Abstract

BackgroundLong 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.ResultsWe 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.ConclusionsAlthough 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.

Highlights

  • Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans

  • We have not yet detected endogenous ORF2p using these approaches; we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers (CRC), in which ORF2p was not found

  • Clinical Proteomics Tumor Analysis Consortium (CPTAC) has generated deep mass spectrometry based proteomics data from treatment naive breast [36] and ovarian [37] tumors using isobaric labeling and extensive prefractionation with alkaline reversed-phase chromatography followed by inline reversed-phase chromatography and Orbitrap mass spectrometry

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Summary

Introduction

Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. Many malignant tissues undergo L1 promoter hypomethylation [13,14,15,16,17] and permit L1 expression and (2020) 11:1 somatic retrotransposition [13, 14, 18,19,20,21,22,23,24,25,26] Both targeted and genome-wide sequencing efforts have identified thousands of de novo insertions that have occurred across hundreds of human cancers. L1 retrotransposition can contribute directly to cellular transformation; in colon cancers, acquired L1 insertions are known to cause driving mutations in the adenomatous polyposis coli (APC) tumor suppressor [13, 29]

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