LINE-1 ORF2p expression is nearly imperceptible in human cancers
Abstract Background Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized...
| Main Authors: | , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2019-12-01
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| Series: | Mobile DNA |
| Online Access: | https://doi.org/10.1186/s13100-019-0191-2 |
| _version_ | 1818611862212706304 |
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| author | Daniel Ardeljan Xuya Wang Mehrnoosh Oghbaie Martin S. Taylor David Husband Vikram Deshpande Jared P. Steranka Mikhail Gorbounov Wan Rou Yang Brandon Sie H. Benjamin Larman Hua Jiang Kelly R. Molloy Ilya Altukhov Zhi Li Wilson McKerrow David Fenyö Kathleen H. Burns John LaCava |
| author_facet | Daniel Ardeljan Xuya Wang Mehrnoosh Oghbaie Martin S. Taylor David Husband Vikram Deshpande Jared P. Steranka Mikhail Gorbounov Wan Rou Yang Brandon Sie H. Benjamin Larman Hua Jiang Kelly R. Molloy Ilya Altukhov Zhi Li Wilson McKerrow David Fenyö Kathleen H. Burns John LaCava |
| author_sort | Daniel Ardeljan |
| collection | DOAJ |
| description | Abstract Background Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines. Results We report mass spectrometry-based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743. Conclusions Although somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth. |
| first_indexed | 2024-12-16T15:37:04Z |
| format | Article |
| id | doaj.art-20049d0c7d7043acb41589f76709b424 |
| institution | Directory Open Access Journal |
| issn | 1759-8753 |
| language | English |
| last_indexed | 2024-12-16T15:37:04Z |
| publishDate | 2019-12-01 |
| publisher | BMC |
| record_format | Article |
| series | Mobile DNA |
| spelling | doaj.art-20049d0c7d7043acb41589f76709b4242022-12-21T22:26:09ZengBMCMobile DNA1759-87532019-12-0111111910.1186/s13100-019-0191-2LINE-1 ORF2p expression is nearly imperceptible in human cancersDaniel Ardeljan0Xuya Wang1Mehrnoosh Oghbaie2Martin S. Taylor3David Husband4Vikram Deshpande5Jared P. Steranka6Mikhail Gorbounov7Wan Rou Yang8Brandon Sie9H. Benjamin Larman10Hua Jiang11Kelly R. Molloy12Ilya Altukhov13Zhi Li14Wilson McKerrow15David Fenyö16Kathleen H. Burns17John LaCava18McKusick Nathans Department of Genetic Medicine, Johns Hopkins University School of MedicineInstitute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU School of MedicineLaboratory of Cellular and Structural Biology, The Rockefeller UniversityDepartment of Pathology, Johns Hopkins University School of MedicineDepartment of Pathology, Johns Hopkins University School of MedicineDepartment of Pathology, Massachusetts General Hospital, Harvard Medical SchoolDepartment of Pathology, Johns Hopkins University School of MedicineDepartment of Pathology, Johns Hopkins University School of MedicineDepartment of Pathology, Johns Hopkins University School of MedicineDepartment of Pathology, Johns Hopkins University School of MedicineDepartment of Pathology, Johns Hopkins University School of MedicineLaboratory of Cellular and Structural Biology, The Rockefeller UniversityLaboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller UniversityMoscow Institute of Physics and TechnologyInstitute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU School of MedicineInstitute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU School of MedicineInstitute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, NYU School of MedicineMcKusick Nathans Department of Genetic Medicine, Johns Hopkins University School of MedicineLaboratory of Cellular and Structural Biology, The Rockefeller UniversityAbstract Background Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines. Results We report mass spectrometry-based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743. Conclusions Although somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth.https://doi.org/10.1186/s13100-019-0191-2 |
| spellingShingle | Daniel Ardeljan Xuya Wang Mehrnoosh Oghbaie Martin S. Taylor David Husband Vikram Deshpande Jared P. Steranka Mikhail Gorbounov Wan Rou Yang Brandon Sie H. Benjamin Larman Hua Jiang Kelly R. Molloy Ilya Altukhov Zhi Li Wilson McKerrow David Fenyö Kathleen H. Burns John LaCava LINE-1 ORF2p expression is nearly imperceptible in human cancers Mobile DNA |
| title | LINE-1 ORF2p expression is nearly imperceptible in human cancers |
| title_full | LINE-1 ORF2p expression is nearly imperceptible in human cancers |
| title_fullStr | LINE-1 ORF2p expression is nearly imperceptible in human cancers |
| title_full_unstemmed | LINE-1 ORF2p expression is nearly imperceptible in human cancers |
| title_short | LINE-1 ORF2p expression is nearly imperceptible in human cancers |
| title_sort | line 1 orf2p expression is nearly imperceptible in human cancers |
| url | https://doi.org/10.1186/s13100-019-0191-2 |
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