Involvement of Conserved Amino Acids in the C-Terminal Region of LINE-1 ORF2p in Retrotransposition

Abstract

Long interspersed element 1 (L1) is the only currently active autonomous retroelement in the human genome. Along with the parasitic SVA and short interspersed element Alu, L1 is the source of DNA damage induced by retrotransposition: a copy-and-paste process that has the potential to disrupt gene function and cause human disease. The retrotransposition process is dependent upon the ORF2 protein (ORF2p). However, it is unknown whether most of the protein is important for retrotransposition. In particular, other than the Cys motif, the C terminus of the protein has not been intensely examined in the context of retrotransposition. Using evolutionary analysis and the Alu retrotransposition assay, we sought to identify additional amino acids in the C terminus important for retrotransposition. Here, we demonstrate that Gal4-tagged and untagged C-terminally truncated ORF2p fragments possess residual potential to drive Alu retrotransposition. Using sight-directed mutagenesis we identify that while the Y1180 amino acid is important for ORF2p- and L1-driven Alu retrotransposition, a mutation at this position improves L1 retrotransposition. Even though the mechanism of the contribution of Y1180 to Alu and L1 mobilization remains unknown, experimental evidence rules out its direct involvement in the ability of the ORF2p reverse transcriptase to generate complementary DNA. Additionally, our data support that ORF2p amino acids 1180 and 1250-1262 may be involved in the reported ORF1p-mediated increase in ORF2p-driven Alu retrotransposition.

A novel role of TRIM28 B box domain in L1 retrotransposition and ORF2p-mediated cDNA synthesis

“…In this study using previously reported ORF2p fragments ( 18 , 34 , 78 ) and TRIM28 fragments ( 56 , 68 ), we show that TRIM28 increases L1 retrotransposition in its oligomer-forming B box domain-dependent manner. TRIM28 B box as well as B boxes from TRIM24 and TRIM33 are sufficient to increase L1 retrotransposition.…”

“…In short, HeLa cells co-transfected with T7 tagged ORF2p expression plasmid and a plasmid expressing either TRIM28 WT, BB WT, BB 3M, TRIM28 3M, or empty plasmid were harvested 48 h post transfection for ORF2 RNPs purification. Following ultracentrifugation of the lysates through a sucrose cushion, ORF2 mRNA was reverse transcribed by the ORF2p present in the purified RNPs in a LEAP reaction using ORF2 mRNA specific primer as described ( 34 , 78 ). To analyze the resulting cDNA, we used a series of ORF2 sequence-specific, stepwise primer sets designed to amplify cDNA products varying in length from 353 bp to 684 bp (Figure 7A ).…”

“…The ORF2 constructs were generated as previously described ( 18 , 34 , 78 ). Specifically, Cryptic-T7 (ORF2 AA 247–380) was tagged with T7 on the C-terminus.…”

Structures, functions and adaptations of the human LINE-1 ORF2 protein

“…The wrist domain (863–1061) has not been previously recognized, although experiments deleting large portions of the wrist and the subsequent CTD have shown that both domains are required for efficient retrotransposition 37 . Scanning mutagenesis also has shown numerous wrist regions required for retrotransposition 38 .…”

New Understanding of the Relevant Role of LINE-1 Retrotransposition in Human Disease and Immune Modulation

“…High expression of endonuclease induces double-strand DNA breakage that can aggravate DNA damage repair and increase genomic instability (Kines et al, 2014). Reverse transcriptase activation can promote cell proliferation and differentiation and also alter the non-coding RNA transcription spectrum and other epigenetic phenotypes, resulting in alterations in cell regulatory networks, tumor development, and other important pathological processes (Rodic and Burns, 2013;Burns, 2017;Christian et al, 2017). ORF2 can express early in the tumorigenesis process, as it can be detected by a highly specific monoclonal antibody (mAb chA1-L1) in both transitional colon mucosa and prostate intraepithelial neoplasias (De Luca et al, 2016).…”