprecipitation of tagged ORF1p detected by α-FLAG antibody.
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Related In: Results  -  Collection

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gkt512-F3: Ectopically expressed and endogenous proteins associate with L1 complexes in multiple cell lines. (A) V5-, 6xMyc- or GFP-tagged proteins exogenously expressed in 293T cells specifically co-immunoprecipitate with pc-L1-1FH, but not empty vector (pcDNA6 myc/his B) [IP: α-FLAG affinity gel, western blotting (WB): α-V5, α-Myc or α-GFP]. IP reactions were in the presence or absence of 15 μg/ml RNase (lanes 3–5). Lysate input samples are also shown (lanes 1 and 2). Several protein panels are reproduced from Goodier et al. (29). GFP-mIGF2BP1 is derived from mouse.The bottom-most panel is representative of tagged ORF1p in the input and IP fractions and confirms that RNase treatment does not affect ORF1p immunoprecipitation on α-FLAG agarose. Molecular weights shown include the epitope tag. The protein standard is See Blue Plus 2 (Invitrogen). (B) Co-IP of endogenous ORF1p from 2102Ep cells by selected V5-tagged proteins (IP: α-V5/IgG affinity gel). Upper rows: detection of ORF1p (WB: α-ORF1 AH40). Asterisk indicates proteins that strongly co-IP endogenous ORF1p. ‘o’ marks proteins that clearly associate with ORF1p on gel overexposure. Lower rows confirm successful IP of the test proteins (WB: α-V5). Exposure times are not necessarily the same for each lane. Input lysate fractions are shown in Supplementary Figure S1. (C) Co-IP of selected endogenous proteins by pc-L1-1FH from 293T cells (IP: α-FLAG affinity gel, WB: various antibodies). The antibody name is followed by the expected protein molecular weight. NCL has an expected weight of 77 kDa, but observed molecular weight of ∼100 kDa. As previously reported (27), an antibody against DDX39B [UAP56; (50)] detects a dominant band of 55 kDa in cytoplasmic lysates, and a smaller isoform of 49 kDa (the expected size for DDX39B) that co-IPs with tagged ORF1p. In total, 12 antibodies were tested; α-PCBP2 (Abnova), α-FBL (Santa Cruz) and α-TOP1 (Spring) failed to detect their endogenous targets in pc-L1-1FH immunoprecipitates. The bottom-most panel shows efficient immunoprecipitation of tagged ORF1p detected by α-FLAG antibody.
Mentions: For each co-immunoprecipitation of Figure 3A, extracts from 3.5 × 106 293T cells transfected with L1 and test protein constructs in T75 flasks were prepared in 370 μl of buffer A supplemented as aforementioned. Lysates containing test proteins of predominantly nuclear localization were sonicated. Reactions transfected with pc-L1-1FH were immunoprecipitated with 30 μl of anti-FLAG M2 affinity gel and eluted as aforementioned. RNase inhibitors were omitted from samples treated with 15 μg/ml DNase-free RNase (Roche). To detect their interaction with endogenous ORF1p, V5-tagged proteins were transfected in 7 × 106 2102Ep embryonal carcinoma cells (Figure 3B), immunoprecipitated on Protein G Agarose/Salmon Sperm DNA (Upstate) and eluted in SDS loading buffer with heating at 95°C, followed by addition of β-mercaptoethanol.

Bottom Line: These elements have significant effects on gene organization and expression.By co-immunoprecipitation of tagged L1 constructs and mass spectrometry, we identified proteins associated with the L1 ORF1 protein and its ribonucleoprotein.We also assayed the effects of these proteins on cell culture retrotransposition and found strong inhibiting proteins, including some that control HIV and other retroviruses.

View Article: PubMed Central - PubMed

Affiliation: McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University School of Medicine.

ABSTRACT
LINE1s occupy 17% of the human genome and are its only active autonomous mobile DNA. L1s are also responsible for genomic insertion of processed pseudogenes and >1 million non-autonomous retrotransposons (Alus and SVAs). These elements have significant effects on gene organization and expression. Despite the importance of retrotransposons for genome evolution, much about their biology remains unknown, including cellular factors involved in the complex processes of retrotransposition and forming and transporting L1 ribonucleoprotein particles. By co-immunoprecipitation of tagged L1 constructs and mass spectrometry, we identified proteins associated with the L1 ORF1 protein and its ribonucleoprotein. These include RNA transport proteins, gene expression regulators, post-translational modifiers, helicases and splicing factors. Many cellular proteins co-localize with L1 ORF1 protein in cytoplasmic granules. We also assayed the effects of these proteins on cell culture retrotransposition and found strong inhibiting proteins, including some that control HIV and other retroviruses. These data suggest candidate cofactors that interact with the L1 to modulate its activity and increase our understanding of the means by which the cell coexists with these genomic 'parasites'.

Show MeSH
Related in: MedlinePlus