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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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pc-L1-1FH immunoprecipitates basal L1 RNP complexes from 293T cell lysates after α-FLAG agarose purification. (A) Structure of FLAG-HA-tagged pc-L1-1FH cloned in vector pcDNA6 myc/his B. RT: ORF2 reverse transcriptase domain; EN: endonuclease domain; PCMV: CMV promoter; BGH An: bovine growth hormone polyadenylation signal. (B) FLAG-tagged ORF1p expressed from the construct pc-L1-1FH binds α-FLAG agarose independent of RNase digestion (lanes 5 and 8), but untagged ORF1p (construct pc-L1-RP) will not bind (lane 6). (C and D) Detection of L1 proteins in the RNP IP. Lanes 1–4: input lysates; lanes 6–9: immunoprecipitates; lanes 1, 2, 6 and 7: cytoplasmic fractions; lanes 3, 4, 8 and 9: nuclear fractions. (C) FLAG-HA-tagged ORF1p, detected by α-FLAG antibody. Putative ORF1p dimer and trimer bands are visible in IP samples (lanes 7 and 9). The reason for their absence in lysate samples is unclear (lanes 2 and 4). IP purification factors were determined for cytoplasmic (lanes 2 versus 7, 26-fold) and nuclear (lanes 4 versus 9, 42-fold) fractions and are presented in Supplementary Table S3. Lane labels are at the bottom of panel D. (D) ORF2p detected by α-ORF2-N (154–167) antibody (lanes 7 and 9). ORF2p in nuclear lysate samples is below the level of detection (lanes 3 and 4). (E) ORF2p reverse transcriptase activity detected in both nuclear and cytoplasmic IP reactions containing pc-L1-1FH (lanes 3 and 5), but not in reactions with the empty vector (lanes 2 and 4). RT- control: the RT incubation step was omitted and 2 µl of pc-L1-1FH immunoprecipitate was added directly to the PCR reaction. No PCR product was detected (lanes 1 and 6). The assay is described in Kulpa and Moran (51). (F) L1 RNA detected by RT–PCR (lanes 3 and 5). RT-: RT enzyme was omitted from the cDNA synthesis step using pc-L1-1FH immunoprecipitates (lanes 1 and 6). (G) FLAG-HA-tagged ORF2p is detected in nuclear and cytoplasmic extracts after IP of pc-L1-2FH. (H) The purity of nuclear and cytoplasmic whole-cell lysate fractions is shown by western blotting. α-HDAC1 is a strictly nuclear protein (54) and α-MEK1/2 is cytoplasmic (55). (I) Immunoprecipitated samples resolved on silver-stained polyacrylamide gels. To support protein identification data from complex IP samples, selected prominent band regions were excised for additional MS sequencing. Both cytoplasmic (left) and nuclear (right) IP fractions are shown.
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gkt512-F1: pc-L1-1FH immunoprecipitates basal L1 RNP complexes from 293T cell lysates after α-FLAG agarose purification. (A) Structure of FLAG-HA-tagged pc-L1-1FH cloned in vector pcDNA6 myc/his B. RT: ORF2 reverse transcriptase domain; EN: endonuclease domain; PCMV: CMV promoter; BGH An: bovine growth hormone polyadenylation signal. (B) FLAG-tagged ORF1p expressed from the construct pc-L1-1FH binds α-FLAG agarose independent of RNase digestion (lanes 5 and 8), but untagged ORF1p (construct pc-L1-RP) will not bind (lane 6). (C and D) Detection of L1 proteins in the RNP IP. Lanes 1–4: input lysates; lanes 6–9: immunoprecipitates; lanes 1, 2, 6 and 7: cytoplasmic fractions; lanes 3, 4, 8 and 9: nuclear fractions. (C) FLAG-HA-tagged ORF1p, detected by α-FLAG antibody. Putative ORF1p dimer and trimer bands are visible in IP samples (lanes 7 and 9). The reason for their absence in lysate samples is unclear (lanes 2 and 4). IP purification factors were determined for cytoplasmic (lanes 2 versus 7, 26-fold) and nuclear (lanes 4 versus 9, 42-fold) fractions and are presented in Supplementary Table S3. Lane labels are at the bottom of panel D. (D) ORF2p detected by α-ORF2-N (154–167) antibody (lanes 7 and 9). ORF2p in nuclear lysate samples is below the level of detection (lanes 3 and 4). (E) ORF2p reverse transcriptase activity detected in both nuclear and cytoplasmic IP reactions containing pc-L1-1FH (lanes 3 and 5), but not in reactions with the empty vector (lanes 2 and 4). RT- control: the RT incubation step was omitted and 2 µl of pc-L1-1FH immunoprecipitate was added directly to the PCR reaction. No PCR product was detected (lanes 1 and 6). The assay is described in Kulpa and Moran (51). (F) L1 RNA detected by RT–PCR (lanes 3 and 5). RT-: RT enzyme was omitted from the cDNA synthesis step using pc-L1-1FH immunoprecipitates (lanes 1 and 6). (G) FLAG-HA-tagged ORF2p is detected in nuclear and cytoplasmic extracts after IP of pc-L1-2FH. (H) The purity of nuclear and cytoplasmic whole-cell lysate fractions is shown by western blotting. α-HDAC1 is a strictly nuclear protein (54) and α-MEK1/2 is cytoplasmic (55). (I) Immunoprecipitated samples resolved on silver-stained polyacrylamide gels. To support protein identification data from complex IP samples, selected prominent band regions were excised for additional MS sequencing. Both cytoplasmic (left) and nuclear (right) IP fractions are shown.

Mentions: The L1 expresses a 6-kb bicistronic RNA that encodes the 40 kDa Open Reading Frame-1 RNA-binding protein (ORF1p) of essential but uncertain function, and a 150 kDa ORF2 protein with endonuclease and reverse transcriptase (RT) activities (Figure 1A). Retrotransposition is a complex process involving transcription of the L1, transport of its RNA to the cytoplasm, translation of the bicistronic RNA, formation of a ribonucleoprotein (RNP) particle, its re-import to the nucleus and target-primed reverse transcription at the integration site.Figure 1.


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

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

pc-L1-1FH immunoprecipitates basal L1 RNP complexes from 293T cell lysates after α-FLAG agarose purification. (A) Structure of FLAG-HA-tagged pc-L1-1FH cloned in vector pcDNA6 myc/his B. RT: ORF2 reverse transcriptase domain; EN: endonuclease domain; PCMV: CMV promoter; BGH An: bovine growth hormone polyadenylation signal. (B) FLAG-tagged ORF1p expressed from the construct pc-L1-1FH binds α-FLAG agarose independent of RNase digestion (lanes 5 and 8), but untagged ORF1p (construct pc-L1-RP) will not bind (lane 6). (C and D) Detection of L1 proteins in the RNP IP. Lanes 1–4: input lysates; lanes 6–9: immunoprecipitates; lanes 1, 2, 6 and 7: cytoplasmic fractions; lanes 3, 4, 8 and 9: nuclear fractions. (C) FLAG-HA-tagged ORF1p, detected by α-FLAG antibody. Putative ORF1p dimer and trimer bands are visible in IP samples (lanes 7 and 9). The reason for their absence in lysate samples is unclear (lanes 2 and 4). IP purification factors were determined for cytoplasmic (lanes 2 versus 7, 26-fold) and nuclear (lanes 4 versus 9, 42-fold) fractions and are presented in Supplementary Table S3. Lane labels are at the bottom of panel D. (D) ORF2p detected by α-ORF2-N (154–167) antibody (lanes 7 and 9). ORF2p in nuclear lysate samples is below the level of detection (lanes 3 and 4). (E) ORF2p reverse transcriptase activity detected in both nuclear and cytoplasmic IP reactions containing pc-L1-1FH (lanes 3 and 5), but not in reactions with the empty vector (lanes 2 and 4). RT- control: the RT incubation step was omitted and 2 µl of pc-L1-1FH immunoprecipitate was added directly to the PCR reaction. No PCR product was detected (lanes 1 and 6). The assay is described in Kulpa and Moran (51). (F) L1 RNA detected by RT–PCR (lanes 3 and 5). RT-: RT enzyme was omitted from the cDNA synthesis step using pc-L1-1FH immunoprecipitates (lanes 1 and 6). (G) FLAG-HA-tagged ORF2p is detected in nuclear and cytoplasmic extracts after IP of pc-L1-2FH. (H) The purity of nuclear and cytoplasmic whole-cell lysate fractions is shown by western blotting. α-HDAC1 is a strictly nuclear protein (54) and α-MEK1/2 is cytoplasmic (55). (I) Immunoprecipitated samples resolved on silver-stained polyacrylamide gels. To support protein identification data from complex IP samples, selected prominent band regions were excised for additional MS sequencing. Both cytoplasmic (left) and nuclear (right) IP fractions are shown.
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Related In: Results  -  Collection

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Show All Figures
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gkt512-F1: pc-L1-1FH immunoprecipitates basal L1 RNP complexes from 293T cell lysates after α-FLAG agarose purification. (A) Structure of FLAG-HA-tagged pc-L1-1FH cloned in vector pcDNA6 myc/his B. RT: ORF2 reverse transcriptase domain; EN: endonuclease domain; PCMV: CMV promoter; BGH An: bovine growth hormone polyadenylation signal. (B) FLAG-tagged ORF1p expressed from the construct pc-L1-1FH binds α-FLAG agarose independent of RNase digestion (lanes 5 and 8), but untagged ORF1p (construct pc-L1-RP) will not bind (lane 6). (C and D) Detection of L1 proteins in the RNP IP. Lanes 1–4: input lysates; lanes 6–9: immunoprecipitates; lanes 1, 2, 6 and 7: cytoplasmic fractions; lanes 3, 4, 8 and 9: nuclear fractions. (C) FLAG-HA-tagged ORF1p, detected by α-FLAG antibody. Putative ORF1p dimer and trimer bands are visible in IP samples (lanes 7 and 9). The reason for their absence in lysate samples is unclear (lanes 2 and 4). IP purification factors were determined for cytoplasmic (lanes 2 versus 7, 26-fold) and nuclear (lanes 4 versus 9, 42-fold) fractions and are presented in Supplementary Table S3. Lane labels are at the bottom of panel D. (D) ORF2p detected by α-ORF2-N (154–167) antibody (lanes 7 and 9). ORF2p in nuclear lysate samples is below the level of detection (lanes 3 and 4). (E) ORF2p reverse transcriptase activity detected in both nuclear and cytoplasmic IP reactions containing pc-L1-1FH (lanes 3 and 5), but not in reactions with the empty vector (lanes 2 and 4). RT- control: the RT incubation step was omitted and 2 µl of pc-L1-1FH immunoprecipitate was added directly to the PCR reaction. No PCR product was detected (lanes 1 and 6). The assay is described in Kulpa and Moran (51). (F) L1 RNA detected by RT–PCR (lanes 3 and 5). RT-: RT enzyme was omitted from the cDNA synthesis step using pc-L1-1FH immunoprecipitates (lanes 1 and 6). (G) FLAG-HA-tagged ORF2p is detected in nuclear and cytoplasmic extracts after IP of pc-L1-2FH. (H) The purity of nuclear and cytoplasmic whole-cell lysate fractions is shown by western blotting. α-HDAC1 is a strictly nuclear protein (54) and α-MEK1/2 is cytoplasmic (55). (I) Immunoprecipitated samples resolved on silver-stained polyacrylamide gels. To support protein identification data from complex IP samples, selected prominent band regions were excised for additional MS sequencing. Both cytoplasmic (left) and nuclear (right) IP fractions are shown.
Mentions: The L1 expresses a 6-kb bicistronic RNA that encodes the 40 kDa Open Reading Frame-1 RNA-binding protein (ORF1p) of essential but uncertain function, and a 150 kDa ORF2 protein with endonuclease and reverse transcriptase (RT) activities (Figure 1A). Retrotransposition is a complex process involving transcription of the L1, transport of its RNA to the cytoplasm, translation of the bicistronic RNA, formation of a ribonucleoprotein (RNP) particle, its re-import to the nucleus and target-primed reverse transcription at the integration site.Figure 1.

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