1. A 3′ Poly(A) Tract Is Required for LINE-1 Retrotransposition
Aurélien J. Doucet, Jeremy E. Wilusz, Tomoichiro Miyoshi, Ying Liu, John V. Moran 
Molecular Cell 
Volume 60, Issue 5, Pages (December 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 60, 728-741DOI: (10.1016/j.molcel.2015.10.012)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1
L1/MALAT RNAs Accumulate in Cells and Support L1 Protein Expression
(A) Engineered human L1 constructs. The 3′ ends of RC-L1s in the pCEP4 episomal expression vector containing either conventional polyadenylation signals (left, L1pA and SV40pA) or a 174 nt fragment from the 3′ end of the mouse MALAT1 locus (MALAT) are shown. The MALAT cassette contains two U-rich motifs (U1 and U2, red), an A-rich tract (green), and a tRNA-like structure (orange). RNase P cleaves the tRNA-like structure, leading to the production of mascRNA and a mature L1 transcript lacking a poly(A) tail. The U1, U2, and the A-rich tract form a triple helical structure that stabilizes the 3′ end of L1/MALAT RNA. The oligonucleotide probes used for RNA detection (1 and 2 for TAP-1 and TAP-2, respectively) and sizes of L1 RNA fragments after RNase H treatment are indicated below the processed L1 RNAs. The expression vectors contain the Hygromycin resistance marker (HygroR) (see Figure S1A for details).
(B and C) Full-length and RNase H-treated L1 RNA detection. The full-length L1 RNAs (B) or RNase H-treated L1 RNAs (C) detected with TAP-2 probe (see A) are shown. ACTB (β-actin) and Hygro (B), and RNU6-1 (U6 snRNA) (C) are loading controls. The cells transfected with pCEP-GFP are the negative control (Figure S1A). At least three biological replicates were analyzed by northern blot for each sample.
(D) Western blot detection of the L1-encoded proteins. The ORF1p and ORF2p proteins are detected using antibodies against epitope tags at the carboxyl-termini of ORF1p or ORF2p (T7 gene10 or TAP, respectively). TUBA4A (tubulin) and EIF3C (p110) are the loading controls. The ORF1p or ORF2p levels were normalized to both the loading control and the ORF1p or ORF2p levels in pAT2nn-SV and are shown as mean ± SD. The cells transfected with pCEP-GFP are the negative control. At least three biological replicates were analyzed by western blot for each sample.
See also Figure S1 and Tables S1 and S2.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 An L1 3′ Poly(A) Tract Is Required for Retrotransposition
(A) Schematic of L1 retrotransposition assay. The pAT2-MALAT expression plasmid contains retrotransposition indicator (mneoI) and MALAT cassettes. L1 retrotransposition leads to an L1 insertion at a new genomic DNA location (gray bars). The expression of NEO protein (purple oval) confers G418-resistance to HeLa cells.
(B) Results from retrotransposition assay. The schematics of engineered L1 constructs (left) and results of retrotransposition assay (right) are shown. The x axis shows the construct names and G418-resistant foci from a representative experiment. The y axis shows the relative retrotransposition efficiency. The pAT2-SV construct is the positive control (set at 100%). The pAD135 construct (an RT- allele) is the negative control. The error bars represent the SD in a representative experiment containing three technical replicates. Three biological replicates were performed for each experiment.
See also Figure S2 and Table S1.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Expression of L1/MALAT RNAs Ending in 3′ Tails of a Defined Sequence
(A) Schematic of nucleotide addition in MALAT sequence. The L1 expression vectors containing MALAT (left) or modified MALAT (right) sequence with extra nucleotides (NNNNN) immediately upstream of the RNase P cleavage site. After RNase P cleavage, the mature L1 RNA ends in either a triple helix (left) or a triple helix followed by extra nucleotides (right).
(B and C) Full-length and RNase H-treated L1 RNA detection by northern blot. The extra nucleotides in the modified MALAT constructs are noted by bold lettering in the construct names. Full-length L1 RNAs (B) or RNase H-treated L1 RNAs (C) detected with TAP-2 probe as described in Figures 1B and 1C are shown. For each sample, at least three biological replicates were analyzed.
(D) Western blot detection of L1-encoded proteins. ORF1p and ORF2p detected as noted in Figure 1D are shown. For each sample, at least three biological replicates were analyzed. The data are shown as mean ± SD.
See also Figure S3 and Tables S1 and S2.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Addition of a 3′ Poly(A) Tract to MALAT Cassette Restores L1 Retrotransposition in Cis
(A) Results from the retrotransposition assay. The x axis shows the construct names and G418-resistant foci from a representative experiment. The y axis shows the relative retrotransposition efficiency. The pAT2-SV construct is the positive control (set at 100%). The pAD135 construct (an RT- L1 allele) is the negative control. The error bars represent the SD in the representative experiment containing three technical replicates. Three biological replicates were performed for each experiment.
(B) Structural hallmarks of L1 retrotransposition events. The names of constructs and G418-resistant foci from a representative experiment (left) are shown. The columns indicate clone name, chromosomal location, size of TSD, L1 EN cleavage site, size of the 3′ A-tract, and description of the 3′ end of L1 RNA. All of the recovered insertions contained a 3′ poly(A) sequence.
See also Figure S4 and Table S1.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
A 3′ Poly(A) Tract Mediates Recruitment of ORF2p to Retrotransposition-Defective L1 RNAs in Trans
(A) Engineered L1 constructs used in trans-complementation assay. The driver L1 plasmid (pTMO2F3) expresses a version of L1 ORF2p that contains a carboxyl-terminal triple-FLAG epitope tag (3×FLAG). The reporter plasmids express an RT- L1 that contains either a conventional polyadenylation signal (pAD135) or derivatives of a MALAT sequence (pAD135-MALAT plasmid series), and the reporter plasmids contain the mneoI retrotransposition indicator cassette. The locations of primers (TM187 and TM188) used for the RNA-IP experiment are shown.
(B) Results of trans-complementation assay. The x axis shows construct names and G418-resistant foci from a representative experiment. The y axis shows the relative retrotransposition efficiency. The cells co-transfected with pTMO2F3 and pAD135 (SV40pA) are the positive control (set at 100%). The cells co-transfected with pCEP-GFP and pAD135 (SV40pA) are the negative control. The data are shown as mean ± SD of three biological replicates.
(C and D) Flow chart and results of RNA-IP experiments. Quantitative RT-PCR was used to measure the association of FLAG-tagged ORF2p with L1 RNA. The x axis shows the driver and reporter plasmids co-transfected into HeLa-JVM cells. The y axis shows the enrichment of the reporter plasmid L1 RNA in the IP fraction relative to the input fraction. The cells co-transfected with pTMO2F3 plus pAD135 serve as the positive control (set at 100%). The error bars represent the SD in a representative experiment containing three technical replicates. There were two biological replicates that were performed for each experiment.
See also Figure S5 and Table S1.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Alu Mobilization Is Increased when L1 RNA Lacks a 3′ Poly(A) Tract
(A) Rationale of Alu retrotransposition assay. The driver L1 expression plasmids that contain either a conventional polyadenylation signal or a version of the MALAT cassette were co-transfected into HeLa-HA cells with the Alu reporter plasmid containing the retrotransposition indicator cassette. The assay measures the ability of the L1 ORF2p (blue oval) to retrotranspose Alu RNA in trans.
(B) Results of Alu retrotransposition assay. The x axis shows construct names and G418-resistant foci from a representative experiment. The y axis shows the relative retrotransposition efficiency. The cells co-transfected with pAT2nn-SV and pAlu-neoTet are the positive control (set at 100%). The cells co-transfected with an RT- L1 allele (pAD135-NT) and pAlu-neoTet are the negative control. The error bars represent the SD in a representative experiment containing three technical replicates. Three biological replicates were performed for each experiment. See also Figure S6 and Table S1.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Model for the Role of L1 3′ Poly(A) Tract in Retrotransposition Dynamics
ORF2p (blue oval) expressed from L1 RNA containing either a 3′ poly(A) tail (left) or a MALAT sequence supplemented with an A-rich tract at its 3′ end (right) can bind to either L1 or Alu RNA. When L1 RNA lacks a terminal 3′ poly(A) sequence (center), ORF2p cannot efficiently bind L1 RNA; thus, more ORF2p binds Alu RNA, leading to more efficient retrotransposition.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions