LEAP: L1 Element Amplification Protocol

Abstract

Long INterspersed Element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that are required for retrotransposition. The L1 element amplification protocol (LEAP) assays the ability of L1 ORF2p to reverse transcribe L1 RNA in vitro. Ultracentrifugation or immunoprecipitation is used to isolate L1 ribonucleoprotein particle (RNP) complexes from cultured human cells transfected with an engineered L1 expression construct. The isolated RNPs are incubated with an oligonucleotide that contains a unique sequence at its 5' end and a thymidine-rich sequence at its 3' end. The addition of dNTPs to the reaction allows L1 ORF2p bound to L1 RNA to generate L1 cDNA. The resultant L1 cDNAs then are amplified using polymerase chain reaction (PCR) and the products are visualized by gel electrophoresis. Sequencing the resultant PCR products then allows product verification. The LEAP assay has been instrumental in determining how mutations in L1 ORF1p and ORF2p affect L1 reverse transcriptase (RT) activity. Furthermore, the LEAP assay has revealed that the L1 ORF2p RT can extend a DNA primer with mismatched 3' terminal bases when it is annealed to an L1 RNA template. As the LINE-1 biology field gravitates toward studying cellular proteins that regulate LINE-1, molecular genetic and biochemical approaches such as LEAP, in conjunction with the LINE-1-cultured cell retrotransposition assay, are essential to dissect the molecular mechanism of L1 retrotransposition.

Keywords: L1 element amplification protocol (LEAP); LINE-1; Reverse transcriptase (RT); Ribonucleoprotein particle (RNP).

Figure 1. Target site-primed reverse transcription (TPRT)

The L1 RNA (grey-yellow-blue-grey line), ORF1p (yellow circles), and ORF2p (blue circle) minimally constitute the L1 RNP. The L1 RNP targets genomic DNA (black bold lines) and nicks one T-rich strand exposing a free 3′OH. L1 ORF2p RT uses the T-rich DNA strand as a primer for reverse transcription of the L1 RNA template. After initial priming, L1 ORF2p RT synthesizes a complementary L1 cDNA strand (green arrow) using L1 RNA as a template. Second strand synthesis and completion of integration remain to be elucidated from L1 retrotransposition but may follow the Bombyx mori R2 retrotransposon model of integration (52).

Figure 2. Enrichment of the L1 RNP

(A) A timeline of the LEAP assay is depicted and described in the Methods. Days of the protocol are noted above and the corresponding days post-transfection (d0-14) are noted below. (B) The pJM101/L1.3 L1 construct contains the L1.3 element (accession no. L19088). The pCEP4 plasmid (Life Technologies) backbone encodes for the EBNA-1 (EBNA-1) viral protein and contains an origin of viral replication (oriP), a hygromycin B-resistance gene (HYG^R), the cytomegalovirus (CMV) promoter (large black triangle) and an SV40 polyadenylation signal (large black lollipop) for plasmid replication, hygromycin-selection, and transcription, respectively, in mammalian cultured cells. The pCEP4 backbone also has a bacterial origin of replication (ori) and an ampicillin-resistance gene (AMP^R) for replication and ampicillin-selection (respectively) in E.coli. (For details of the mneoI reporter cassette, please see the “LINE-1 Cultured Cell Retrotransposition Assay” chapter in this volume.) After transfection and hygromycin B-selection in HeLa-JVM cells, the cells are lysed and subjected to high-speed centrifugation through a sucrose cushion. After centrifugation, cellular RNPs are enriched in the pellet fraction. This fraction contains the L1 RNA bound by L1 ORF1p and ORF2p, which minimally constitutes the L1 RNP (see Figure 1). (C) The pES2TE1 L1 construct (18), like pJM101/L1.3, contains the L1.3 element in a pCEP4 (Life Technologies) backbone. Unlike pJM101/L1.3, the pES2TE1 construct encodes a T7-tagged (orange diamond) ORF1p and a FLAG-HA-tagged (purple diamond) ORF2p (18). After transfection and hygromycin B selection, HeLa cells are lysed and subjected to immunoprecipitation using an anti-FLAG antibody conjugated to beads (red circle with black “Y”). Immunoprecipitated complexes contain L1 ORF1p and ORF2p bound to its encoding RNA, which minimally constitutes the L1 RNP.

Figure 3. The LEAP Assay

(A) The L1 RNP minimally consists of L1 ORF1p (yellow circles) and ORF2p (blue circle) bound to the L1 RNA (multicolored line). The L1 RNP is incubated with a 5′-RACE-T₁₂VN-3′ primer and dNTPs. The L1 RT activity (green arrow) of ORF2p initiates L1 cDNA (green line) synthesis. Subsequently, the L1 cDNA is amplified using an engineered LINE-1 construct-specific primer (SENSE) and a RACE primer (red arrows), resulting in the LEAP product (double red line). (B) LEAP products can be resolved by electrophoresis and visualized by staining. LEAP products (top panel), L1 RNA present in RNPs (MMLV-RT; middle panel), and GAPDH RNA levels (GAPDH; lower panel) are shown. A standard LEAP assay includes a water control PCR reaction (H₂0); a control reaction without any RT (No RNP/RT); a reaction using RNPs from empty vector-transfected cells (pCEP4); a reaction with wild type L1 RNPs (WT LINE-1); and a reaction with RT-mutant L1 RNPs (RT mutant LINE-1). The molecular weight (MW) ladder sizes are shown in base pairs (bp). L1 ORF1p and ORF2p protein levels can also be detected by western blot analyses (not shown) (18, 28).