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. Author manuscript; available in PMC: 2016 Dec 3.
Published in final edited form as: Mol Cell. 2015 Nov 12;60(5):728–741. doi: 10.1016/j.molcel.2015.10.012

A 3′ poly(A) tract is required for LINE-1 retrotransposition

Aurélien J Doucet 1,*,+, Jeremy E Wilusz 4, Tomoichiro Miyoshi 1, Ying Liu 3, John V Moran 1,2,3,*
PMCID: PMC4671821  NIHMSID: NIHMS730039  PMID: 26585388

Summary

L1 retrotransposons express proteins (ORF1p and ORF2p) that preferentially mobilize their encoding RNA in cis, but they also can mobilize Alu RNA and, more rarely, cellular mRNAs in trans. Although these RNAs differ in sequence, each ends in a 3′ polyadenosine (poly(A)) tract. Here, we replace the L1 polyadenylation signal with sequences derived from a non-polyadenylated long noncoding RNA (MALAT1), which can form a stabilizing triple helix at the 3′ end of an RNA. L1/MALAT RNAs accumulate in cells, lack poly(A) tails, and are translated; however, they cannot retrotranspose in cis. Remarkably, the addition of a 16 or 40 base poly(A) tract downstream of the L1/MALAT triple helix restores retrotransposition in cis. The presence of a poly(A) tract also allows ORF2p to bind and mobilize RNAs in trans. Thus, a 3′ poly(A) tract is critical for the retrotransposition of sequences that comprise approximately one billion base pairs of human DNA.

Introduction

Long INterspersed Element-1 (L1) retrotransposons comprise ~17% of the human genome (Lander et al., 2001). The vast majority of L1s cannot mobilize (i.e., retrotranspose) because they are 5′ truncated or have accumulated mutations (Grimaldi et al., 1984). However, an average human genome contains ~80-100 retrotransposition-competent L1s (RC-L1s) (Beck et al., 2010; Brouha et al., 2003; Sassaman et al., 1997). A subset of RC-L1s, termed ‘hot L1s’, is responsible for the bulk of ongoing retrotransposition in man and continues to generate both intra- and inter-individual genetic variation (reviewed in (Beck et al., 2011; Richardson et al., 2015)). L1-mediated retrotransposition events also can lead to disease-producing mutations (Kazazian et al., 1988) (reviewed in (Hancks and Kazazian, 2012)).

Full-length human RC-L1s are ~6 kb. They contain a 5′ untranslated region (UTR), two open reading frames (ORF1 and ORF2) that are separated by a 63 nucleotide (nt) inter-ORF region, and a 3′ UTR that ends in a polyadenosine-rich (poly(A)) tract (Dombroski et al., 1991; Scott et al., 1987). ORF1 encodes an ~40 kDa protein (ORF1p) that has RNA binding and nucleic acid chaperone activities (Hohjoh and Singer, 1996; Holmes et al., 1992; Khazina et al., 2011; Martin and Bushman, 2001). ORF2 encodes an ~150 kDa protein (ORF2p) (Alisch et al., 2006; Doucet et al., 2010; Ergun et al., 2004) that has DNA endonuclease (EN) and reverse transcriptase (RT) activities (Feng et al., 1996; Mathias et al., 1991). Both ORF1p and ORF2p are required for efficient L1 retrotransposition (Feng et al., 1996; Moran et al., 1996).

The production of a full-length, polyadenylated L1 RNA begins a round of L1 retrotransposition (reviewed in (Beck et al., 2011)). An internal RNA polymerase II promoter within the L1 5′ UTR directs transcription to start at or near the first base of a RC-L1 (Athanikar et al., 2004; Becker et al., 1993; Swergold, 1990). A canonical RNA polymerase II polyadenylation signal, located at the end of the L1 3′ UTR or in 3′ flanking genomic DNA sequences (Belancio et al., 2007; Holmes et al., 1994; Moran et al., 1999), directs the addition of a poly(A) tail to L1 RNA. The resultant L1 RNA is exported to the cytoplasm, where it undergoes translation to produce ORF1p and ORF2p (Alisch et al., 2006; Dmitriev et al., 2007; Leibold et al., 1990; McMillan and Singer, 1993).

ORF1p and ORF2p preferentially associate with their encoding RNA in cis (Esnault et al., 2000; Kulpa and Moran, 2006; Wei et al., 2001), leading to the assembly of a ribonucleoprotein particle (RNP) that is a necessary retrotransposition intermediate (Doucet et al., 2010; Hohjoh and Singer, 1996; Kulpa and Moran, 2005; Martin, 1991). Components of the L1 RNP can gain access to the nucleus where retrotransposition likely occurs by target-site primed reverse transcription (TPRT) (Cost et al., 2002; Feng et al., 1996; Luan et al., 1993). During TPRT, ORF2p-encoded EN makes a single-strand endonucleolytic nick in genomic DNA at a degenerate consensus sequence (e.g., 5′-TTTT/A-3′ or variants of that sequence) (Cost and Boeke, 1998; Cost et al., 2002; Feng et al., 1996; Morrish et al., 2002). The resultant 3′ hydroxyl group is used by the L1 RT to prime L1 cDNA synthesis, which generally begins within the L1 RNA 3′ poly(A) tail (Cost et al., 2002; Kulpa and Moran, 2006; Monot et al., 2013). Subsequent steps of L1 integration (i.e., second-strand DNA cleavage and (+) strand cDNA synthesis) require elucidation. TPRT ultimately leads to the integration of L1 at a new genomic location that generally contains the following structural hallmarks: integration at an L1 EN consensus cleavage site, frequent 5′ truncation, a 3′ poly(A) tract, and flanking variably-sized target-site duplications (TSDs) (reviewed in (Beck et al., 2011)).

L1 ORF1p and/or ORF2p also can act in trans to promote the retrotransposition of Short INterspersed Element (SINE) RNAs (e.g., Alu and SINE-R/VNTR/Alu (SVA) elements) (Dewannieux et al., 2003; Hancks et al., 2011; Raiz et al., 2012), as well as cellular mRNAs, which leads to processed pseudogene formation (Esnault et al., 2000; Wei et al., 2001). Although L1, SINE, and cellular mRNAs differ in sequence, they all contain a 3′ poly(A) tail or 3′ adenosine-rich tract (Boeke, 1997; Richardson et al., 2015). As a 50 base poly(A) tract at the 3′ end of Alu RNA is required for efficient Alu retrotransposition (Dewannieux et al., 2003; Dewannieux and Heidmann, 2005), we hypothesized that the L1 poly(A) tail also plays a critical role in retrotransposition. However, this hypothesis has been difficult to test, as the removal of the L1 poly(A) tail likely would lead to destabilization and/or degradation of L1 RNA by cellular exonucleases (reviewed in (Houseley et al., 2006)).

Here, we replace the polyadenylation signal at the 3′ end of a human RC-L1 with a sequence derived from the 3′ end of the long noncoding RNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) (Brown et al., 2014; Brown et al., 2012; Wilusz et al., 2008; Wilusz et al., 2012). The MALAT cassette comprises a sequence capable of forming a triple helical structure followed by a tRNA-like sequence. Cleavage by RNase P releases the tRNA-like sequence, allowing the triple helical structure to form at the 3′ end of the mature L1/MALAT RNA. Previous work revealed that the MALAT triple helix can prevent the degradation of a green fluorescent protein (GFP)/MALAT reporter RNA and that GFP is efficiently translated from GFP/MALAT RNA despite its lack of a poly(A) tail (Wilusz et al., 2012).

We report that processed, non-polyadenylated L1/MALAT RNA accumulates in cells and serves as a template for ORF1p and ORF2p translation. Intriguingly, the mature L1/MALAT RNA is severely compromised for retrotransposition in cis. However, the addition of either 16 or 40 adenosine residues immediately downstream of the triple helix in the L1/MALAT primary transcript (upstream of the RNase P cleavage site) restores efficient retrotransposition in cis. Genetic and biochemical assays reveal that a poly(A) tract allows ORF2p to bind and mobilize RNAs in trans. Finally, we find that ORF2p produced from L1/MALAT RNA enhances Alu retrotransposition in trans. Thus, a poly(A) tract is a critical RNA sequence required for efficient L1-mediated retrotransposition.

Results

L1/MALAT RNAs lack a 3′ poly(A) tract and are stably expressed in cultured human cells

We replaced the polyadenylation signals at the 3′ end of an engineered human RC-L1 expression vector (Figure 1A; left panel, pAT2nn-SV, and Figure S1A) with a 174-nt sequence cassette derived from the long noncoding RNA MALAT1 (Figure 1A; right panel, pAT2nn-MALAT, Figure S1A, and Table S1). MALAT1 is transcribed by RNA polymerase II, but the canonical cleavage/polyadenylation machinery is not used to generate its mature 3′ end (Wilusz et al., 2008; Wilusz et al., 2012). Instead, a tRNA-like structure present at the 3′ end of the MALAT1 primary transcript is cleaved by RNase P to simultaneously generate the mature 3′ end of the MALAT1 transcript and the 5′ end of a small tRNA-like transcript (Figure 1A; right panel). Additional tRNA biogenesis enzymes process the tRNA-like transcript to generate a 61 nt MALAT1-associated small cytoplasmic RNA (mascRNA) (Wilusz et al., 2008). After RNase P cleavage, the two uracil-rich motifs (U1 and U2) and an adenosine-rich (A-rich) tract form a triple helical structure that protects the 3′ end of MALAT1 from 3′-5′ exonucleolytic degradation (Figure 1A; right panel) (Brown et al., 2014; Brown et al., 2012; Wilusz et al., 2012). This 174-nt MALAT sequence cassette (nucleotides 6581-6754 of mouse MALAT1 comprising the U1, U2, A-rich tract, and tRNA-like sequence) can substitute for a canonical polyadenylation signal and stabilize a mature RNA polymerase II transcript (Brown et al., 2012; Wilusz et al., 2012).

Figure 1. L1/MALAT RNAs accumulate in cells and support L1 protein expression.

Figure 1

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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). 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. U1, U2, and the A-rich tract form a triple helical structure that stabilizes the 3′ end of L1/MALAT RNA. 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 processed L1 RNAs. 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: Full-length L1 RNAs (B) or RNase H-treated L1 RNAs (C) detected with TAP-2 probe (see panel A). β-actin and Hygro (panel B), and U6 (panel C) are loading controls. Cells transfected with pCEP-GFP, 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: ORF1p and ORF2p detected using antibodies against epitope tags at carboxyl-termini of ORF1p or ORF2p (T7 gene10 or TAP, respectively). Tubulin and p110, loading controls. 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. Cells transfected with pCEP-GFP, negative control. At least three biological replicates were analyzed by Western blot for each sample. See also Figure S1 and Tables S1 and S2.

HeLa-JVM cells were transfected with either pAT2nn-SV or pAT2nn-MALAT and selected with hygromycin B. Total cellular RNAs isolated 10 days post-transfection were subjected to Northern blot analysis, using a probe complementary to sequences specific to the human L1 constructs (Figure 1A; TAP-2 probe). The pAT2nn-SV and pAT2nn-MALAT RNAs were the expected sizes and were stably expressed at comparable levels (Figures 1B and S1B). Mutating the U- and A-rich motifs (Figure S1C) to destabilize the triple helical structure of the L1/MALAT transcript drastically reduced steady state L1/MALAT RNA levels (Figures 1B, S1B, and Table S1; pAT2nn-MmutCG).

We next conducted RNase H cleavage assays to determine if the RNAs generated from the above constructs were properly processed at their 3′ ends. A DNA oligonucleotide complementary to sequences near the 3′ end of the engineered L1 RNAs (Figure 1A; TAP-1 probe) was hybridized to total cellular RNAs isolated from transfected HeLa-JVM cells. The resultant RNA/DNA hybrids were subjected to RNase H digestion and were analyzed by Northern blot (Figure 1A; TAP-2 probe and Figure 1C). RNAs derived from pAT2nn-SV exhibited products of the expected size (~306 nt plus the size of the variable length poly(A) tail). Ligation-mediated 3′ rapid amplification of cDNA ends (3′-RACE; see Methods) confirmed the presence of short (<23 nt) poly(A) tracts on the resultant RNAs (Table S2). By comparison, a shorter band of the expected size (~234 nt) was observed for RNAs derived from the pAT2nn-MALAT and pAT2nn-MmutCG expression constructs (Figures 1C and S1D), confirming efficient RNase P cleavage. As above, we observed a dramatic reduction in the pAT2nn-MmutCG RNA levels when compared to RNAs derived from pAT2nn-SV and pAT2nn-MALAT (Figures 1C, S1B, and S1D). Ligation-mediated 3′-RACE verified that the L1 RNAs were processed at the RNase P cleavage sequence, although some exhibited evidence of post-cleavage degradation (Table S2). Northern blotting was unable to detect mascRNA expression at either 24 hours (Figure S1E) or 10 days post-transfection (data not shown), which likely reflects the short half-life (<4 hours) of mascRNA (Wilusz et al., 2008). Furthermore, the engineered L1 transcripts appear to be expressed at reduced levels when compared to previously analyzed expression constructs (Figure S1E) (Wilusz et al., 2012). Nevertheless, L1/MALAT RNAs accumulate in cells and lack a 3′ poly(A) tail.

L1/MALAT RNAs lacking a 3′ poly(A) tract are efficiently translated in cultured cells

To determine if L1/MALAT RNA could undergo translation, whole cell lysates derived from hygromycin resistant HeLa-JVM cells transfected with pAT2nn-SV, pAT2nn-MALAT, or pAT2nn-MmutCG were subjected to Western blot analyses using antibodies to epitope tags at the carboxyl terminus of either ORF1p (T7 gene10) or ORF2p (TAP) (Figure S1A) (Doucet et al., 2010; Kulpa and Moran, 2005). ORF1p and ORF2p were detected in pAT2nn-MALAT extracts, albeit at reduced levels when compared to the pAT2nn-SV control (Figures 1D and S1F). ORF1p and ORF2p also were detected at low levels in pAT2nn-MmutCG extracts (Figures 1D and S1F); thus, a subset of mutant pAT2nn-MmutCG/MALAT RNAs underwent translation.

L1 RNAs lacking a 3′ poly(A) tract cannot retrotranspose efficiently in cis

We next introduced a retrotransposition indicator cassette into the 3′ UTR of the above L1 expression vectors to create pAT2-SV, pAT2-MALAT, and pAT2-MmutCG (Figure 2). The mneoI indicator cassette consists of a backward copy of the neomycin phosphotransferase gene (Neo) equipped with a heterologous promoter and a polyadenylation signal (Moran et al., 1996). The Neo gene is interrupted by an intron that is in the same transcriptional orientation as the L1 RNA. This arrangement ensures that Neo expression only becomes activated upon a successful round of L1 retrotransposition. Thus, a quantitative assessment of L1 retrotransposition efficiency is obtained by counting the resultant number of G418-resistant foci (Figure 2A) (Moran et al., 1996).

Figure 2. A L1 3′ poly(A) tract is required for retrotransposition.

Figure 2

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A. Schematic of L1 retrotransposition assay: The pAT2-MALAT expression plasmid contains retrotransposition indicator (mneoI) and MALAT cassettes. Retrotransposition leads to an L1 insertion at a new genomic DNA location (gray bars). Expression of NEO protein (purple oval) confers G418-resistance to HeLa cells.

B. Results from retrotransposition assay: Schematics of engineered L1 constructs (left) and results of retrotransposition assay (right). X-axis: construct names and G418-resistant foci from a representative experiment. Y-axis: relative retrotransposition efficiency. pAT2-SV, positive control (set at 100%). pAD135 construct (an RT- allele), negative control. Error bars, standard deviation in a representative experiment containing three technical replicates. Three biological replicates were performed for each experiment. See also Figure S2 and Table S1.

HeLa-JVM cells were transfected with pAT2-SV, pAT2-MALAT, or pAT2-MmutCG and subjected to G418 selection three days post-transfection. L1 RNAs derived from the pAT2-SV construct were retrotransposition-competent, whereas L1 RNAs derived from a mutant construct that contains a point mutation in the ORF2p reverse transcriptase active site (pAD135 (RT-)) were retrotransposition-defective (Figure 2B) (Doucet et al., 2010). Although L1/MALAT RNAs derived from pAT2-MALAT stably accumulated in cells and were translated, we did not detect G418-resistant foci, suggesting that L1/MALAT RNAs cannot retrotranspose in cis (Figure 2B). Moreover, while RNAs derived from the pAT2-MmutCG mutant construct were present only at low levels (Figures 1C, S1B, and S1D), we detected G418-resistant foci at ~3% the level of the pAT2-SV control (Figure 2B), suggesting a small subset of pAT2-MmutCG RNAs remains retrotransposition-competent.

The above data suggest that the L1 poly(A) tail is required for efficient retrotransposition in cis or that the MALAT cassette impairs L1 retrotransposition. To discriminate between these possibilities, we inserted the MALAT triple helical sequence (i.e., the U1, U2, and A-rich sequences, but not the tRNA-like sequence) into the 3′ UTR of an RC-L1 upstream of a canonical SV40 polyadenylation signal (Figure S2; pAT2-MP-SV). L1 RNAs derived from pAT2-MP-SV retrotransposed at ~60% of the pAT2-SV control (Figure S2); thus, the MALAT triple helix sequence in pAT2-MP-SV-derived L1 RNA only weakly compromises retrotransposition. These data indicate that a 3′ poly(A) tract is critical for efficient L1 retrotransposition in cis.

Addition of a 3′ poly(A) tract downstream of the MALAT triple helix restores L1 retrotransposition in cis

We next asked if the addition of adenosine tracts to the 3′ end of mature L1/MALAT RNAs could restore retrotransposition (Figure 3A). We generated constructs that contain either 16 adenosines (pAT2nn-Mextra16A), 40 adenosines (pAT2nn-Mextra40A), 16 cytidines (pAT2nn-Mextra16C), or four copies of a CAAA repeat sequence (pAT2nn-Mextra4CAAA) directly between the 3′ end of the triple helix sequence and the RNase P processing site (Figures 3A, S3A, and Table S1). Northern blot analyses revealed that the extra nucleotides did not notably affect L1/MALAT RNA levels (Figures 3B and S3B) and RNase H cleavage assays confirmed proper processing of the 3′ ends (Figure 3C) (e.g., note the mobility shift of RNAs in the pAT2nn-Mextra16A vs. pAT2nn-Mextra40A constructs). Ligation-mediated 3′ RACE experiments revealed that some of the extra nucleotides were removed from the resultant transcripts (likely by 3′-5′ exonucleases) (Table S2). In addition, some of the encoded A tracts were extended (likely by a cellular poly(A) polymerase), whereas other RNAs ended in untemplated uridine residues (Table S2). Uridylation was associated with the degradation of GFP/MALAT (Wilusz et al., 2012) and other cellular RNAs (Lee et al., 2014). That being stated, 92% of the sequenced clones retained extra nucleotides (Table S2). Finally, Western blot analyses revealed that ORF1p and ORF2p were expressed from L1/MALAT RNAs containing extra nucleotides (Figure 3D). The steady state levels of ORF1p and ORF2p expressed from the wild-type polyadenylated construct or from constructs containing either 16 or 40 adenosines were more abundant when compared to constructs containing the MALAT sequence alone or the MALAT sequence supplemented with 16 cytidines or four copies of the CAAA repeat (Figures 3D and S3C). These data suggest that a 3′ poly(A) tract may be critical for efficient L1 translation.

Figure 3. Expression of L1/MALAT RNAs ending in 3′ tails of a defined sequence.

Figure 3

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A. Schematic of nucleotide addition in MALAT sequence: L1 expression vectors containing MALAT (left) or modified MALAT (right) sequence with extra nucleotides (NNNNN) immediately upstream of 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: Extra nucleotides in modified MALAT constructs noted by bold lettering in 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. 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. For each sample, at least three biological replicates were analyzed. Data are shown as mean ± SD. See also Figure S3 and Tables S1 and S2.

To test if the above L1s were capable of retrotransposition, we tagged the modified MALAT sequences with the mneoI indicator cassette (Figure 4A). As above (Figure 2B), L1 RNAs derived from pAT2-MALAT could not retrotranspose (Figure 4A). The addition of either 16 or 40 adenosines to the MALAT sequence restored retrotransposition to ~22% and ~26% of the polyadenylated pAT2-SV control, respectively (Figure 4A). By comparison, the addition of 16 cytidines or four copies of the CAAA repeat only led to a minor enhancement of retrotransposition (Figure 4A; ~5% the level of the pAT2-SV control). Thus, the addition of adenosine residues at the 3′ end of the L1/MALAT RNA rescues L1 retrotransposition in cis.

Figure 4. Addition of a 3′ poly(A) tract to MALAT cassette restores L1 retrotransposition in cis.

Figure 4

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A. Results from the retrotransposition assay: X-axis: construct names and G418-resistant foci from representative experiment. Y-axis: relative retrotransposition efficiency. pAT2-SV, positive control (set at 100%). pAD135 construct (an RT- L1 allele), negative control. Error bars, standard deviation in representative experiment containing three technical replicates. Three biological replicates were performed for each experiment.

B. Structural hallmarks of L1 retrotransposition events: Names of constructs and G418-resistant foci from a representative experiment (left). Columns indicate: clone name, chromosomal location, size of target site duplication (TSD), L1 endonuclease cleavage site, size of the 3′ A-tract, and description of the 3′ end of L1 RNA. All recovered insertions contained a 3′ poly(A) sequence. See also Figure S4 and Table S1.

Retrotransposition events exhibit typical L1 structural hallmarks

We next used inverse PCR to characterize L1 retrotransposition events derived from pAT2-MALAT, pAT2-Mextra16A, and pAT2-MmutU1/U2 (a construct that contains destabilizing mutations in the MALAT triple helix (Wilusz et al., 2012)) (Figure 4B and Table S1). While the transfection of pAT2-MALAT into HeLa-JVM cells did not lead to the generation of G418-resistant foci when retrotransposition assays were conducted in 6-well tissue culture plates (Figure 2B), rare G418-resistant foci were detected when retrotransposition assays were conducted in 15-cm plates (Figures 4B and S4A). Comparisons between the pre- and post-integration sites of 16 L1 retrotransposition events revealed the following L1 TPRT structural hallmarks: 1) integration at L1 EN consensus cleavage sites; 2) 5′ truncations; 3) 3′ poly(A) tracts; and 4) flanking target-site duplications (reviewed in (Beck et al., 2011)). Notably, all the retrotransposition events ended in a poly(A) sequence; 15/16 contained adenosine residues added to the A-rich tract present at the 3′ end of the L1/MALAT triple helix (Figures 4B and S4B). The remaining L1/MALAT insertion was derived from a prematurely terminated RNA that ended in a poly(A) tract (Figure S4C). Thus, all of the 16 L1 retrotransposition events were derived from an L1 RNA that contained a 3′ poly(A) tract.

A 3′ poly(A) tract is required for the retrotransposition of defective L1 RNAs in trans

The L1-encoded proteins occasionally can promote the mobility of retrotransposition-defective L1 (RD-L1) RNAs by a process termed trans-complementation (Wei et al., 2001). To determine if a 3′ poly(A) tract is required for trans-complementation, we adapted the rationale from a previously developed genetic assay (Wei et al., 2001). We co-transfected HeLa-JVM cells with a “driver” plasmid that expresses a triple-FLAG tagged version of L1 ORF2p and lacks the mneoI retrotransposition indicator cassette (pTMO2F3), along with a series of “reporter” plasmids that express an RD-L1 RNA harboring a D702A missense mutation in the ORF2p RT active site (Figure 5A). The “reporter” plasmids contain the mneoI retrotransposition indicator cassette and end with either conventional polyadenylation signals (pAD135) or a version of the MALAT cassette (pAD135-MALAT series) (Figures 5A and 5B). This assay design ensures that G418-resistant foci arise only if ORF2p produced from the “driver” plasmid acts to retrotranspose the “reporter” RD-L1 RNA in trans (Figure S5A). Trans-complementation only occurs at ~0.7% of the level of L1 retrotransposition in cis (Wei et al., 2001); however, the two-to-three orders of magnitude signal-to-noise ratio of the L1 retrotransposition assay allows the ready detection of these rare L1 retrotransposition events (Figure 5B) (Alisch et al., 2006; Wei et al., 2001).

Figure 5. A 3′ poly(A) tract mediates recruitment of ORF2p to retrotransposition-defective L1 RNAs in trans.

Figure 5

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A. Engineered L1 constructs used in trans-complementation assay: “Driver” L1 plasmid (pTMO2F3) expresses a version of L1 ORF2p that contains a carboxyl-terminal triple-FLAG epitope tag (3xFLAG). “Reporter” plasmids express an RT- L1 that contains either a conventional polyadenylation signal (pAD135) or derivatives of a MALAT sequence (pAD135-MALAT plasmid series); reporter plasmids contain the mneoI retrotransposition indicator cassette. Locations of primers (TM187 and TM188) used for RNA-IP experiment are shown.

B. Results of trans-complementation assay: X-axis: construct names and G418-resistant foci from a representative experiment. Y-axis: relative retrotransposition efficiency. Cells co-transfected with pTMO2F3 and pAD135 (SV40pA), positive control (set at 100%). Cells co-transfected with pCEP-GFP and pAD135 (SV40pA), negative control. Data are shown as mean ± SD of three biological replicates.

C. and D. Flow chart and results of RNA-immunoprecipitation (RNA-IP) experiments: Quantitative RT-PCR was used to measure association of FLAG-tagged ORF2p with L1 RNA. X-axis: “driver” and “reporter” plasmids co-transfected into HeLa-JVM cells. Y-axis: enrichment of “reporter” plasmid L1 RNA in IP fraction relative to the input fraction. Cells co-transfected with pTMO2F3 plus pAD135 serve as positive control (set at 100%). Error bars, standard deviation in a representative experiment containing three technical replicates. Two biological replicates were performed for each experiment. See also Figure S5 and Table S1.

To establish the dynamic range of the trans-complementation assay, the ORF2p “driver” plasmid (pTMO2F3) was co-transfected with an RT-defective “reporter” plasmid (pAD135). The resultant G418-resistant foci represent the trans-complementation efficiency (Figure 5B; set at 100%). As a negative control, a “driver” plasmid lacking L1 sequences (pCEP-GFP) was co-transfected with the RT-defective “reporter” plasmid (pAD135). The small number of G418-resistant foci (Figure 5B; less than 1% the level of the positive control) represents the background signal, which likely results from the ability of endogenous L1 proteins to mobilize pAD135 L1 RNA at a low level in trans (Wei et al., 2001). Co-transfection of the ORF2p “driver” plasmid (pTMO2F3) with an RT-defective L1/MALAT “reporter” plasmid (pAD135-MALAT) did not enhance the trans-complementation above background levels (Figure 5B). The addition of either 16 or 40 adenosine residues to the L1/MALAT “reporter” restored trans-complementation to ~27% or ~46% of the positive control, respectively (Figure 5B; pAD135-Mextra16A vs. pAD135 and pAD135-Mextra40A vs. pAD135). By comparison, the addition of 16 cytidine residues or four copies of the CAAA repeat only resulted in a minor enhancement in the trans-complementation efficiency (Figure 5B; ~8 to 9% of the positive control). We confirmed that ORF2p was expressed from our “driver” plasmid at similar levels in each experimental condition (Figure S5B). Thus, similar to the cis-based retrotransposition assay, the 3′ end of L1 reporter RNAs requires a poly(A) tract to mediate efficient trans-complementation.

A 3′ poly(A) tract is required for efficient association of ORF2p with L1 RNA

To test if the L1 poly(A) tract is required to bind ORF2p, we used the trans-complementation assay in conjunction with RNA co-immunoprecipitation (Figures 5C and S5A). We co-transfected a “driver” plasmid that expresses a version of L1 ORF2p containing (pTMO2F3) or lacking (pAD001) a triple-FLAG (3xFLAG) epitope tag with an RT-defective “reporter” plasmid that either contains (pAD135) or lacks (pAD135-MALAT series) conventional polyadenylation signals (Figure S5A). Ten days post-transfection, we performed immunoprecipitation (IP) reactions using an anti-FLAG antibody followed by quantitative RT-PCR to determine the extent to which ORF2-3xFLAGp associated with a “reporter” RNA in trans (Figures 5C and S5A).

The co-transfection of pTMO2F3 with pAD135 led to an enrichment of L1 RNA in the ORF2-3xFLAGp IP fraction when compared to the negative control (Figure 5D; cells co-transfected with pAD001 and pAD135). Replacing the polyadenylation signal on pAD135 with the MALAT cassette (pAD135-MALAT) led to significantly less L1 RNA being detected in the IP fraction (Figure 5D; ~6% of the positive control). The introduction a poly(A) tract downstream of the MALAT triple helix (pAD135-Mextra16A) led to a restoration of L1 RNA associated with ORF2p (Figure 5D; ~41% of the positive control). In contrast, the addition of 16 cytidine residues (pAD135-Mextra16C) resulted in significantly less restoration (Figure 5D; ~14% of the positive control). Thus, a 3′ poly(A) tract plays a critical role in allowing the association of ORF2p to an RD-L1 RNA in trans.

The L1 3′ poly(A) tract and Alu 3′ poly(A) tract compete for the recruitment of ORF2p

We next asked whether the proteins encoded by an L1 lacking a 3′ poly(A) tract could act in trans to retrotranspose a non-autonomous SINE RNA (i.e., an Alu element). HeLa-HA cells (Hulme et al., 2007) were co-transfected with an L1 “driver” plasmid and an Alu “reporter” plasmid. The Alu plasmid contains an active Alu that is interrupted by an RNA polymerase III compatible retrotransposition indicator cassette (Figure 6A; pAlu-neoTet) (Dewannieux et al., 2003). As in the trans-complementation assay, G418-resistant foci only arise if ORF2p produced from the “driver” plasmid can retrotranspose the Alu “reporter” RNA in trans (Figure 6A).

Figure 6. Alu mobilization is increased when L1 RNA lacks a 3′ poly(A) tract.

Figure 6

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A. Rationale of Alu retrotransposition assay: “Driver” L1 expression plasmids that contain either a conventional polyadenylation signal or a version of MALAT cassette were co-transfected into HeLa-HA cells with the Alu “reporter” plasmid containing retrotransposition indicator cassette. The assay measures ability of L1 ORF2p (blue oval) to retrotranspose Alu RNA in trans.

B. Results of Alu retrotransposition assay: X-axis: construct names and G418-resistant foci from a representative experiment. Y-axis: relative retrotransposition efficiency. Cells co-transfected with pAT2nn-SV and pAlu-neoTet, positive control (set at 100%). Cells co-transfected with an RT- L1 allele (pAD135-NT) and pAlu-neoTet, negative control. Error bars, standard deviation in a representative experiment containing three technical replicates. Three biological replicates were performed for each experiment. See also Figure S6 and Table S1.

As a positive control, we co-transfected a “driver” L1 plasmid that contains a conventional polyadenylation signal (pAT2nn-SV) with the Alu “reporter” plasmid. The resultant number of G418-resistant foci was set at 100% (Figure 6B; pAlu-neoTet with AT2nn-SV). As a negative control, we co-transfected a “driver” L1 plasmid that contains a missense mutation in the ORF2p RT active site (pAD135-NT) with the Alu “reporter” plasmid. This experiment only yielded rare G418-resistant foci (Figure 6B). Remarkably, we observed an increase in Alu retrotransposition when the “driver” L1 plasmid (pAT2nn-MALAT) lacked a 3′ poly(A) tract (Figure 6B; ~160% the level of the pAT2nn-SV positive control). The addition of 16 (pAT2nn-Mextra16A) or 40 (pAT2nn-Mextra40A) adenosines to the MALAT sequence of the “driver” L1 plasmid restored Alu retrotransposition to the level of the positive control (Figure 6B; ~97% and 100% the level of the pAT2nn-SV, respectively). By comparison, the addition of 16 cytidines (pAT2nn-Mextra16C) or four copies of the CAAA repeat sequence (pAT2nn-Mextra4CAAA) to the MALAT sequence of the “driver” plasmid increased Alu retrotransposition to ~149% and 143% of the pAT2nn-SV positive control, respectively (Figure 6B). Thus, the absence of a 3′ poly(A) sequence on the “driver” L1 RNA leads to an enhancement of Alu retrotransposition (see Discussion).

Notably, the lack of a 3′ poly(A) tract on L1 “driver” plasmids did not lead to enhanced trans-complementation from a second “reporter” plasmid that resembles a Half-L1 (HAL1) retrotransposon (Figure S6A; pL1.3/ORF1mneoI) (Bao and Jurka, 2010; Jurka et al., 2005; Smit, 1999). Instead, the efficiency of L1.3 ORF1mneoI trans-complementation generally correlated with the steady state level of ORF2p expressed from the respective “driver” plasmids (Figures S3C and S6B). Thus, trans-complementation likely occurs by a different mechanism than Alu retrotransposition (see Discussion).

Discussion

We provide multiple lines of evidence that a 3′ poly(A) tract is required for efficient L1-mediated retrotransposition. First, although L1/MALAT RNAs lack a 3′ poly(A) tract, stably accumulate in cells, and are translated, they cannot retrotranspose in cis (Figures 1 and 2). Second, the addition of a 16 or 40 base adenosine tract downstream of the stabilizing triple helix sequence in L1/MALAT RNA leads to an increase in retrotransposition when compared to L1/MALAT RNAs lacking a 3′ poly(A) tract (Figures 3 and 4). Third, rare retrotransposition events derived from engineered L1/MALAT expression constructs always contained added 3′ poly(A) tracts (Figure 4). Thus, the post-transcriptional extension of the A-rich tract at the 3′ end of the MALAT triple helix by a cellular poly(A) polymerase, or perhaps the use of a cryptic polyadenylation signal, can render L1/MALAT RNAs retrotransposition-competent. Fourth, a 3′ poly(A) tract is required for ORF2p to mediate trans-complementation (Figures 5A and B). Finally, RNA co-immunoprecipitation experiments indicate that ORF2p preferentially associates with RNAs containing a 3′ poly(A) tract (Figures 5C and 5D).

ORF2p binding to the L1 3′ poly(A) tail mediates cis-preference

Non-LTR retrotransposons have evolved different strategies to retrotranspose in eukaryotic genomes (Malik et al., 1999). For “stringent” LINEs such as UnaL2 from the eel genome, the LINE-encoded reverse transcriptase binds to sequences in its 3′ UTR to mediate retrotransposition (Hayashi et al., 2014; Kajikawa and Okada, 2002). Although the human genome contains “stringent” LINEs (e.g., LINE-2 retrotransposons), they are mutated and are no longer retrotransposition-competent (Smit, 1996). In contrast, L1s are considered “relaxed” LINEs and their 3′ UTR is dispensable for retrotransposition in cultured cells (Moran et al., 1996). The finding that ORF2p preferentially associates with L1 RNAs containing a 3′ poly(A) tract provides a mechanistic explanation both for how ORF2p mediates the retrotransposition of its encoding RNA and how L1 RNAs harboring 3′ transduced sequences can retrotranspose to new genomic locations (Richardson et al., 2015).

Our data are consistent with biochemical data that demonstrated that ORF2p initiates reverse transcription within the 3′ L1 poly(A) tract (Cost et al., 2002; Kulpa and Moran, 2006; Monot et al., 2013). We speculate that ORF2p co-translationally binds to the L1 3′ poly(A) tract. In addition, it remains possible that cellular factors, such as poly(A) binding proteins (Taylor et al., 2013; West et al., 2002), also may influence ORF2p binding to the L1 3′ poly(A) tract. Notably, the presence of 16 or 40 adenosines on the 3′ end of L1/MALAT RNA did not result in the same level of retrotransposition as a canonically polyadenylated L1 RNA (Figure 4A). This result likely is due to a combination of factors, including reduced binding of ORF2p to L1 RNA (Figure 5D), as well as weak inhibition of reverse transcription by the MALAT triple helical structure (Figure S2). Nevertheless, we conclude that the L1 3′ poly(A) tract is a cis-acting sequence required for “relaxed” LINE retrotransposition. Indeed, this 3′ poly(A) tract requirement might ensure the preferential amplification of RC-L1s in the human genome.

A poly(A) tract is required for efficient trans-complementation

Our data provide an intuitive explanation for how ORF2p occasionally can act in trans to mobilize retrotransposition-deficient L1 RNAs (Figure 5) and RNAs derived from a reporter construct (pL1.3/ORF1mneoI) to new genomic locations (Figure S6A). The finding that a 3′ poly(A) tract is required for L1-mediated trans-complementation provides evidence that a 3′ poly(A) tract is critical to recruit ORF2p to other cellular RNAs in trans. Moreover, the finding that L1.3 ORF1mneoI trans-complementation generally correlates with the amount of ORF2p encoded by the L1 “driver” plasmids (Figure S6B) is consistent with the idea that ORF2p, on occasion, can bypass cis-preference to promote the retrotransposition of mutant L1 RNAs (Wei et al., 2001). These findings also could explain, in part, how ORF1p and ORF2p retrotranspose cellular RNAs containing 3′ poly(A) tracts to generate processed pseudogenes (Esnault et al., 2000; Wei et al., 2001; Zhang et al., 2003).

The L1 and Alu 3′ poly(A) sequences compete for the binding of ORF2p

Alu elements comprise ~11% of human genomic DNA and require ORF2p to mediate their retrotransposition (Dewannieux et al., 2003). The sheer mass of Alu elements in the genome indicates that they have evolved effective strategies to parasitize L1 ORF2p to mediate their retrotransposition (Lander et al., 2001). As opposed to “stringent” LINE/SINE relationships, where SINE RNAs have co-opted cis-acting reverse transcriptase binding sequences from a “stringent” LINE to mediate their retrotransposition (Kajikawa and Okada, 2002; Ohshima, 2012; Ohshima et al., 1996), the only sequence shared between L1 and Alu RNAs is a 3′ poly(A) tract (Boeke, 1997). Previous studies revealed that the deletion of the Alu 3′ poly(A) sequence or its substitution with a poly(C) or a poly(G) sequence drastically reduced Alu retrotransposition in cultured cells (Dewannieux et al., 2003; Dewannieux and Heidmann, 2005). As in our study, Alu retrotransposition was restored by the addition of variably-sized poly(A) tracts, and was most efficient when the poly(A) tract was ~50 bases in size (Dewannieux and Heidmann, 2005).

We found that the 3′ poly(A) tracts present in L1 and Alu RNAs compete to bind ORF2p. When a 3′ poly(A) tract is present in L1 RNA, ORF2p can bind to either L1 or Alu RNA (Figure 7, left). However, when L1 RNA lacks a 3′ poly(A) tract but is stabilized by the MALAT triple helix, ORF2p cannot efficiently be recruited to L1 RNA in cis, leading to a 1.6-fold higher level of Alu retrotransposition (Figure 7, center). The introduction of a 16 or 40 base adenosine tract downstream of the L1/MALAT triple helix restores the ability of ORF2p to bind to the L1 RNA, restoring Alu retrotransposition to the level of a wild-type L1 control containing an SV40 polyadenylation signal (Figure 7, right). The increase in Alu retrotransposition when L1 RNA lacks a 3′ poly(A) tract suggests an intimate association between L1 and Alu RNA in the cell and that other sequences within L1 RNA are not sufficient to recruit ORF2p.

Figure 7. Model for the role of L1 3′ poly(A) tract in retrotransposition dynamics.

Figure 7

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ORF2p (blue oval) expressed from L1 RNA containing either a 3′ poly(A) tail (left panel) or a MALAT sequence supplemented with an A-rich tract at its 3′ end (right panel) can bind to either L1 or Alu RNA. When L1 RNA lacks a terminal 3′ poly(A) sequence (center panel), ORF2p cannot efficiently bind L1 RNA; thus, more ORF2p binds Alu RNA, leading to more efficient retrotransposition.

How does Alu parasitize ORF2p to mediate its retrotransposition? As suggested in the poly(A) connection model (Boeke, 1997), the association of Alu RNA with signal recognition particle 9 and 14 proteins (SRP9/14), its localization to the ribosome (Bennett et al., 2008; Labuda and Striker, 1989; Sinnett et al., 1991), and the presence of a 3′ poly(A) tract may allow Alu to effectively recruit ORF2p to mediate its retrotransposition. We propose that the co-translational binding of ORF2p to the L1 3′ poly(A) tract mediates cis-preference, and that the Alu RNA 3′ poly(A) tract allows it to compete for binding nascent ORF2p. Indeed, the accompanying paper by Ahl et al. reveals a structural basis for how both L1 and Alu RNA can be present at the same ribosome to ensure such an intimate association. This proximity-based mechanism also could explain why Alu RNA, when in an RNP complex with SRP9/14, more effectively competes for ORF2p than RD-L1 RNAs and other cellular mRNAs (Figures 6 and S6) (Esnault et al., 2000; Wei et al., 2001). In sum, our studies provide compelling evidence for the poly(A) connection model proposed over 15 years ago (Boeke, 1997). The preferential association of nascent ORF2p with the 3′ poly(A) tracts of RNAs could help explain the evolutionary success and abundance of L1 and Alu retrotransposons, as well as the formation of processed pseudogenes, in the human genome.

Experimental Procedures

Cell lines and culture conditions

HeLa-JVM cells (Moran et al., 1996) and HeLa-HA cells (Hulme et al., 2007) were cultured using standard cell culture procedures (see Supplemental Methods). Short Tandem Repeat DNA profiling (Cell Line Genetics, Inc., Madison, WI) authenticated the cell lines. Cells were checked using the PCR-based Venor™GeM Mycoplasma detection kit (Sigma, St. Louis, MO) to ensure they were mycoplasma-free.

Plasmid constructs

Detailed descriptions of the plasmids used in this study are in the Supplemental Methods.

Northern Blot detection of full-length and RNase H treated RNA samples

Northern analyses were conducted using established protocols (Wilusz et al., 2012). Briefly, 15 μg of total RNA from transfected HeLa-JVM cells were separated by 1% formaldehyde agarose gel electrophoresis and capillary transferred overnight to a Hybond N membrane (GE Healthcare), following the manufacturer’s instructions. The membranes were cross-linked with a Stratalinker (Stratagene), using the Optimum Crosslink setting, and incubated overnight with radiolabeled oligonucleotide probes in ULTRAhyb-Oligo Hybridization Buffer (Life Technologies). Blots were washed two times with 2X SSC, 0.5% SDS, and visualized with the GE Healthcare Typhoon Trio imaging system (GE Healthcare). Detailed protocols and probe sequences are in the Supplemental Methods section.

For RNase H treatments, 20 μg of total RNA were mixed with 20 pmol of the TAP-1 antisense oligonucleotide (see Supplemental Methods) and heated for 10 minutes at 65°C. After annealing by slow cooling, the RNA was treated with RNase H (New England BioLabs) for 30 minutes at 37°C and subjected to Northern blot analysis using an 8% polyacrylamide gel. RNAs were electroblotted using the Trans-Blot SD Semi-Dry Transfer cell (Bio-Rad, Hercules, CA) to Hybond N+ membrane (GE Healthcare), UV cross-linked, and incubated with the labeled TAP-2 oligonucleotide probe in ULTRAhyb-Oligo Hybridization Buffer (Life Technologies).

Ligation-Mediated 3′ Rapid Amplification of cDNA Ends

The microRNA Cloning Linker 3 (Integrated DNA Technologies, Coralville, IA) was ligated to the 3′ ends of total RNA as described previously (Wilusz et al., 2008). RNAs were reverse transcribed using SuperScript III (Life Technologies) and a primer complementary to the ligated linker (5′-GACTAGCTGGAATTCGCGGTTAAA). PCR amplification was performed using AmpliTaq (Life Technologies) for 30 cycles (94°C for 30 sec, 58°C for 30 sec, 72°C for 45 sec) with the following primers: forward (5′-CGAAAGTAGACGCTAATTAG) and reverse (5′-GACTAGCTGGAATTCGCGGTTAAA). PCR products subsequently were ligated into pGEM-T Easy (Promega, Madison, WI) and subjected to DNA sequencing at the MIT Koch Institute Biopolymers core facility. At least 10 clones were sequenced for each distinct 3′ end.

Western blot detection of protein expression

Western blots were performed with protein samples obtained from the same transfected cells for which total RNAs were analyzed by Northern blot (see above). Thirty micrograms of total protein were separated by electrophoresis. The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-FL PVDF, Millipore, Billerica, MA). The proteins of interest were detected with the following antibodies: anti-T7 Epitope Tag antibody (Millipore), TAP Tag polyclonal antibody (Thermo Scientific), FLAG-M2 antibody (Agilent Technologies, Santa Clara, CA), monoclonal anti-α-Tubulin (Sigma), eIF3 p110 (B-6) antibody (Santa Cruz Biotechnology, Dallas, TX), anti-Mouse IRDye 680LT (LI-COR Biosciences, Lincoln, NE) and anti-Rabbit IRDye 800CW (LI-COR Biosciences). Fluorescent secondary antibody signals were detected with an Odyssey CLx (LI-COR Biosciences) and quantified with Image Studio software (LI-COR Biosciences). Detailed protocols are in the Supplemental Methods.

Cultured cell retrotransposition assays

The L1 retrotransposition assay was conducted as described previously in HeLa-JVM cells (Moran et al., 1996) and is summarized in Figure 2A. The trans-complementation assay was conducted as described previously in HeLa-JVM cells (Wei et al., 2001) and is summarized in Figures 5, S5, and S6. The Alu retrotransposition assay was conducted as previously described in HeLa-HA cells (Dewannieux et al., 2003; Hulme et al., 2007) and is summarized in Figure 6A. Details are provided in the Supplemental Methods.

Characterization of L1 insertions by inverse PCR

The inverse PCR protocol was conducted as described previously (Morrish et al., 2002). Briefly, genomic DNA was extracted from individual G418 resistant colonies obtained after retrotransposition assay in HeLa-JVM cells. The DNA then was subjected to inverse PCR using primers specific to the mneoI reporter followed by cloning and Sanger sequencing. Details are provided in the Supplemental Methods.

RNA-IP assay

RNA immunoprecipitation (RNA-IP) experiments were performed as described previously with some modifications (Doucet et al., 2010; Moser et al., 2009). Briefly, HeLa-JVM cells were co-transfected with “driver” pTMO2F3 plasmid expressing a FLAG-tagged ORF2p and “reporter” plasmids expressing L1 with various 3′ ends. After hygromycin selection, whole cell lysis and FLAG immunoprecipitation, quantitative RT-PCR was used to measure the amount of L1 RNA associated with ORF2-FLAGp. Details are provided in the Supplemental Methods.

Supplementary Material

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NIHMS730039-supplement-1.pdf (1,018.8KB, pdf)
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3

Acknowledgements

We thank Nancy Leff for help editing the manuscript and Drs. Phillip Sharp and José Luis Garcia-Perez and members of the Moran Lab for helpful discussions. We thank Dr. Thierry Heidmann for the pAlu-neoTet plasmid. We thank the University of Michigan DNA Sequencing Core for technical support. AJD was supported in part by a fellowship from the Fondation pour la Recherche Médicale (FRM). Early stages of this research were conducted at MIT by JEW and were supported in part by NIH grants R01-GM34277 and R01-CA133404 to Phillip Sharp, JEW’s postdoctoral advisor. JEW is supported by NIH grant R00-GM104166 and is a Rita Allen Foundation Scholar. TM was supported by fellowships from the Japan Society for the Promotion of Science (JSPS), the Uehara Memorial Foundation, and the Kanae Foundation. JVM is an Investigator of the Howard Hughes Medical Institute and is supported by NIH grant GM060518.

Footnotes

Author Contributions

AJD, JEW, TM, and JVM designed experiments and analyzed the data. AJD, JEW, TM, and YL performed experiments. AJD and JVM wrote the manuscript. All authors edited the manuscript.

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NIHMS730039-supplement-1.pdf (1,018.8KB, pdf)
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