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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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Many L1-associated proteins co-localize with EGFP-ORF1p in cytoplasmic granules of unstressed 293T cells. (A–W) Construct ORF1-EGFP-L1-RP was co-transfected with V5-tagged proteins in all cases except (B) FL-CSDA, (G) RFP-HNRNPA1 and (H) mouse GFP-mIGF2BP1 (the latter being co-transfected with pc-L1-1FH, which was detected by α-FLAG antibody). Only overlaid confocal micrographs are shown. (X) Endogenous LARP1 protein co-localizes with ORF1-EGFP-L1-RP in 293T cells. (Y) Endogenous ORF1p and PCBP2 co-localize in some cytoplasmic granules of 2102Ep cells (shown by arrows). (Z) Endogenous ORF1p and fibrillarin (FBL) co-localize in nucleoli of 2102Ep cells. ORF1p is typically found in nucleoli of only a minor percentage of cells (53). Enlargement of two nucleoli are shown. In Y and Z endogenous, ORF1p is detected by α-ORF1 AH40.1 antibody.
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gkt512-F5: Many L1-associated proteins co-localize with EGFP-ORF1p in cytoplasmic granules of unstressed 293T cells. (A–W) Construct ORF1-EGFP-L1-RP was co-transfected with V5-tagged proteins in all cases except (B) FL-CSDA, (G) RFP-HNRNPA1 and (H) mouse GFP-mIGF2BP1 (the latter being co-transfected with pc-L1-1FH, which was detected by α-FLAG antibody). Only overlaid confocal micrographs are shown. (X) Endogenous LARP1 protein co-localizes with ORF1-EGFP-L1-RP in 293T cells. (Y) Endogenous ORF1p and PCBP2 co-localize in some cytoplasmic granules of 2102Ep cells (shown by arrows). (Z) Endogenous ORF1p and fibrillarin (FBL) co-localize in nucleoli of 2102Ep cells. ORF1p is typically found in nucleoli of only a minor percentage of cells (53). Enlargement of two nucleoli are shown. In Y and Z endogenous, ORF1p is detected by α-ORF1 AH40.1 antibody.

Mentions: As described earlier in the text, a majority of proteins we tested directly co-IP with the ORF1p-tagged L1 (Figure 3). We next co-transfected in 293T cells tagged cDNA constructs and ORF1-EGFP-L1-RP, a plasmid containing CMV promoter, ORF1 C-terminally tagged with EGFP, followed by intact downstream L1 sequence (34). One-third of the tagged proteins tested (23/69) at least partially co-localized with ORF1p-EGFP in cytoplasmic granules in the absence of any applied stress (Figure 5A–W). Panels X and Y show co-localization in granules of ORF1p with endogenous LARP1 and PCBP2. Similarly, we previously reported that endogenous ORF1p co-localizes with endogenous HNRNPA1 and PABPC1 in granules of chemically stressed 2102Ep cells (27). Several additional proteins we report here as associated with the ORF1p RNP have also been detected in cytoplasmic granules by other studies but not this one, perhaps because of differences in cell type (Supplementary Table S5). Indeed, many protein components of SGs and PBs have been identified [reviewed in (68,69)]. To the best of our knowledge, seven proteins of Figure 5 have not been previously reported in cytoplasmic granules, including C22orf28, FAM98A, HNRNPAB, IVNS1APB1, PRPF31, RIOK1 and STK38.Figure 5.


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

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

Many L1-associated proteins co-localize with EGFP-ORF1p in cytoplasmic granules of unstressed 293T cells. (A–W) Construct ORF1-EGFP-L1-RP was co-transfected with V5-tagged proteins in all cases except (B) FL-CSDA, (G) RFP-HNRNPA1 and (H) mouse GFP-mIGF2BP1 (the latter being co-transfected with pc-L1-1FH, which was detected by α-FLAG antibody). Only overlaid confocal micrographs are shown. (X) Endogenous LARP1 protein co-localizes with ORF1-EGFP-L1-RP in 293T cells. (Y) Endogenous ORF1p and PCBP2 co-localize in some cytoplasmic granules of 2102Ep cells (shown by arrows). (Z) Endogenous ORF1p and fibrillarin (FBL) co-localize in nucleoli of 2102Ep cells. ORF1p is typically found in nucleoli of only a minor percentage of cells (53). Enlargement of two nucleoli are shown. In Y and Z endogenous, ORF1p is detected by α-ORF1 AH40.1 antibody.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3753637&req=5

gkt512-F5: Many L1-associated proteins co-localize with EGFP-ORF1p in cytoplasmic granules of unstressed 293T cells. (A–W) Construct ORF1-EGFP-L1-RP was co-transfected with V5-tagged proteins in all cases except (B) FL-CSDA, (G) RFP-HNRNPA1 and (H) mouse GFP-mIGF2BP1 (the latter being co-transfected with pc-L1-1FH, which was detected by α-FLAG antibody). Only overlaid confocal micrographs are shown. (X) Endogenous LARP1 protein co-localizes with ORF1-EGFP-L1-RP in 293T cells. (Y) Endogenous ORF1p and PCBP2 co-localize in some cytoplasmic granules of 2102Ep cells (shown by arrows). (Z) Endogenous ORF1p and fibrillarin (FBL) co-localize in nucleoli of 2102Ep cells. ORF1p is typically found in nucleoli of only a minor percentage of cells (53). Enlargement of two nucleoli are shown. In Y and Z endogenous, ORF1p is detected by α-ORF1 AH40.1 antibody.
Mentions: As described earlier in the text, a majority of proteins we tested directly co-IP with the ORF1p-tagged L1 (Figure 3). We next co-transfected in 293T cells tagged cDNA constructs and ORF1-EGFP-L1-RP, a plasmid containing CMV promoter, ORF1 C-terminally tagged with EGFP, followed by intact downstream L1 sequence (34). One-third of the tagged proteins tested (23/69) at least partially co-localized with ORF1p-EGFP in cytoplasmic granules in the absence of any applied stress (Figure 5A–W). Panels X and Y show co-localization in granules of ORF1p with endogenous LARP1 and PCBP2. Similarly, we previously reported that endogenous ORF1p co-localizes with endogenous HNRNPA1 and PABPC1 in granules of chemically stressed 2102Ep cells (27). Several additional proteins we report here as associated with the ORF1p RNP have also been detected in cytoplasmic granules by other studies but not this one, perhaps because of differences in cell type (Supplementary Table S5). Indeed, many protein components of SGs and PBs have been identified [reviewed in (68,69)]. To the best of our knowledge, seven proteins of Figure 5 have not been previously reported in cytoplasmic granules, including C22orf28, FAM98A, HNRNPAB, IVNS1APB1, PRPF31, RIOK1 and STK38.Figure 5.

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