MicroRNA miR-128 represses LINE-1 (L1) retrotransposition by downregulating the nuclear import factor TNPO1

Repetitive elements, including LINE-1 (L1), comprise approximately half of the human genome. These elements can potentially destabilize the genome by initiating their own replication and reintegration into new sites (retrotransposition). In somatic cells, transcription of L1 elements are repressed by distinct molecular mechanisms including DNA methylation and histone modifications to repress transcription. Under conditions of hypomethylation (e.g. in tumor cells) a window of opportunity for L1 de-repression arises and additional restriction mechanisms become crucial. We recently demonstrated that the microRNA miR-128 represses L1 activity by directly binding to L1 ORF2 RNA. In this study, we tested whether miR-128 can also control L1 activity by repressing cellular proteins important for L1 retrotransposition. We found that miR-128 targets the 3’UTR of the nuclear import factor transportin 1 (TNPO1) mRNA. Manipulation of miR-128 and TNPO1 levels demonstrated that induction or depletion of TNPO1 affects L1 retrotransposition and nuclear import of an L1-RNP complex (using L1-encoded ORF1p as a proxy for L1-RNP complexes). Moreover, TNPO1 overexpression partially reversed the repressive effect of miR-128 on L1 retrotransposition. Our study represents the first description of a protein factor involved in nuclear import of the L1 element and demonstrates that miR-128 controls L1 activity in somatic cells through two independent mechanisms: direct binding to L1 RNA, and regulating a cellular factor necessary for L1 nuclear import and retrotransposition.


INTRODUCTION
Repetitive elements make up approximately half of the mammalian genomes. A substantial portion of repetitive elements are derived from retrotransposons (long terminal repeats (LTR)-containing and non-LTR), which transpose to new chromosomal locations by reverse transcription of the RNA into DNA, followed by integration of the copied DNA into a new chromosomal location. Retrotransposition of these elements in germ cells lead to integration of new retrotransposons in the genomes of progeny, and since there is no mechanism for excision, they accumulate over evolutionary time scales (1)(2).
Long-interspaced nuclear elements-1 (LINE-1 or L1) are the only autonomous transposable elements that are currently active in humans and have directly or indirectly contributed to ~ 17% of the human genome (1). Intact, active L1 is ~6 kilobase pairs (kb) in length and contain a 5'UTR, three openreading frames -ORF1, ORF2 and ORF0 -and a short 3'UTR. The 5'UTR has promoter activity in both the sense and antisense direction (3)(4)(5)(6). ORF1 encodes a protein with RNA-binding and nucleic acid chaperone activity and ORF2 encodes a protein with endonuclease and reverse transcriptase activities (2,(7)(8)(9). ORF0, which is transcribed in the antisense direction, encodes a protein with unknown function, but which enhances L1 activity. L1 mobilizes replicatively from one place in the genome to another by a "copy and paste" mechanism via an RNA intermediate (10)(11). L1-RNP complexes have been described to enter the nucleus during cell division (12)(13). However, recently L1 retrotransposition has been demonstrated also to take place in non-diving cells such as neurons (14)(15). The mechanism by which L1-RNP complexes access the host DNA independently of cell division is unknown.
Integration of retrotransposons at new chromosomal locations can generate new genes and affect expression of already existing genes (16)(17)(18)(19). It has been suggested that retrotransposon activity could contribute to various diseases such as neurological disorders and cancer, as well as developmental defects (20)(21)(22)(23). As a result, multiple mechanisms have evolved to tightly control retrotransposon activity.
The recent discovery of microRNAs (miRNAs or miRs) has revolutionized our understanding of gene control. miRs exemplify the emerging view that non-coding RNAs (ncRNAs) may rival proteins in regulatory importance. The majority of the human transcriptome is believed to be under miR regulation, positioning this post-transcriptional control mechanism to regulate many gene pathways (32,34). miRs function as 21-24-nucleotide (nt) guides that regulate the expression of mRNAs containing complementary sequences. The mature miR is loaded onto specific Argonaute (Ago) proteins, which are then referred to as a miR-inducing silencing complex (miRISC) (34). In animals, partial pairing between a miR and an mRNA target site usually results in reduced protein expression through a variety of mechanisms that involve mRNA degradation and translational repression (35)(36). The best-characterized feature determining miR-target recognition are six nucleotide "seed" sites in the 3'UTR of mRNA targets, which perfectly complement the 5' end of the miR (positions 2-7) (35).
We recently discovered that miR-128 represses activity of L1 retrotransposons in somatic cells, analogous to the role of piRNAs in germ cells. We found a novel mechanism for this regulation in that miR-128 binds directly to L1 RNA in the ORF2 coding region sequence (CRS), resulting in L1 repression (37). In contrast, miRs typically are thought to repress multiple cellular mRNAs by binding to homologous target sequences; the proteins of these target mRNAs often work in concert, so miRs can fine-tune specific cellular networks (38)(39)(40)(41).
In this study, we explored if miR-128 also regulates L1 activity in somatic cells by repressing cellular proteins important for its retrotransposition. Here we report that miR-128 significantly represses retrotransposition, by targeting the nuclear import factor Transportin-1 (TNPO-1). TNPO1, also referred to as Karyopherin-ß2 or Importin-ß2, acts by binding to diverse nuclear localization sequences including (PY-NLSs) (37)(38)(39). TNPO1-mediated nuclear import requires RanGTP for cargo delivery into the nucleus (46) and known TNPO1 cargoes include viral, ribosomal and histone proteins (45)(46). We have determined that miR-128 targets the TNPO1 3'UTR and represses expression of TNPO1 mRNA and protein. In addition, we find that TNPO1 facilitates L1 mobilization and that miR-128-induced TNPO1 deficiency represses L1 retrotransposition, by inhibiting nuclear import of L1-RNP (using ORF1p as a proxy for L1-RNP complexes). This represents the first description of a cellular host factor likely to be involved in nuclear import of L1. Thus, in summary we have discovered a dual mechanism by which miR-128 controls L1 mobilization in somatic cells.

miR-128 repress L1 activity.
We recently determined that miR-128 directly targets L1 RNA and represses de novo retrotransposition and integration in somatic cells including cancer cells, cancer initiating cells (CICs) and induced pluripotent stem cells (iPSCs), which are all characterized by global demethylation and enhanced opportunity for L1 de-repression (37). After demonstrating an important role for miR-128 in the control of L1 retrotransposition in a panel of different cell lines and in iPSCs, we wished to further characterize the mechanism(s) of miR-128-induced restriction of L1 mobilization.
First we initiated analysis to dissect the direct (L1 RNA) versus potential indirect (cellular factors) effects of miR-128 on L1 retrotransposition. We performed colony formation assays using different variants of a neomycin reporter constructs encoding the full length L1 mRNA and a retrotransposition indicator cassette. Briefly, one construct consists of a neomycin gene in the antisense orientation relative to a full-length L1 element, which is disrupted by an intron in the sense orientation (see Supplemental Figure S1A). The neomycin (neo) protein can be translated into a functional enzyme only after L1 transcription and splicing of the mRNA, reverse transcription followed by integration of the spliced variant into the genome, thus allowing the quantification of cells with new retrotransposition events in culture. In addition, we generated a miR-128 resistant variant of the L1 plasmid, by introducing a silent mutation in the miR-128 binding site (in the ORF2 sequence) attenuating miR-128 binding, but allowing L1 to retrotranspose (as described in (37) and see Supplemental Figure S1B). A third variant of the L1 plasmid described by (51) encodes a L1 RNA harboring a D702A mutation in the RT domain of the ORF2 protein, rendering the encoded L1 RT deficient (RT dead). This plasmid variant was used as a negative control (see Supplemental Figure S1C). miR-128, anti-miR-128 or miR control shRNAs were cloned into the pMIR-ZIP plasmid, packaged into high-titer lentiviruses, and HeLa cells were transduced, puromycin selected and modulation of miR-128 expression levels were verified by miR specific qRT-PCR (Supplemental Figure   S2A). miR expressing HeLa cell lines were transfected with either the wildtype (WT), the miR-128 resistant L1 (Mutant) or the RT deficient L1 (RT-dead) neomycin reporter and selected for 14 days with neomycin replenished daily. We verified that the L1 plasmid was introduced into miR-expressing HeLa cells at equal levels by quantifying levels of a neomycin-expressing construct (see Supplemental Figure   S2B). We then compared the effect of miR-128 and anti-miR-128 on L1 mobilization to a panel of miR controls (miR-control (miR-control 1), anti-miR-control (miR-control 2) and miR-127, which does not affect L1 retrotransposition (miR-control 3)). In agreement with our previous findings, we observed a significant decrease in the number of neomycin-resistant colonies in cells overexpressing miR-128 and conversely a significant increase in neomycin resistant colonies in anti-miR-128 overexpressing cells (in which endogenously expressed miR-128 is neutralized), relative to HeLa cells with endogenous miR-128 levels, indicating lower versus higher rates of active retrotransposition of WT L1, in cells where miR-128 is either overexpressed or neutralized ( Figure 1A left panel) (32). Next analysis of miR-128's regulation of miR-128-resistant L1 retrotransposition (Mutant) was performed to evaluate potential indirect regulation of L1 by miR-128. We found that miR-128-induction significantly repressed mobilization of miR-128-resistant L1, and that miR-128 neutralization by anti-miR-128, significantly enhanced mobilization of miR-128 resistant L1, relative to miR control ( Figure 1A, middle panel).
Importantly, miR-modulated HeLa cells encoding RT-deficient L1 (RT-dead) resulted in no neomycinresistant colonies, demonstrating that colonies obtained upon wild-type and miR-128-resistant L1 plasmid transfections and neo selection are the consequence of a round of de novo L1 ( Figure 1A, right panel) (48,51). These results support the idea that miR-128 functions through direct binding of L1 RNA and by regulating cellular co-factors, which L1 is dependent on for successful mobilization.

Identification of miR-128 targets involved in regulation of L1 retrotransposition.
miRs often exert their regulatory roles of complex cellular functions by repressing multiple targets in the same signaling pathway. As such miRs can be thought of as master RNA regulators, similar to transcription factors, which are DNA regulators. We have employed different strategies to identify miR-128 targets, which may work in synergy with direct L1 RNA targeting, to limit L1 mobilization. We performed an unbiased screen to validate bioinformatically predicted miR-128 targets by using PicTar and TargetScan (47,52) ( Figure S3).
An area of L1 biology which the literature is conflicted about, deals with whether L1 ribonuclear (L1-RNP) complexes are dependent on cell division for nuclear import or not (12)(13)15). Interestingly, Macia et al. recently demonstrated that L1 can retrotranspose efficiently in mature nondividing neuronal cells, however the mechanism responsible for active nuclear import is unknown (14). With this in mind, we were excited to identify Transportin-1 (TNPO1) as a potential miR-128 target, as TNPO1 functions in nuclear import of a variety of RNA binding proteins critical for various steps in gene expression (56, 59,).
We determined that stably transduced HeLa cells expressing anti-miR-128 exhibit significantly higher levels of TNPO1 mRNA relative to the control sequence ( Figure 1C, left panel), in contrast to miR-128 overexpressing HeLa cells in which TNPO1 mRNA was significantly reduced ( Figure 1C, left panel). To rule out the possibility that the observed miR-128 effect was an artifact stemming from genomic integration of lentiviral encoded miRs, we transiently transfected miR-128, anti-miR-128 or control miR mimic oligonucleotides into HeLa cells, as an alternative approach and verified the effect of miR-128 and anti-miR-128, relative to miR controls ( Figure 1C, right panel and Figure 1B, bottom panel). Next we determined that miR-128 versus anti-miR-128 regulated the protein level of TNPO1 correlating with the observed changes in expression levels of TNPO1 mRNA ( Figure 1D, top panel, quantifications top right panel) and that these changes were accompanied by significant ORF1p reductions versus increases ( Figure 1D, bottom panel, quantification bottom right panels and (37)).
Finally, to exclude the possibility that miR-128 exclusively targets TNPO1 mRNA in HeLa cells we tested a teratoma cell line (Tera-1) and an induced pluripotent stem (iPS) cell line (IMR90). We found that TNPO1 mRNA expression levels were significantly changed in Tera-1 and IMR90 cells, in addition to HeLa cells ( Figure 1E and Figure 1C). These combined results show that miR-128 regulates the expression levels of TNPO1 in different cell types.

miR-128 interacts with a target sequence in the 3'UTR of TNPO1 mRNA.
Next we wished to examine whether miR-128 indirectly regulates TNPO1 expression or directly interacts with TNPO1 mRNA. Bioinformatics analyses identified 3 potential seed matches in TNPO1 mRNA ( Figure 2A). TNPO1 3'UTR or coding reading frame sequence including the three potential miR-128 binding sites, were cloned into a luciferase-based miR-binding site reporter construct. In addition, a perfect 23 nt miR-128 sequence (positive control) luciferase construct was generated. HeLa cells were transfected with one of the TNPO1 binding site-encoding plasmids in addition to mature miR-128 or miR control mimics. Luciferase activity was significantly reduced in cells transfected with miR-contrast miR-128 expression did not substantially reduce luciferase activity in cells encoding binding site #2 or #3. These results indicate that miR-128 preferentially targets TNPO1 mRNA by binding to site #1.
Next, mutations were introduced into the putative miR-128 binding site in the TNPO1 mRNA encoding site #1 in 3'UTR ( Figure 2A), to determine if this sequence is responsible for the interaction with miR-128 ( Figure 2C Furthermore, Argonaute (Ago) complexes containing miRs and target mRNAs were isolated by immuno-purification and assessed for relative complex occupancy by the TNPO1 mRNA to validate that miR-128 directly targets TNPO1 mRNA in cells ( Figure 2D, top) in miR-128-versus anti-miR-128overexpressing HeLa cells, as previously described (37). The relative level of TNPO1 mRNA was significantly lower in cells stably overexpressing miR-128 when compared to those expressing anti-miR-128 constructs, as expected ( Figure 2D middle, Input). Despite the increased levels of TNPO1 mRNA (because of lower miR-128 expression levels), which may underestimate the scale of the effect, the relative fraction of Ago-bound TNPO1 mRNA significantly increased when miR-128 was overexpressed ( Figure 2D, IP). When correcting for the lower expression level of TNPO1 mRNA, the increase in miR-128 bound TNPO1 mRNA was even more significant ( Figure 2D, top right panel). In contrast, miR-128 did not repress GAPDH mRNA expression levels or immuno-purified gapdh mRNA, as expected ( Figure 2E). We interpret this to mean that high levels of miR-128 lead to higher levels of TNPO1 mRNA being bound and regulated directly by miR-128. These data support the conclusion that miR-128 represses TNPO1 expression via a direct interaction with the target site located in the 3'UTR of the TNPO1 mRNA.

TNPO1 modulation regulates L1 activity and de novo retrotransposition.
TNPO1 functions by interacting with nuclear localization sequences on protein cargoes and facilitates nuclear import (55)(56)(57)(58). We hypothesized that L1-RNP may utilize TNPO1-dependent active transport, in addition to accessing the host DNA during cell division.
First we wished to evaluate if TNPO1 directly plays a role in L1 mobilization. For this purpose we generated TNPO1 constructs expressing TNPO1 shRNA (to obtain TNPO1 knock-down HeLa cells), or encoding the full-length TNPO1 mRNA transcript harboring the 5.6 kb 3'UTR including the miR-128 binding site (to generate HeLa cells overexpressing TNPO1) and control plasmids. We verified that TNPO1 shRNA or overexpression plasmids significantly reduced versus increased mRNA of TNPO1, relative to controls ( Figure 3A). We also evaluated whether TNPO1 knockdown or overexpression are toxic to cells or affect cell proliferation. Morphological and cell proliferation analysis of TNPO1 modulated HeLa cells showed that TNPO1 knockdown or overexpression, is not toxic to HeLa cells, which proliferate at a similar rate relative to plasmid control HeLa cells (Supplemental Figure S4A).
Since HeLa cells express low levels of endogenous L1 activity, we transiently transfected a construct encoding the full-length wild-type (WT) L1 and monitored the effects of TNPO1 depletion on artificially expressed L1 mRNA. We then performed colony formation assays to determine a possible requirement of TNPO1 on new L1 retrotransposition events. We verified that the L1 plasmid was introduced into TNPO1-expressing HeLa cells at similar levels by quantifying levels of neo-encoding expression plasmid (Supplemental Figure S2C and S2D). We then determined that cells deficient in TNPO1 exhibited a significantly lower number of neomycin-resistant colonies, versus cells overexpressing TNPO1, which showed a significant increase in neomycin-resistant colonies relative to controls ( Figure 3B). This is consistent with lower versus higher rates of de novo retrotransposition and genomic integration ( Figure 3B, shown as colony counts (%) and colony counts). TNPO1-modulated HeLa cells encoding RT-deficient L1 (RT-dead) resulted in no neomycin-resistant colonies, demonstrating that colonies obtained upon wild-type L1 plasmid transfections and neo selection are the consequence of a round of de novo L1 (data not shown). Next, protein lysates from TNPO1 deficient cells and TNPO1 overexpressing cells were prepared and TNPO1 and ORF1p protein levels were found to be significantly reduced in TNPO1 deficient cells, and increased in TNPO1-induced cells, as compared to controls (3C, left panel, quantification, right panel). The amount of L1 mRNA (ORF2) was regulated by TNPO1 consistent with the observed effect on L1 protein (ORF1p) abundance (data not shown). We noticed that the global amount of L1 protein changes when TNPO1 levels changes. This may be a consequence of accelerated degradations of L1-RNP components, caused by dysregulated nuclear transport of L1.
These combined data support the conclusion that TNPO1 neutralization or overexpression results in a corresponding decrease or increase in new retrotransposition events and establish a role of TNPO1 as a novel and specific modulator of L1 activity.
Reminiscent of the generally accepted role which TNPO3 plays in nuclear import of the pre-integration complex (PIC) of HIV-1 (60)(61)(62)(63)(64)(65)(66), we next decided to explore whether TNPO1 functions in a similar manner by assisting in the nuclear import of the L1-RNP complex. Faced with the difficulties of investigating RNA and proteins encoded by endogenous L1s, we developed a construct expressing a tagged protein of L1 containing HA (ORF1p-HA) and used localization of ORF1p as a proxy to reflects localization of L1-RNP, keeping in mind the limitations of this approach. We generated stable TNPO1 overexpressing and TNPO1 knock-down HeLa cell lines, which were transiently co-transfected with full-length wild-type (WT) L1 and ORF1p-HA or control vector and ORF1p localization was visualized and quantified by immunofluorescence confocal analysis. As the FL-TNPO1 plasmid co-expresses GFP, an alternate secondary antibody was used to visualize ORF1p in TNPO1-induced HeLa cell lines (AlexaFluor 568). We determined that TNPO1 reduction (shTNPO1) resulted a significant reduction of  Figure   S4C). As a positive control, a known TNPO1 interaction partner, TBP associated factor 15 (TAF15), was also analyzed (67). As expected, TNPO1 knock-down decreased the nuclear localization of TAF15, which was, instead, found in the cytoplasm and at the plasma membrane (see Supplemental Figure S4D).
Next we subjected TNPO1 modulated HeLa cell lines, which were transiently co-transfected with full-length wild-type (WT) L1 and ORF1p-HA, to subcellular fractionation analysis, keeping in mind the limitation of this approach. Nuclear and cytoplasmic fractionations were evaluated by determining the expression levels of α-tubulin (cytoplasmic) and Lamin A/C (nuclear) ( Figure 4B, Western blots) and TNPO1 knock-down and overexpression were verified by qRT-PCR (Supplemental Figure S4B). We next examined the effect of TNPO1 modulation on encoded L1 protein (ORF1p) as an indirect measure of L1-RNP localization. Analyzing the ORF1p levels in nuclear versus cytoplasmic fractions from TNPO1 knock-down HeLa cells lines (shTNPO1) (shown in Figure 3C, left panel), showed a substantial decrease in ORF1p levels in the nucleus, relative to controls ( Figure 4A, top panels). Overexpression of TNPO1 (FL-TNPO1) resulted in an increased nuclear L1 ORF1p expression as compared to controls ( Figure 4B, bottom panels). We did not observe a significant change in ORF1p levels in the cytoplasmic fractions. This is not too surprising as the vast majority of ORF1p is localized in the cytoplasm, thus changes in expression levels might not be measurable, as opposed to expression levels in the nucleus. We noted that ORF1p levels as determined by western blot analysis following subcellular fractionation surprisingly showed a ratio of less ORF1p in the cytoplasm versus nucleus.
This finding was in contrast to our confocal analysis of ORF1p localization. This difference is possibly due to a much more dilute cytoplasmic fraction, as compared to nucleic fraction. However, even with these limitations in mind, the combined results from the confocal and subcellular fractionation analysis indicate that TNPO1 is facilitating L1's access to host DNA. Furthermore, we performed immunoprecipitation analysis to evaluate if TNPO1 interacts with ORF1p. HeLa cells were transduced with a tagged version of ORF1 (ORF1p-HA), protein-containing lysates were prepared and ORF1p immunoprecipitations were performed and blotted for TNPO1 and HA. The ORF1p co-immunoprecipitations results suggest that ORF1p (L1-RNP complex) and TNPO1 interact ( Figure 4C). However, further studies are needed to determine whether this interaction is direct or indirect through an RNA bridge, as previously demonstrated for many ORF1p binding partners (68).
Finally, we evaluated the effect of miR-128-induced TNPO1 repression on L1 nuclear import.
miR-128, anti-miR-128 or control miR HeLa cells were transfected with ORF1p-HA expression plasmids and localization of L1 was analyzed by confocal analysis, as described above. miR-128mediated TNPO1 repression (verified and shown in Figure 1D

TNPO1 is a functional target of miR-128-induced L1 repression
We have previously demonstrated that miR-128 targets L1 RNA and represses L1 activity by a direct interaction, similar to how miRs represses replication of RNA virus (69,70). In addition, we have now determined that miR-128 is capable of repressing miR-128 resistant L1 (using a L1 mutant vector), by an indirect mechanism ( Figure 1A). With this in mind we wished to evaluate the significance of TNPO1 as a functional mediator of miR-128-induced L1 repression.
We utilized the L1 mutant vector, in which the miR-128 binding site had been mutated and miR-128 is no longer able to bind (miR-128 resistant L1). In addition, in order to perform TNPO1 rescue experiments, we needed to overexpress a miR-128-resistant version of the TNPO1 vector, as miR-128 may otherwise be able to bind to WT TNPO1 plasmid and could in theory function as a miR-128 sponge. We generated a miR-128 resistant full-length TNPO1 vector, in which the miR-128 binding Site#1 in the 3'UTR had been mutated according to our mutation analysis and miR-128 was no longer able to bind ( Figure 2C) (FL-TNPO1mut).
We found that overexpression of TNPO1 (WT and miR-128 resistant) in miR-128 overexpressing HeLa cells were able to partially but significantly, rescue miR-128-induced repression of L1 retrotransposition and genomic integration as determined by colony formation assays, relative to  Figure 5E). These combined results strongly support the idea that TNPO1 is a functional target for miR-128 and play an important role in L1 retrotransposition, possibly by affecting nuclear import of L1.

DISCUSSION
Our present data now provides additional mechanistic context for our earlier report that miRs have adopted part of piRNAs role in somatic cells in order to function as genomic gatekeepers by directly repressing L1 retrotransposon mobilization (37). In addition we show for the first time that a cellular factor (TNPO1) is involved in L1 mobilization by facilitating nuclear import of some L1-RNP complexes and thus gaining access to host DNA. Our results are in alignment with previous reports describing that TNPO1 function in nuclear transport of cargoes including viral proteins (45,46), and suggests that mobile DNA elements such as L1 elements are part of TNPO1 cargoes. Furthermore, recent data by Marcia et al. demonstrates that L1 efficiently can transposes in non-diving cells (15). We propose that TNPO1 may be involved in active nuclear import of L1-RNP complexes in all cells, but may be crucial for L1 mobilization in non-dividing cells such as neurons. It is possible that TNPO1 functions in a similar fashion during L1-RNP nuclear import as TNPO3 has been demonstrated to assist in nuclear import of the pre-integration complex of HIV-1 (60)(61)(62)(63)(64)(65)(66). In summary, we propose a model for miR-128-induced L1 repression in which miR-128 acts by directly targeting L1 RNA (37), as well as indirectly reducing L1 mobilization by repressing a cellular factor involved in nuclear import of some L1-RNP complex (TNPO1) (see Figure 6). We speculate that a dual mechanism helps secure L1 restriction and thus L1-induced retrotransposition and genomic integration in somatic cells.
Interestingly, both TNPO1 and L1 ORF1p have previously, independently been found to interact with the heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which contains nuclear localization signals (NLSs) required for shuttling between the cytoplasm and the nucleus (45, 55-58, 68, 71, 72). We have now obtained results, which supports the idea that TNPO1 and ORF1p are also binding partners, either directly or through an RNA bridge (L1 RNA), suggesting a possible scenario in which nuclear import of the L1-RNP complex is assisted through ORF1p, TNPO1 and hnRNPA1 interactions. Another possible scenario is that ORF1p and/or ORF2p could be direct cargoes of TPNO1. While a PY-NLS relies on structure, there is a weak consensus based on characterized motifs (R/H/KX2-5PY).
Interestingly, both proteins contain 'PY' motifs within the protein sequence, which fits the consensus (perfectly for ORF1p and partly for ORF2p). Future studies will determine whether these motifs are critical for L1 retrotransposition and binding to TPNO1.
The family of TNPO proteins (TNPO1, -2 and TNPO3), all function in nuclear import (44, 55-58, 73, 74). Interestingly, miR-128 harbors predicted binding sites in all three TNPO mRNAs and our preliminary results show that miR-128 down-regulates the expressing level of TNPO1, TNPO2 and TNPO3 mRNAs. This finding has important implications, as TNPO3 is a demonstrated cellular cofactor, which HIV-1 is dependent on for nuclear import of HIV-1 and viral replication (61,62,(64)(65)(66). We anticipate that miR-128-induced TNPO3 repression could have significant effects on the viral life cycle of HIV-1.
Finally, it is interesting to note that the brain expresses ~70% of all mature miRs, that miR-128 is highly enriched in the brain as compared to other human tissue (81,82) and that L1 retrotransposition surprisingly have been found derepressed in neuronal progenitor, leading to somatic brain mosaicism and enhanced plasticity (83,84). These finding suggest a potential important role for miR-128 in the regulation of genomic instability and plasticity in the human brain.
In conclusions, our results show that increased miR-128 expression reduces nuclear import of L1 (ORF1p) and significantly inhibit L1 mobilization; meanwhile, upregulation of TNPO1, a direct and functional target of miR-128, can markedly enhance levels of nuclear L1 (ORF1p) and de novo L1 retrotransposition. This newly identified miR-128/TNPO1 module provides a new avenue to an understanding of the L1 life cycle, especially, how some L1-RNP complexes may access host DNA, independently of cell division. Finally, the fact that TNPO1 can partially rescue the miR-128's inhibitory effect, suggests that miR-128 may repress additional cellular factors, which L1 is dependent on for optimal genomic mobilization.
Images were acquired with a Zeiss LSM 700 Confocal microscope in the Optical Biology Core at UC Irvine. Co-localization of ORF1p-HA with the nucleus was calculated as: Percent L1 ORF1p in nucleus = (L1 ORF1p signal co-localized with nucleus/ Total L1 ORF1p signal) X 100. CellProfiler software (47) was utilized to automatically segment nuclei, determine the area of positive staining for L1 ORF1p, the area of positive staining for nuclei (DAPI) and the co-localized area of L1 ORF1p and nuclear staining. Amount of ORF1p in control nuclei was set to 100% and the levels in the experimental nuclei are shown as a percentage of the controls.

qPCR screen for additional cellular targets of miR-128
As part of a effort to incorporate authentic research experiences into undergraduate labs at UC Irvine, a basic screen to identify bioinformatically determined cellular targets of miR-128 was performed.
Briefly, HeLa cells were transfected with 60 pmol of miR control or miR-128 mimics (GE Dharmacon) using Dharmafect1 (ThermoFisher). After 24 hours, cells were transfected a second time and incubated for another 24 hours after which cells were pelleted and snap frozen in LN2. Control or miR-128 transfected pellets were provided to undergraduate students who isolated RNA and made cDNA using (GeneJET RNA purification kit, ThermoFisher). qPCR was performed using student-designed primers to detect bioinformatically-defined targets of miR-128 (targetscan, pictar). Graduate students further tested differentially expressed targets in independent biological replicates.

Site directed mutagenesis
Reverse transcriptase incompetent PJM101/L1 plasmid was made using Q5 Site-directed mutagenesis Kit (E0554S, New England Biolabs) and mutation strategy described in Morrish et al (48) where D702A mutation in L1 ORF2 resulted in an incompetent reverse transcriptase. were used as loading controls, validation can be found on the manufacturer websites. Secondary HRPconjugated anti-rat (ab102172, Abcam), HRP-conjugated anti-rabbit (GE), and HRP-conjugated anti-mouse (GE) was used at 1:5000. ECL substrate (32106, ThermoFisher) was added and visualized on a BioRad ChemiDoc imager. Since many proteins were similar in size, blots were not cut but developed sequentially. After developing, each blot was washed three times in 1X TBST (TBS, #BP24711, FisherSci + 0.05% Tween 20, #BP337-500, FisherSci) prior to incubation with the next primary antibody.

Argonaute RNA Immunopurifications (Ago RIP)
Immunopurification of Argonaute from HeLa cell extracts was performed using the 4F9 antibody [4F9, Santa Cruz Biotechnology] as described previously (49,50). Briefly, 10mm plates of 80% confluent       Cartoon representation of miR-128-induced repression of L1 retrotransposition and genomic integration. miR-128 inhibits L1 activity by directly targeting L1 RNA, as well as indirectly by repressing the levels of the cellular cofactor TNPO1, which L1 is dependent on for nuclear import and replication.