Identification of L1 ORF2p sequence important to retrotransposition using Bipartile Alu retrotransposition (BAR)

Abstract

Long Interspersed Element 1 (LINE-1 or L1) is capable of causing genomic instability through the activity of the L1 ORF2 protein (ORF2p). This protein contains endonuclease (EN) and reverse transcriptase (RT) domains that are necessary for the retrotransposition of L1 and the Short Interspersed Element (SINE) Alu. The functional importance of approximately 50% of the ORF2p molecule remains unknown, but some of these sequences could play a role in retrotransposition, or be necessary for the enzymatic activities of the EN and/or RT domains. Conventional approaches using the full-length, contiguous ORF2p make it difficult to study the involvement of these unannotated sequences in the function of L1 ORF2p. Our lab has developed a Bipartile Alu Retrotransposition (BAR) assay that relies on separate truncated ORF2p fragments: an EN-containing and an RT-containing fragment. We validated the utility of this method for studying the ORF2p function in retrotransposition by assessing the effect of expression levels and previously characterized mutations on BAR. Using BAR, we identified two pairs of amino acids important for retrotransposition, an FF and a WD. The WD appears to play a role in cDNA synthesis by the ORF2p molecule, despite being outside the canonical RT domain.

The importance of L1 ORF2p cryptic sequence to ORF2p fragment-mediated cytotoxicity

“…It may be the case that Cryptic, in addition to harboring amino acids necessary for RT function, possesses amino acids that affect EN activity. Interestingly, the amino acids identified here (aa 270–274) within the Cryptic region of the ORF2p overlap with a putative PCNA binding site that has been shown to be important for Alu retrotransposition (FF 273–274) 9 . Our data suggest that it is possible that PCNA may play a role in the localization or nuclear retention of these longer fragments.…”

“…The area of the ORF2p that may be involved in this phenomenon (between the EN and Z domains) was previously unannotated and served no known function. However, our lab has recently discovered that this region (called Cryptic) of the ORF2p has several amino acids and putative motifs important to retrotransposition 9 . Our data presented here show that there are amino acids in the Cryptic region of the ORF2p molecule that may be modulating EN function.…”

“…The same phenomenon was observed with other EN-containing fragments of various lengths. The Cryptic area of the ORF2p contains amino acids important for retrotransposition 9 . We hypothesized that this area of the ORF2p also contains the amino acid sequence that influences the subcellular localization of the ORF2p fragments and the different cytotoxic potential of these fragments.…”

Comprehensive scanning mutagenesis of human retrotransposon LINE-1 identifies motifs essential for function

“…L1 insertion at the target genomic locus occurs via target-primed reverse transcription (TPRT) (Luan et al 1993;Feng et al 1996;Cost et al 2002). While some key amino acid sequences have been elucidated (Mathias et al 1991;Feng et al 1996;Weichenrieder et al 2004;Khazina et al 2011;Christian et al 2016;Ade et al 2018;Khazina and Weichenrieder 2018), there is still much more that remains to be understood about the various L1 protein motifs and how they contribute to the L1 life cycle.…”

“…The most thoroughly understood regions functionally are the enzymatic endonuclease (EN) and reverse transcriptase (RT) domains (Mathias et al 1991;Feng et al 1996). Other less functionally defined motifs include the recently described Cryptic (Cry) sequence (Christian et al 2016), the Z domain region (Clements and Singer 1998), and the carboxy-terminal segment (CTS), which harbors a cysteine rich motif (Fanning and Singer 1987) that is important for retrotransposition. There is a crystal structure of the EN domain (Weichenrieder et al 2004) but the remainder of ORF2p remains structurally uncharacterized.…”