Retrotransposons are genetic elements that replicate themselves within a host genome in a process known as retrotransposition by a ‘copy and paste’ mechanism that utilizes an RNA intermediate. The Long Interspersed Nuclear Element 1 (LINE-1 or L1) family of retrotransposons comprises 17% of the human genome by sequence and is the only autonomously active retrotransposon in the human genome, encoding proteins necessary for its own transposition. L1 also drives propagation of non-autonomous retrotransposons and processed pseudogenes, which make up an additional 21% of the human genome (Lander et al., 2001; Jurka, 1997; Dewannieux et al., 2003; Kazazian and Moran, 2017). While the majority of genomic human L1s have undergone truncations and mutations that have rendered them inactive, full-length, retrotransposition-competent L1s are 6 kilobases (kb) in length and have a 5’ untranslated region (UTR) containing a bidirectional promoter, two open reading frames (ORFs), ORF1 and ORF2, separated by a short inter-ORF linker, and a 3’ UTR with a weak polyadenylation signal (Figure 1A; Speek, 2001; Swergold, 1990; Dombroski et al., 1991; Doucet et al., 2015; Burns and Boeke, 2012).

Figure 1 with 1 supplement see all

Download asset Open asset

Figure 1—source data 1

Cytoplasmic and nuclear ORF1 puncta tracking data.

Output data matrix from GEMspa with tracking data for all tracked ORF1 particle trajectories in HeLa cells expressing the reporter L1 for 24 hr; associated with Figure 1D.

https://cdn.elifesciences.org/articles/82991/elife-82991-fig1-data1-v2.zip

Download elife-82991-fig1-data1-v2.zip

Figure 1—source data 2

ORF1-ORF1 colocalization data.

Output data matrices from the spot detection algorithm run on the ORF1-Halo and ORF1-mNG2 colocalization data, with intensity information for the detected intracellular JF549+ spots and randomly assigned intracellular spots; associated with Figure 1F.

https://cdn.elifesciences.org/articles/82991/elife-82991-fig1-data2-v2.zip

Download elife-82991-fig1-data2-v2.zip

Figure 1—source data 3

ORF1-L1 reporter RNA colocalization data.

Output data matrices from the spot detection algorithm run on the ORF1-Halo and reporter L1 RNA FISH colocalization data, with intensity information for the detected Halo+ spots and randomized cytoplasmic ‘Halo’ spots, as well as detected RNA+ spots and randomized cytoplasmic ‘RNA’ spots; associated with Figure 1G.

https://cdn.elifesciences.org/articles/82991/elife-82991-fig1-data3-v2.zip

Download elife-82991-fig1-data3-v2.zip

To undergo retrotransposition, the genomic L1 must first be transcribed by RNA polymerase II, polyadenylated, and exported into the cytoplasm where the two encoded proteins, ORF1 protein (ORF1p) and ORF2 protein (ORF2p), are translated. These proteins are both necessary for retrotransposition and exhibit cis preference, a phenomenon in which they are more likely to mobilize the mRNA from which they were expressed than a co-expressed L1 mRNA (Wei et al., 2001). While the mechanism for cis preference remains unclear, a prevalent model is that ORF1p and ORF2p bind to the mRNA from which they were translated cotranslationally or immediately after translation (Boeke, 1997). The two proteins are translated non-stoichiometrically, with a large excess of ORF1p, and co-assemble with L1 mRNA to form the L1 ribonucleoprotein (RNP), which is the functional unit of L1 (Hohjoh and Singer, 1996; Taylor et al., 2013). ORF2p has at least two crucial enzymatic activities required for retrotransposition, the reverse transcriptase (RT) required to produce dsDNA from the RNA template (Feng et al., 1996; Cost et al., 2002; Mathias et al., 1991) and the endonuclease (EN), which defines the target site in genomic DNA (Feng et al., 1996; Cost et al., 2002; Mathias et al., 1991). Once assembled, the L1 RNP must translocate to the nucleus where ORF2p uses its endonuclease activity to create a single-stranded nick in the DNA, and reverse transcribes the L1 mRNA into DNA. The ORF2p-encoded reverse transcriptase uses the free 3’-hydroxyl of the nicked DNA as a primer, directly synthesizing the L1 complementary DNA (cDNA) into the host genome in a mechanism called target-primed reverse transcription (TPRT) (Feng et al., 1996; Cost et al., 2002; Mathias et al., 1991; Luan et al., 1993).

ORF1p is a 40 kDa nucleic acid binding protein that assembles into a homotrimer, and has nucleic acid binding activity that is necessary for L1 retrotransposition (Martin and Bushman, 2001; Martin et al., 2005; Kulpa and Moran, 2005). ORF1p contains three structured domains, a coiled coil (CC) that mediates trimerization, an RNA recognition motif (RRM), and a C-terminal domain (CTD) that cooperates with the RRM to bind nucleic acids (Figure 1A–B; Januszyk et al., 2007; Khazina and Weichenrieder, 2009; Khazina et al., 2011). The N-terminal region (NTR) is the first 52 residues of ORF1p. This region is unstructured and contains phosphorylation sites and a basic charged patch that are necessary for retrotransposition (Adney et al., 2019; Cook et al., 2015; Khazina and Weichenrieder, 2018).

Many RNA-binding proteins participate in the formation of membraneless organelles, including nucleoli, stress granules, RNA processing bodies, and mRNA transport granules (Brangwynne et al., 2009; Brangwynne et al., 2011; Feric et al., 2016; Zhang et al., 2015; Wheeler et al., 2016; Lee et al., 2020). These biomolecular condensates demix from the surrounding cytoplasm or nucleoplasm and serve specific functions by selectively including proteins and nucleic acids based on their biochemical properties (Hyman et al., 2014). The process by which these membraneless compartments form is known as biomolecular condensation. One mechanism of condensation is phase separation, a density transition in which the constituent molecules exceed a critical concentration and separate into two coexisting phases, one with a higher density of the molecules of interest and one that is relatively depleted (Flory, 1942; Huggins, 1942). An alternative mechanism of condensation is gelation or percolation, which is a networking transition in which a dispersed solution of monomers and oligomers (a sol) switches into a system-spanning network (a gel) when a concentration threshold known as the percolation threshold is exceeded (Broadbent and Hammersley, 1957; Harmon et al., 2017). Recent conceptual work has proposed that phase separation and percolation can be coupled in certain cases, such that phase separation drives the formation of a dense phase that then undergoes percolation as the molecular concentration in the dense phase exceeds the percolation threshold (Mittag and Pappu, 2022). Phase separation coupled to percolation (PSCP) allows for the formation of dense phases with viscoelastic material properties at higher concentrations while also driving oligomerization and the emergence of pre-percolation clusters in subsaturated solutions (Harmon et al., 2017; Kar et al., 2022; Mittag and Pappu, 2022). Importantly, whether these condensed phases behave more like viscous liquids or elastic solids depends on the timescales on which they are observed/perturbed and the densities and lifetimes of the molecular interactions that serve as physical crosslinks (Alshareedah et al., 2021a; Alshareedah et al., 2021b; Mittag and Pappu, 2022).

Across the numerous characterized phase-separating RNA-binding proteins, shared molecular features such as multivalency, intrinsically disordered domains, and structured RNA binding have been shown to be important for condensation (Sanders et al., 2020; Yang et al., 2020; Molliex et al., 2015). Given that ORF1p exhibits all three of these molecular features of phase-separating proteins, we hypothesized that ORF1p undergoes condensation to carry out its roles in L1 RNP formation and chaperoning of L1 machinery. Recent work has confirmed that purified ORF1p is able to form a liquid-like condensed phase in vitro and that a truncated protein containing only the NTR and CC is sufficient for condensation (Newton et al., 2021). Here, we demonstrate that ORF1p expressed from a full-length active L1 element rapidly forms cytoplasmic condensates in cells that exhibit cis preference. We found that structured and unstructured charged residues and coiled coil flexibility were all necessary for cellular condensation. Biochemical reconstitution experiments revealed that the material properties of the reconstituted ORF1p condensates and their response to differing protein-RNA stoichiometries predicted their propensity to assemble in cells. ORF1p condensation in vivo correlated with L1 retrotransposition activity: disruption of nucleic-acid binding motifs or a critical flexibility motif led to loss of condensation and abrogated retrotransposition, while orthogonal restoration of trimer dynamics rescued both condensation and retrotransposition. Together these results indicate that condensation is critical for L1 retrotransposition. We propose that the biochemical properties of ORF1p are tuned to efficiently nucleate L1 RNP condensates cotranslationally, while also allowing for dynamic interactions between fully assembled L1 RNPs and host proteins and nucleic acids.

Given the established importance of RNP formation for L1 retrotransposition and the recent appreciation of the contribution of dynamic interactions to the function of RNA-binding proteins, we investigated how the properties of ORF1p assemblies contribute to L1 retrotransposition. Although ORF1p’s major role in retrotransposition is thought to involve the formation of an RNP with the L1 mRNA and ORF2p in the cytoplasm, the diversity of ORF1p mutations that abrogate retrotransposition suggests the possibility of additional roles, including mediating RNP interactions with host proteins and participation in the downstream steps of nuclear translocation, DNA search for integration sites, or reverse transcription and integration (Martin et al., 2005; Kulpa and Moran, 2005; Khazina et al., 2011; Taylor et al., 2013; Khazina and Weichenrieder, 2018; Taylor et al., 2018; Adney et al., 2019). Using a well-characterized L1 expression system and modifying it to allow for live-cell imaging of ORF1p is a significant first step towards relating the physical properties of higher order ORF1p assemblies to L1 function in cells. Expanding the use of this approach to investigate the role of ORF1p in cells and tissues with high endogenous L1 expression, including germline tissues and neoplastic tissues, will likely provide further insights into ORF1p interactions with host cell physiology (Branciforte and Martin, 1994; Rodić et al., 2014; Ardeljan et al., 2017).

High spatiotemporal resolution imaging of ORF1p in live cells allowed us to investigate long-standing hypotheses regarding L1 RNP assembly. In particular, the ability of L1-encoded machinery to specifically drive retrotransposition of L1 mRNA while minimizing transposition of off-target substrates was hypothesized to occur through cotranslational binding of ORF1p and ORF2p to their encoding L1 mRNA in cis (Boeke, 1997). This idea was substantiated with data demonstrating that two coexpressed L1 constructs exhibit only minimal trans complementation when one has a loss-of-function mutation in ORF1p or ORF2p (Wei et al., 2001; Kulpa and Moran, 2006). Our work adds additional evidence supporting the cis preference model. The minimal mixing observed for co-expressed fluorescent ORF1p puncta suggests that the physical properties of these assemblies minimize fusion events and protein exchange with the surrounding cytoplasm, providing a biophysical mechanism for L1 cis preference (Figure 1E–G, Figure 4D–E). This type of kinetically arrested condensate has been described in simulations and has been proposed as a mechanism for the coexistence of many small condensates at low, endogenous concentrations, rather than the thermodynamic expectation that the condensates would coalesce into a single, large equilibrium-like assembly (Ranganathan and Shakhnovich, 2020; Mittag and Pappu, 2022). A mechanism involving reversible co-condensation of ORF1p together with the L1 RNA from which it was translated would allow for cis substrate preference without sequence-specific RNA binding (Kolosha and Martin, 2003). Recent work has provided cellular evidence that disrupting the spatiotemporal regulation of expression of an RNA-binding protein and the RNAs it condenses with can abrogate the formation of compositionally distinct condensates, emphasizing the importance of dynamical control of protein-RNA co-condensation in cells (Lin et al., 2023).

We propose that ORF1p condensation in cells occurs cotranslationally, creating a viscous condensate with the L1 mRNA. Since this condensation must occur with the L1 mRNA from which it was translated for successful propagation, it requires a protein that is exquisitely sensitive to very low concentrations of RNA, which we found to be a biochemical characteristic shared by the ORF1p variants that robustly formed punctate assemblies in cells (Figure 4D). Cotranslational condensation of ORF1p with its cis RNA would also require initial low concentrations of protein to favor protein-protein assembly while limiting protein-RNA condensation to avoid incorporating non-template RNA molecules in the L1 RNP, which we found to be another shared feature of the ORF1p variants that underwent condensation in cells (Figure 4E). Cis-preferential L1 RNP assembly driven by dynamical control of ORF1p co-condensation with its source RNA is an attractive model due to the necessary spatiotemporal association between the L1 RNA and ORF1p during translation, but further work is required to understand how this type of cotranslational assembly would function on a mechanistic level. This proposed role of ORF1p condensation in L1 RNP assembly would further imply that L1s expressing ORF1p defective in condensation may exhibit increased trans mobilization of off-target RNAs. Data supporting this hypothesis has been reported for retrotransposition of the mammalian SINE Alu, which was observed to transpose twice as efficiently when driven by an L1 with ORF1p deleted rather than an intact L1 (Dewannieux et al., 2003).

The behavior of ORF1p condensates in biochemical reconstitution experiments was distinct from the puncta properties observed in cells. In particular, the extensive droplet-like phase and the droplet fusion events seen in reconstituted ORF1p contrasted with the uniform punctate morphology and lack of mixing seen in cells. We found that reducing protein concentrations and titrating RNA concentrations allowed us to better approximate the punctate condensates seen in cells (Figure 2B, Figure 4—figure supplement 5A). However, our reconstitution system did not fully recapitulate our observations in cells, in which WT ORF1p assembled but K3A/K4A and R261A did not (Figure 3B and F, Figure 4—figure supplement 5A). Our simple biochemical reconstitution experiments involve equilibrium mixing of protein and RNA, allowing for nucleation, growth, and fusion of condensates in the absence of additional perturbations. While we posit that the discrepancies we observe are due to a dynamical control mechanism in cells that is not reflected in our reconstituted system (Lin et al., 2023), there are many other buffer parameters and non-equilibrium processes in cells that could play a role in modifying ORF1p assembly kinetics, including macromolecular crowding, ATP hydrolysis, and post-translational modifications (Khan et al., 2018; Delarue et al., 2018; Brangwynne et al., 2011; Brangwynne, 2011; Nott et al., 2015; Aumiller and Keating, 2016; Carlson et al., 2020). Given this array of cellular parameters that may affect condensation of proteins in cells, we strove to ensure that we could characterize the behavior and properties of ORF1p assemblies in a cellular expression system that allowed us to simultaneously assess protein function. The ORF1 mutants in this study indicate that ORF1p condensation is critical for L1 retrotransposition. We are now poised to further leverage this system to more precisely elucidate the emergent properties conferred to L1 RNPs as a result of condensation and how they contribute to ORF1p’s essential roles in retrotransposition.

A recent study also described phase separation behavior of ORF1p in biochemical reconstitution experiments (Newton et al., 2021). That study characterized the formation of an ORF1p condensed phase in vitro that is mediated by electrostatic interactions. The authors additionally demonstrated that a truncated ORF1p containing just the disordered NTR and coiled coil (residues 1–152; NTR-CC) is sufficient for condensation. This description of NTR-CC sufficiency for condensation in reconstitution experiments is in agreement with our characterization of R261A, which has greatly reduced structured RNA-binding activity (Khazina et al., 2011) but is still able to condense in vitro (Figure 3A–B). We predict that both ORF1p R261A and the truncated NTR-CC protein undergo condensation in vitro through a mechanism similar to complex coacervation, which requires only unstructured charge-charge interactions (Aumiller and Keating, 2016; Boeynaems et al., 2019). Our data suggest that the ORF1p RRM participates in protein-protein interactions that drive condensation in the absence of RNA, while the unstructured NTR plays a smaller role in protein-only condensation (Figure 3A–C, Figure 3—figure supplement 2). However, the NTR charge patch allows for stark modulation of the physical properties of the condensate in response to low stoichiometries of RNA (Figure 4D), which is critical for condensation in cells (Figure 3E–F). The RRM has lesser contributions to the physical properties of ORF1p-RNA condensates but appears to downregulate the incorporation of RNA into the co-condensates (Figure 3—figure supplement 3), and we find that the combination of protein-protein and protein-RNA interactions of both the NTR basic patch and the RRM drives the formation of a condensate that is highly sensitive to very low RNA concentrations (Figure 4D–E). Furthermore, the flexibility of the coiled coil that separates these two motifs appears to be critical to the roles of the NTR and RRM in condensation, since the StammerDel mutant that has a more rigid coiled coil (Khazina and Weichenrieder, 2018) exhibits starkly reduced assembly in cells (Figure 4A–B). In this way, we propose that two distinctive basic motifs and a flexible coiled-coil scaffold allow ORF1p to undergo rapid, dynamically controlled condensation with cis RNA within the complex environment of the cell.

The L1 system characterized in this work employs a uniquely powerful combination of biochemical reconstitution, live-cell imaging, and functional phenotyping in cells. In vitro reconstitution allows us to study the biophysical properties of condensates in a minimal and controllable system. We can use live-cell imaging to observe assembly in cells and identify factors and mutations that modulate condensate formation and behavior in the cellular milieu. A well-characterized retrotransposition assay enables us to correlate changes in cellular condensation with functional effects on the entire retrotransposon life-cycle. Further characterization of this system will reveal additional biophysical determinants of L1 retrotransposition, which will deepen our understanding of functional biomolecular condensation within the complex cellular environment.

All data generated or analysed during this study are included in the manuscript and supporting files.

1. 1. Adney EM
   2. Ochmann MT
   3. Sil S
   4. Truong DM
   5. Mita P
   6. Wang X
   7. Kahler DJ
   8. Fenyö D
   9. Holt LJ
   10. Boeke JD

   (2019) Comprehensive scanning mutagenesis of human retrotransposon LINE-1 identifies motifs essential for function

   Genetics 213:1401–1414.

   https://doi.org/10.1534/genetics.119.302601

   • PubMed
   • Google Scholar
2. 1. Alberti S
   2. Gladfelter A
   3. Mittag T

   (2019) Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates

   Cell 176:419–434.

   https://doi.org/10.1016/j.cell.2018.12.035

   • PubMed
   • Google Scholar
3. 1. Alshareedah I
   2. Kaur T
   3. Banerjee PR

   (2021a) Methods for characterizing the material properties of biomolecular condensates

   Methods in Enzymology 646:143–183.

   https://doi.org/10.1016/bs.mie.2020.06.009

   • PubMed
   • Google Scholar
4. 1. Alshareedah I
   2. Moosa MM
   3. Pham M
   4. Potoyan DA
   5. Banerjee PR

   (2021b) Programmable viscoelasticity in protein-RNA condensates with disordered sticker-spacer polypeptides

   Nature Communications 12:6620.

   https://doi.org/10.1038/s41467-021-26733-7

   • PubMed
   • Google Scholar
5. 1. An W
   2. Dai L
   3. Niewiadomska AM
   4. Yetil A
   5. O’Donnell KA
   6. Han JS
   7. Boeke JD

   (2011) Characterization of a synthetic human LINE-1 retrotransposon orfeus-hs

   Mobile DNA 2:2.

   https://doi.org/10.1186/1759-8753-2-2

   • PubMed
   • Google Scholar
6. 1. Ardeljan D
   2. Taylor MS
   3. Ting DT
   4. Burns KH

   (2017) The human long interspersed element-1 retrotransposon: an emerging biomarker of neoplasia

   Clinical Chemistry 63:816–822.

   https://doi.org/10.1373/clinchem.2016.257444

   • PubMed
   • Google Scholar
7. 1. Aumiller WM
   2. Keating CD

   (2016) Phosphorylation-Mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles

   Nature Chemistry 8:129–137.

   https://doi.org/10.1038/nchem.2414

   • PubMed
   • Google Scholar
8. 1. Boeke JD

   (1997) Lines and alus -- the Polya connection

   Nature Genetics 16:6–7.

   https://doi.org/10.1038/ng0597-6

   • PubMed
   • Google Scholar
9. 1. Boeynaems S
   2. Holehouse AS
   3. Weinhardt V
   4. Kovacs D
   5. Van Lindt J
   6. Larabell C
   7. Van Den Bosch L
   8. Das R
   9. Tompa PS
   10. Pappu RV
   11. Gitler AD

   (2019) Spontaneous driving forces give rise to protein-RNA condensates with coexisting phases and complex material properties

   PNAS 116:7889–7898.

   https://doi.org/10.1073/pnas.1821038116

   • PubMed
   • Google Scholar
10. 1. Boija A
    2. Klein IA
    3. Sabari BR
    4. Dall’Agnese A
    5. Coffey EL
    6. Zamudio AV
    7. Li CH
    8. Shrinivas K
    9. Manteiga JC
    10. Hannett NM
    11. Abraham BJ
    12. Afeyan LK
    13. Guo YE
    14. Rimel JK
    15. Fant CB
    16. Schuijers J
    17. Lee TI
    18. Taatjes DJ
    19. Young RA

    (2018) Transcription factors activate genes through the phase-separation capacity of their activation domains

    Cell 175:1842–1855.

    https://doi.org/10.1016/j.cell.2018.10.042

    • PubMed
    • Google Scholar
11. 1. Branciforte D
    2. Martin SL

    (1994) Developmental and cell type specificity of LINE-1 expression in mouse testis: Implications for transposition

    Molecular and Cellular Biology 14:2584–2592.

    https://doi.org/10.1128/mcb.14.4.2584-2592.1994

    • PubMed
    • Google Scholar
12. 1. Brangwynne CP
    2. Eckmann CR
    3. Courson DS
    4. Rybarska A
    5. Hoege C
    6. Gharakhani J
    7. Jülicher F
    8. Hyman AA

    (2009) Germline P granules are liquid droplets that localize by controlled dissolution/condensation

    Science 324:1729–1732.

    https://doi.org/10.1126/science.1172046

    • Google Scholar
13. 1. Brangwynne CP

    (2011) Soft active aggregates: mechanics, dynamics and self-assembly of liquid-like intracellular protein bodies

    Soft Matter 7:3052.

    https://doi.org/10.1039/c0sm00981d

    • Google Scholar
14. 1. Brangwynne CP
    2. Mitchison TJ
    3. Hyman AA

    (2011) Active liquid-like behavior of nucleoli determines their size and shape in Xenopus laevis oocytes

    PNAS 108:4334–4339.

    https://doi.org/10.1073/pnas.1017150108

    • PubMed
    • Google Scholar
15. 1. Broadbent SR
    2. Hammersley JM

    (1957) Percolation processes: I. crystals and mazes

    Mathematical Proceedings of the Cambridge Philosophical Society 53:629–641.

    https://doi.org/10.1017/S0305004100032680

    • Google Scholar
16. 1. Brouha B
    2. Schustak J
    3. Badge RM
    4. Lutz-Prigge S
    5. Farley AH
    6. Moran JV
    7. Kazazian HH

    (2003) Hot L1s account for the bulk of retrotransposition in the human population

    PNAS 100:5280–5285.

    https://doi.org/10.1073/pnas.0831042100

    • PubMed
    • Google Scholar
17. 1. Brown JH
    2. Cohen C
    3. Parry DA

    (1996) Heptad breaks in alpha-helical coiled coils: Stutters and stammers

    Proteins 26:134–145.

    https://doi.org/10.1002/(SICI)1097-0134(199610)26:2<134::AID-PROT3>3.0.CO;2-G

    • PubMed
    • Google Scholar
18. 1. Burns KH
    2. Boeke JD

    (2012) Human transposon Tectonics

    Cell 149:740–752.

    https://doi.org/10.1016/j.cell.2012.04.019

    • PubMed
    • Google Scholar
19. 1. Cabrita LD
    2. Gilis D
    3. Robertson AL
    4. Dehouck Y
    5. Rooman M
    6. Bottomley SP

    (2007) Enhancing the stability and solubility of TEV protease using in silico design

    Protein Science 16:2360–2367.

    https://doi.org/10.1110/ps.072822507

    • PubMed
    • Google Scholar
20. 1. Carlson CR
    2. Asfaha JB
    3. Ghent CM
    4. Howard CJ
    5. Hartooni N
    6. Safari M
    7. Frankel AD
    8. Morgan DO

    (2020) Phosphoregulation of phase separation by the SARS-cov-2 N protein suggests a biophysical basis for its dual functions

    Molecular Cell 80:1092–1103.

    https://doi.org/10.1016/j.molcel.2020.11.025

    • PubMed
    • Google Scholar
21. 1. Carter V
    2. LaCava J
    3. Taylor MS
    4. Liang SY
    5. Mustelin C
    6. Ukadike KC
    7. Bengtsson A
    8. Lood C
    9. Mustelin T

    (2020) High prevalence and disease correlation of autoantibodies against p40 encoded by long interspersed nuclear elements in systemic lupus erythematosus

    Arthritis & Rheumatology 72:89–99.

    https://doi.org/10.1002/art.41054

    • Google Scholar
22. 1. Choi HMT
    2. Schwarzkopf M
    3. Fornace ME
    4. Acharya A
    5. Artavanis G
    6. Stegmaier J
    7. Cunha A
    8. Pierce NA

    (2018) Third-Generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust

    Development 145:dev165753.

    https://doi.org/10.1242/dev.165753

    • PubMed
    • Google Scholar
23. 1. Conway JF
    2. Parry DA

    (1991) Three-Stranded alpha-fibrous proteins: the heptad repeat and its implications for structure

    International Journal of Biological Macromolecules 13:14–16.

    https://doi.org/10.1016/0141-8130(91)90004-e

    • PubMed
    • Google Scholar
24. 1. Cook PR
    2. Jones CE
    3. Furano AV

    (2015) Phosphorylation of ORF1p is required for L1 retrotransposition

    PNAS 112:4298–4303.

    https://doi.org/10.1073/pnas.1416869112

    • PubMed
    • Google Scholar
25. 1. Cost GJ
    2. Feng Q
    3. Jacquier A
    4. Boeke JD

    (2002) Human L1 element target-primed reverse transcription in vitro

    The EMBO Journal 21:5899–5910.

    https://doi.org/10.1093/emboj/cdf592

    • PubMed
    • Google Scholar
26. 1. Delarue M
    2. Brittingham GP
    3. Pfeffer S
    4. Surovtsev IV
    5. Pinglay S
    6. Kennedy KJ
    7. Schaffer M
    8. Gutierrez JI
    9. Sang D
    10. Poterewicz G
    11. Chung JK
    12. Plitzko JM
    13. Groves JT
    14. Jacobs-Wagner C
    15. Engel BD
    16. Holt LJ

    (2018) Mtorc1 controls phase separation and the biophysical properties of the cytoplasm by tuning crowding

    Cell 174:338–349.

    https://doi.org/10.1016/j.cell.2018.05.042

    • PubMed
    • Google Scholar
27. 1. Dewannieux M
    2. Esnault C
    3. Heidmann T

    (2003) LINE-mediated retrotransposition of marked Alu sequences

    Nature Genetics 35:41–48.

    https://doi.org/10.1038/ng1223

    • PubMed
    • Google Scholar
28. 1. Dombroski BA
    2. Mathias SL
    3. Nanthakumar E
    4. Scott AF
    5. Kazazian HH

    (1991) Isolation of an active human transposable element

    Science 254:1805–1808.

    https://doi.org/10.1126/science.1662412

    • PubMed
    • Google Scholar
29. 1. Doucet AJ
    2. Wilusz JE
    3. Miyoshi T
    4. Liu Y
    5. Moran JV

    (2015) A 3’ poly (a) tract is required for LINE-1 retrotransposition

    Molecular Cell 60:728–741.

    https://doi.org/10.1016/j.molcel.2015.10.012

    • PubMed
    • Google Scholar
30. 1. Doucet AJ
    2. Basyuk E
    3. Gilbert N

    (2016) Cellular localization of engineered human LINE-1 RNA and proteins

    Methods in Molecular Biology 1400:281–297.

    https://doi.org/10.1007/978-1-4939-3372-3_18

    • PubMed
    • Google Scholar
31. 1. Eggers J
    2. Lister JR
    3. Stone HA

    (1999) Coalescence of liquid drops

    Journal of Fluid Mechanics 401:293–310.

    https://doi.org/10.1017/S002211209900662X

    • Google Scholar
32. 1. Elbaum-Garfinkle S
    2. Kim Y
    3. Szczepaniak K
    4. Chen CC-H
    5. Eckmann CR
    6. Myong S
    7. Brangwynne CP

    (2015) The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

    PNAS 112:7189–7194.

    https://doi.org/10.1073/pnas.1504822112

    • Google Scholar
33. 1. Feng Q
    2. Moran JV
    3. Kazazian HH
    4. Boeke JD

    (1996) Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition

    Cell 87:905–916.

    https://doi.org/10.1016/s0092-8674(00)81997-2

    • PubMed
    • Google Scholar
34. 1. Feric M
    2. Vaidya N
    3. Harmon TS
    4. Mitrea DM
    5. Zhu L
    6. Richardson TM
    7. Kriwacki RW
    8. Pappu RV
    9. Brangwynne CP

    (2016) Coexisting liquid phases underlie nucleolar subcompartments

    Cell 165:1686–1697.

    https://doi.org/10.1016/j.cell.2016.04.047

    • PubMed
    • Google Scholar
35. 1. Flory PJ

    (1942) Thermodynamics of high polymer solutions

    The Journal of Chemical Physics 10:51–61.

    https://doi.org/10.1063/1.1723621

    • Google Scholar
36. 1. Gibson DG
    2. Young L
    3. Chuang RY
    4. Venter JC
    5. Hutchison CA III
    6. Smith HO

    (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases

    Nature Methods 6:343–345.

    https://doi.org/10.1038/nmeth.1318

    • Google Scholar
37. 1. Goodier JL
    2. Ostertag EM
    3. Engleka KA
    4. Seleme MC
    5. Kazazian HH

    (2004) A potential role for the nucleolus in L1 retrotransposition

    Human Molecular Genetics 13:1041–1048.

    https://doi.org/10.1093/hmg/ddh118

    • PubMed
    • Google Scholar
38. 1. Goodier JL
    2. Zhang L
    3. Vetter MR
    4. Kazazian HH

    (2007) Line-1 ORF1 protein localizes in stress granules with other RNA-binding proteins, including components of RNA interference RNA-induced silencing complex

    Molecular and Cellular Biology 27:6469–6483.

    https://doi.org/10.1128/MCB.00332-07

    • PubMed
    • Google Scholar
39. 1. Goodier JL
    2. Cheung LE
    3. Kazazian HH

    (2012) Mov10 RNA helicase is a potent inhibitor of retrotransposition in cells

    PLOS Genetics 8:e1002941.

    https://doi.org/10.1371/journal.pgen.1002941

    • PubMed
    • Google Scholar
40. 1. Grimm JB
    2. English BP
    3. Chen J
    4. Slaughter JP
    5. Zhang Z
    6. Revyakin A
    7. Patel R
    8. Macklin JJ
    9. Normanno D
    10. Singer RH
    11. Lionnet T
    12. Lavis LD

    (2015) A general method to improve fluorophores for live-cell and single-molecule microscopy

    Nature Methods 12:244–250.

    https://doi.org/10.1038/nmeth.3256

    • PubMed
    • Google Scholar
41. 1. Hampf M
    2. Gossen M

    (2007) Promoter crosstalk effects on gene expression

    Journal of Molecular Biology 365:911–920.

    https://doi.org/10.1016/j.jmb.2006.10.009

    • PubMed
    • Google Scholar
42. 1. Harmon TS
    2. Holehouse AS
    3. Rosen MK
    4. Pappu RV

    (2017) Intrinsically disordered linkers determine the interplay between phase separation and gelation in multivalent proteins

    eLife 6:e30294.

    https://doi.org/10.7554/eLife.30294

    • PubMed
    • Google Scholar
43. 1. Hohjoh H
    2. Singer MF

    (1996)

    Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA

    The EMBO Journal 15:630–639.

    • PubMed
    • Google Scholar
44. 1. Holmes SE
    2. Dombroski BA
    3. Krebs CM
    4. Boehm CD
    5. Kazazian HH

    (1994) A new retrotransposable human L1 element from the LRE2 locus on chromosome 1q produces a chimaeric insertion

    Nature Genetics 7:143–148.

    https://doi.org/10.1038/ng0694-143

    • PubMed
    • Google Scholar
45. Software

    1. Holt L

    (2023a) Gemspa, version swh:1:rev:893e81cca010ace21efecb579da5b55780f10f70

    Software Heritage.

    https://archive.softwareheritage.org/swh:1:dir:161702be2ad1de60f169c59290cac28205a09e64;origin=https://github.com/liamholtlab/GEMspa;visit=swh:1:snp:fc3cdba68124dd5adb37c21bf6aa9fa08136a9e7;anchor=swh:1:rev:893e81cca010ace21efecb579da5b55780f10f70
46. Software

    1. Holt L

    (2023b) Spot_Detection, version swh:1:rev:98130a80bda25f65a67ac2ffa71339a5c0075407

    Software Heritage.

    https://archive.softwareheritage.org/swh:1:dir:3c739b381b72f69657ca312cfc5edf15719dccc7;origin=https://github.com/liamholtlab/spot_detection;visit=swh:1:snp:a5d3bb2d84ce694f225eb8b23d94f039cc6003fb;anchor=swh:1:rev:98130a80bda25f65a67ac2ffa71339a5c0075407
47. 1. Huggins ML

    (1942) Some properties of solutions of long-chain compounds

    The Journal of Physical Chemistry 46:151–158.

    https://doi.org/10.1021/j150415a018

    • Google Scholar
48. 1. Hyman AA
    2. Weber CA
    3. Jülicher F

    (2014) Liquid-Liquid phase separation in biology

    Annual Review of Cell and Developmental Biology 30:39–58.

    https://doi.org/10.1146/annurev-cellbio-100913-013325

    • PubMed
    • Google Scholar
49. 1. Januszyk K
    2. Li PW-L
    3. Villareal V
    4. Branciforte D
    5. Wu H
    6. Xie Y
    7. Feigon J
    8. Loo JA
    9. Martin SL
    10. Clubb RT

    (2007) Identification and solution structure of a highly conserved C-terminal domain within ORF1p required for retrotransposition of long interspersed nuclear element-1

    The Journal of Biological Chemistry 282:24893–24904.

    https://doi.org/10.1074/jbc.M702023200

    • PubMed
    • Google Scholar
50. 1. Jawerth L
    2. Fischer-Friedrich E
    3. Saha S
    4. Wang J
    5. Franzmann T
    6. Zhang X
    7. Sachweh J
    8. Ruer M
    9. Ijavi M
    10. Saha S
    11. Mahamid J
    12. Hyman AA
    13. Jülicher F

    (2020) Protein condensates as aging Maxwell fluids

    Science 370:1317–1323.

    https://doi.org/10.1126/science.aaw4951

    • PubMed
    • Google Scholar
51. 1. Jurka J

    (1997) Sequence patterns indicate an enzymatic involvement in integration of mammalian retroposons

    PNAS 94:1872–1877.

    https://doi.org/10.1073/pnas.94.5.1872

    • PubMed
    • Google Scholar
52. 1. Kaplan N
    2. Darden T
    3. Langley CH

    (1985) Evolution and extinction of transposable elements in Mendelian populations

    Genetics 109:459–480.

    https://doi.org/10.1093/genetics/109.2.459

    • PubMed
    • Google Scholar
53. 1. Kar M
    2. Dar F
    3. Welsh TJ
    4. Vogel LT
    5. Kühnemuth R
    6. Majumdar A
    7. Krainer G
    8. Franzmann TM
    9. Alberti S
    10. Seidel CAM
    11. Knowles TPJ
    12. Hyman AA
    13. Pappu RV

    (2022) Phase-Separating RNA-binding proteins form heterogeneous distributions of clusters in subsaturated solutions

    PNAS 119:e2202222119.

    https://doi.org/10.1073/pnas.2202222119

    • PubMed
    • Google Scholar
54. 1. Kazazian HH
    2. Wong C
    3. Youssoufian H
    4. Scott AF
    5. Phillips DG
    6. Antonarakis SE

    (1988) Haemophilia a resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man

    Nature 332:164–166.

    https://doi.org/10.1038/332164a0

    • PubMed
    • Google Scholar
55. 1. Kazazian HH
    2. Moran JV

    (2017) Mobile DNA in health and disease

    The New England Journal of Medicine 377:361–370.

    https://doi.org/10.1056/NEJMra1510092

    • PubMed
    • Google Scholar
56. 1. Keenen MM
    2. Larson AG
    3. Narlikar GJ

    (2018) Visualization and quantitation of phase-separated droplet formation by human HP1α

    Methods in Enzymology 611:51–66.

    https://doi.org/10.1016/bs.mie.2018.09.034

    • Google Scholar
57. 1. Keenen MM
    2. Brown D
    3. Brennan LD
    4. Renger R
    5. Khoo H
    6. Carlson CR
    7. Huang B
    8. Grill SW
    9. Narlikar GJ
    10. Redding S

    (2021) Hp1 proteins compact DNA into mechanically and positionally stable phase separated domains

    eLife 10:e64563.

    https://doi.org/10.7554/eLife.64563

    • PubMed
    • Google Scholar
58. 1. Khan T
    2. Kandola TS
    3. Wu J
    4. Venkatesan S
    5. Ketter E
    6. Lange JJ
    7. Rodríguez Gama A
    8. Box A
    9. Unruh JR
    10. Cook M
    11. Halfmann R

    (2018) Quantifying nucleation in vivo reveals the physical basis of prion-like phase behavior

    Molecular Cell 71:155–168.

    https://doi.org/10.1016/j.molcel.2018.06.016

    • PubMed
    • Google Scholar
59. 1. Khazina E
    2. Weichenrieder O

    (2009) Non-Ltr retrotransposons encode noncanonical RRM domains in their first open reading frame

    PNAS 106:731–736.

    https://doi.org/10.1073/pnas.0809964106

    • PubMed
    • Google Scholar
60. 1. Khazina E
    2. Truffault V
    3. Büttner R
    4. Schmidt S
    5. Coles M
    6. Weichenrieder O

    (2011) Trimeric structure and flexibility of the L1ORF1 protein in human L1 retrotransposition

    Nature Structural & Molecular Biology 18:1006–1014.

    https://doi.org/10.1038/nsmb.2097

    • Google Scholar
61. 1. Khazina E
    2. Weichenrieder O

    (2018) Human LINE-1 retrotransposition requires a metastable coiled coil and a positively charged N-terminus in L1ORF1p

    eLife 7:e34960.

    https://doi.org/10.7554/eLife.34960

    • PubMed
    • Google Scholar
62. 1. Kolosha VO
    2. Martin SL

    (2003) High-Affinity, non-sequence-specific RNA binding by the open reading frame 1 (ORF1) protein from long interspersed nuclear element 1 (LINE-1)

    The Journal of Biological Chemistry 278:8112–8117.

    https://doi.org/10.1074/jbc.M210487200

    • PubMed
    • Google Scholar
63. 1. Kulpa DA
    2. Moran JV

    (2005) Ribonucleoprotein particle formation is necessary but not sufficient for LINE-1 retrotransposition

    Human Molecular Genetics 14:3237–3248.

    https://doi.org/10.1093/hmg/ddi354

    • PubMed
    • Google Scholar
64. 1. Kulpa DA
    2. Moran JV

    (2006) Cis-preferential LINE-1 reverse transcriptase activity in ribonucleoprotein particles

    Nature Structural & Molecular Biology 13:655–660.

    https://doi.org/10.1038/nsmb1107

    • PubMed
    • Google Scholar
65. 1. Lander ES
    2. Linton LM
    3. Birren B
    4. Nusbaum C
    5. Zody MC
    6. Baldwin J
    7. Devon K
    8. Dewar K
    9. Doyle M
    10. FitzHugh W
    11. Funke R
    12. Gage D
    13. Harris K
    14. Heaford A
    15. Howland J
    16. Kann L
    17. Lehoczky J
    18. LeVine R
    19. McEwan P
    20. McKernan K
    21. Meldrim J
    22. Mesirov JP
    23. Miranda C
    24. Morris W
    25. Naylor J
    26. Raymond C
    27. Rosetti M
    28. Santos R
    29. Sheridan A
    30. Sougnez C
    31. Stange-Thomann Y
    32. Stojanovic N
    33. Subramanian A
    34. Wyman D
    35. Rogers J
    36. Sulston J
    37. Ainscough R
    38. Beck S
    39. Bentley D
    40. Burton J
    41. Clee C
    42. Carter N
    43. Coulson A
    44. Deadman R
    45. Deloukas P
    46. Dunham A
    47. Dunham I
    48. Durbin R
    49. French L
    50. Grafham D
    51. Gregory S
    52. Hubbard T
    53. Humphray S
    54. Hunt A
    55. Jones M
    56. Lloyd C
    57. McMurray A
    58. Matthews L
    59. Mercer S
    60. Milne S
    61. Mullikin JC
    62. Mungall A
    63. Plumb R
    64. Ross M
    65. Shownkeen R
    66. Sims S
    67. Waterston RH
    68. Wilson RK
    69. Hillier LW
    70. McPherson JD
    71. Marra MA
    72. Mardis ER
    73. Fulton LA
    74. Chinwalla AT
    75. Pepin KH
    76. Gish WR
    77. Chissoe SL
    78. Wendl MC
    79. Delehaunty KD
    80. Miner TL
    81. Delehaunty A
    82. Kramer JB
    83. Cook LL
    84. Fulton RS
    85. Johnson DL
    86. Minx PJ
    87. Clifton SW
    88. Hawkins T
    89. Branscomb E
    90. Predki P
    91. Richardson P
    92. Wenning S
    93. Slezak T
    94. Doggett N
    95. Cheng JF
    96. Olsen A
    97. Lucas S
    98. Elkin C
    99. Uberbacher E
    100. Frazier M
    101. Gibbs RA
    102. Muzny DM
    103. Scherer SE
    104. Bouck JB
    105. Sodergren EJ
    106. Worley KC
    107. Rives CM
    108. Gorrell JH
    109. Metzker ML
    110. Naylor SL
    111. Kucherlapati RS
    112. Nelson DL
    113. Weinstock GM
    114. Sakaki Y
    115. Fujiyama A
    116. Hattori M
    117. Yada T
    118. Toyoda A
    119. Itoh T
    120. Kawagoe C
    121. Watanabe H
    122. Totoki Y
    123. Taylor T
    124. Weissenbach J
    125. Heilig R
    126. Saurin W
    127. Artiguenave F
    128. Brottier P
    129. Bruls T
    130. Pelletier E
    131. Robert C
    132. Wincker P
    133. Smith DR
    134. Doucette-Stamm L
    135. Rubenfield M
    136. Weinstock K
    137. Lee HM
    138. Dubois J
    139. Rosenthal A
    140. Platzer M
    141. Nyakatura G
    142. Taudien S
    143. Rump A
    144. Yang H
    145. Yu J
    146. Wang J
    147. Huang G
    148. Gu J
    149. Hood L
    150. Rowen L
    151. Madan A
    152. Qin S
    153. Davis RW
    154. Federspiel NA
    155. Abola AP
    156. Proctor MJ
    157. Myers RM
    158. Schmutz J
    159. Dickson M
    160. Grimwood J
    161. Cox DR
    162. Olson MV
    163. Kaul R
    164. Raymond C
    165. Shimizu N
    166. Kawasaki K
    167. Minoshima S
    168. Evans GA
    169. Athanasiou M
    170. Schultz R
    171. Roe BA
    172. Chen F
    173. Pan H
    174. Ramser J
    175. Lehrach H
    176. Reinhardt R
    177. McCombie WR
    178. de la Bastide M
    179. Dedhia N
    180. Blöcker H
    181. Hornischer K
    182. Nordsiek G
    183. Agarwala R
    184. Aravind L
    185. Bailey JA
    186. Bateman A
    187. Batzoglou S
    188. Birney E
    189. Bork P
    190. Brown DG
    191. Burge CB
    192. Cerutti L
    193. Chen HC
    194. Church D
    195. Clamp M
    196. Copley RR
    197. Doerks T
    198. Eddy SR
    199. Eichler EE
    200. Furey TS
    201. Galagan J
    202. Gilbert JG
    203. Harmon C
    204. Hayashizaki Y
    205. Haussler D
    206. Hermjakob H
    207. Hokamp K
    208. Jang W
    209. Johnson LS
    210. Jones TA
    211. Kasif S
    212. Kaspryzk A
    213. Kennedy S
    214. Kent WJ
    215. Kitts P
    216. Koonin EV
    217. Korf I
    218. Kulp D
    219. Lancet D
    220. Lowe TM
    221. McLysaght A
    222. Mikkelsen T
    223. Moran JV
    224. Mulder N
    225. Pollara VJ
    226. Ponting CP
    227. Schuler G
    228. Schultz J
    229. Slater G
    230. Smit AF
    231. Stupka E
    232. Szustakowki J
    233. Thierry-Mieg D
    234. Thierry-Mieg J
    235. Wagner L
    236. Wallis J
    237. Wheeler R
    238. Williams A
    239. Wolf YI
    240. Wolfe KH
    241. Yang SP
    242. Yeh RF
    243. Collins F
    244. Guyer MS
    245. Peterson J
    246. Felsenfeld A
    247. Wetterstrand KA
    248. Patrinos A
    249. Morgan MJ
    250. de Jong P
    251. Catanese JJ
    252. Osoegawa K
    253. Shizuya H
    254. Choi S
    255. Chen YJ
    256. Szustakowki J
    257. International Human Genome Sequencing Consortium

    (2001) Initial sequencing and analysis of the human genome

    Nature 409:860–921.

    https://doi.org/10.1038/35057062

    • PubMed
    • Google Scholar
66. 1. Lee CYS
    2. Putnam A
    3. Lu T
    4. He S
    5. Ouyang JPT
    6. Seydoux G

    (2020) Recruitment of mRNAs to P granules by condensation with intrinsically-disordered proteins

    eLife 9:e52896.

    https://doi.org/10.7554/eLife.52896

    • PubMed
    • Google Scholar
67. 1. Li X
    2. Zhang J
    3. Jia R
    4. Cheng V
    5. Xu X
    6. Qiao W
    7. Guo F
    8. Liang C
    9. Cen S

    (2013) The MOV10 helicase inhibits LINE-1 mobility

    The Journal of Biological Chemistry 288:21148–21160.

    https://doi.org/10.1074/jbc.M113.465856

    • PubMed
    • Google Scholar
68. Preprint

    1. Lin AZ
    2. Ruff KM
    3. Jalihal A
    4. Dar F
    5. King MR
    6. Lalmansingh JM
    7. Posey AE
    8. Seim I
    9. Gladfelter AS
    10. Pappu RV

    (2023) Dynamical Control Enables the Formation of Demixed Biomolecular Condensates

    bioRxiv.

    https://doi.org/10.1101/2023.01.04.522702

    • Google Scholar
69. 1. Los GV
    2. Encell LP
    3. McDougall MG
    4. Hartzell DD
    5. Karassina N
    6. Zimprich C
    7. Wood MG
    8. Learish R
    9. Ohana RF
    10. Urh M
    11. Simpson D
    12. Mendez J
    13. Zimmerman K
    14. Otto P
    15. Vidugiris G
    16. Zhu J
    17. Darzins A
    18. Klaubert DH
    19. Bulleit RF
    20. Wood KV

    (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis

    ACS Chemical Biology 3:373–382.

    https://doi.org/10.1021/cb800025k

    • PubMed
    • Google Scholar
70. 1. Luan DD
    2. Korman MH
    3. Jakubczak JL
    4. Eickbush TH

    (1993) Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition

    Cell 72:595–605.

    https://doi.org/10.1016/0092-8674(93)90078-5

    • PubMed
    • Google Scholar
71. 1. Martin SL
    2. Bushman FD

    (2001) Nucleic acid chaperone activity of the ORF1 protein from the mouse LINE-1 retrotransposon

    Molecular and Cellular Biology 21:467–475.

    https://doi.org/10.1128/MCB.21.2.467-475.2001

    • PubMed
    • Google Scholar
72. 1. Martin SL
    2. Cruceanu M
    3. Branciforte D
    4. Wai-Lun Li P
    5. Kwok SC
    6. Hodges RS
    7. Williams MC

    (2005) Line-1 retrotransposition requires the nucleic acid chaperone activity of the ORF1 protein

    Journal of Molecular Biology 348:549–561.

    https://doi.org/10.1016/j.jmb.2005.03.003

    • PubMed
    • Google Scholar
73. 1. Mathias SL
    2. Scott AF
    3. Kazazian HH
    4. Boeke JD
    5. Gabriel A

    (1991) Reverse transcriptase encoded by a human transposable element

    Science 254:1808–1810.

    https://doi.org/10.1126/science.1722352

    • PubMed
    • Google Scholar
74. 1. Mita P
    2. Wudzinska A
    3. Sun X
    4. Andrade J
    5. Nayak S
    6. Kahler DJ
    7. Badri S
    8. LaCava J
    9. Ueberheide B
    10. Yun CY
    11. Fenyö D
    12. Boeke JD

    (2018) Line-1 protein localization and functional dynamics during the cell cycle

    eLife 7:e30058.

    https://doi.org/10.7554/eLife.30058

    • PubMed
    • Google Scholar
75. 1. Mita P
    2. Sun X
    3. Fenyö D
    4. Kahler DJ
    5. Li D
    6. Agmon N
    7. Wudzinska A
    8. Keegan S
    9. Bader JS
    10. Yun C
    11. Boeke JD

    (2020) Brca1 and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells

    Nature Structural & Molecular Biology 27:179–191.

    https://doi.org/10.1038/s41594-020-0374-z

    • PubMed
    • Google Scholar
76. 1. Mitchell LA
    2. Cai Y
    3. Taylor M
    4. Noronha AM
    5. Chuang J
    6. Dai L
    7. Boeke JD

    (2013) Multichange isothermal mutagenesis: a new strategy for multiple site-directed mutations in plasmid DNA

    ACS Synthetic Biology 2:473–477.

    https://doi.org/10.1021/sb300131w

    • PubMed
    • Google Scholar
77. 1. Mittag T
    2. Pappu RV

    (2022) A conceptual framework for understanding phase separation and addressing open questions and challenges

    Molecular Cell 82:2201–2214.

    https://doi.org/10.1016/j.molcel.2022.05.018

    • PubMed
    • Google Scholar
78. 1. Molliex A
    2. Temirov J
    3. Lee J
    4. Coughlin M
    5. Kanagaraj AP
    6. Kim HJ
    7. Mittag T
    8. Taylor JP

    (2015) Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization

    Cell 163:123–133.

    https://doi.org/10.1016/j.cell.2015.09.015

    • PubMed
    • Google Scholar
79. 1. Moran JV
    2. Holmes SE
    3. Naas TP
    4. DeBerardinis RJ
    5. Boeke JD
    6. Kazazian HH

    (1996) High frequency retrotransposition in cultured mammalian cells

    Cell 87:917–927.

    https://doi.org/10.1016/S0092-8674(00)81998-4

    • Google Scholar
80. 1. Nanda JS
    2. Lorsch JR

    (2014) Labeling a protein with fluorophores using NHS ester derivitization

    Methods in Enzymology 536:87–94.

    https://doi.org/10.1016/B978-0-12-420070-8.00008-8

    • PubMed
    • Google Scholar
81. 1. Newton JC
    2. Naik MT
    3. Li GY
    4. Murphy EL
    5. Fawzi NL
    6. Sedivy JM
    7. Jogl G

    (2021) Phase separation of the LINE-1 ORF1 protein is mediated by the N-terminus and coiled-coil domain

    Biophysical Journal 120:2181–2191.

    https://doi.org/10.1016/j.bpj.2021.03.028

    • PubMed
    • Google Scholar
82. 1. Nott TJ
    2. Petsalaki E
    3. Farber P
    4. Jervis D
    5. Fussner E
    6. Plochowietz A
    7. Craggs TD
    8. Bazett-Jones DP
    9. Pawson T
    10. Forman-Kay JD
    11. Baldwin AJ

    (2015) Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles

    Molecular Cell 57:936–947.

    https://doi.org/10.1016/j.molcel.2015.01.013

    • PubMed
    • Google Scholar
83. 1. Ostertag EM
    2. Prak ET
    3. DeBerardinis RJ
    4. Moran JV
    5. Kazazian HH

    (2000) Determination of L1 retrotransposition kinetics in cultured cells

    Nucleic Acids Research 28:1418–1423.

    https://doi.org/10.1093/nar/28.6.1418

    • PubMed
    • Google Scholar
84. 1. Pereira GC
    2. Sanchez L
    3. Schaughency PM
    4. Rubio-Roldán A
    5. Choi JA
    6. Planet E
    7. Batra R
    8. Turelli P
    9. Trono D
    10. Ostrow LW
    11. Ravits J
    12. Kazazian HH
    13. Wheelan SJ
    14. Heras SR
    15. Mayer J
    16. García-Pérez JL
    17. Goodier JL

    (2018) Properties of LINE-1 proteins and repeat element expression in the context of amyotrophic lateral sclerosis

    Mobile DNA 9:35.

    https://doi.org/10.1186/s13100-018-0138-z

    • PubMed
    • Google Scholar
85. 1. Ranganathan S
    2. Shakhnovich EI

    (2020) Dynamic metastable long-living droplets formed by sticker-spacer proteins

    eLife 9:e56159.

    https://doi.org/10.7554/eLife.56159

    • PubMed
    • Google Scholar
86. 1. Rodić N
    2. Sharma R
    3. Sharma R
    4. Zampella J
    5. Dai L
    6. Taylor MS
    7. Hruban RH
    8. Iacobuzio-Donahue CA
    9. Maitra A
    10. Torbenson MS
    11. Goggins M
    12. Shih I-M
    13. Duffield AS
    14. Montgomery EA
    15. Gabrielson E
    16. Netto GJ
    17. Lotan TL
    18. De Marzo AM
    19. Westra W
    20. Binder ZA
    21. Orr BA
    22. Gallia GL
    23. Eberhart CG
    24. Boeke JD
    25. Harris CR
    26. Burns KH

    (2014) Long interspersed element-1 protein expression is a hallmark of many human cancers

    The American Journal of Pathology 184:1280–1286.

    https://doi.org/10.1016/j.ajpath.2014.01.007

    • PubMed
    • Google Scholar
87. 1. Sanders DW
    2. Kedersha N
    3. Lee DSW
    4. Strom AR
    5. Drake V
    6. Riback JA
    7. Bracha D
    8. Eeftens JM
    9. Iwanicki A
    10. Wang A
    11. Wei MT
    12. Whitney G
    13. Lyons SM
    14. Anderson P
    15. Jacobs WM
    16. Ivanov P
    17. Brangwynne CP

    (2020) Competing protein-RNA interaction networks control multiphase intracellular organization

    Cell 181:306–324.

    https://doi.org/10.1016/j.cell.2020.03.050

    • PubMed
    • Google Scholar
88. 1. Seim I
    2. Posey AE
    3. Snead WT
    4. Stormo BM
    5. Klotsa D
    6. Pappu RV
    7. Gladfelter AS

    (2022) Dilute phase oligomerization can oppose phase separation and modulate material properties of a ribonucleoprotein condensate

    PNAS 119:e2120799119.

    https://doi.org/10.1073/pnas.2120799119

    • PubMed
    • Google Scholar
89. 1. Sharma R
    2. Rodić N
    3. Burns KH
    4. Taylor MS

    (2016) Immunodetection of human LINE-1 expression in cultured cells and human tissues

    Methods in Molecular Biology 1400:261–280.

    https://doi.org/10.1007/978-1-4939-3372-3_17

    • PubMed
    • Google Scholar
90. Preprint

    1. Shu T
    2. Szórádi T
    3. Kidiyoor GR
    4. Xie Y
    5. Herzog NL
    6. Bazley A
    7. Bonucci M
    8. Keegan S
    9. Saxena S
    10. Ettefa F
    11. Brittingham G
    12. Lemiere J
    13. Fenyö D
    14. Chang F
    15. Delarue M
    16. Holt LJ

    (2021) NucGEMs Probe the Biophysical Properties of the Nucleoplasm

    bioRxiv.

    https://doi.org/10.1101/2021.11.18.469159

    • Google Scholar
91. 1. Speek M

    (2001) Antisense promoter of human L1 retrotransposon drives transcription of adjacent cellular genes

    Molecular and Cellular Biology 21:1973–1985.

    https://doi.org/10.1128/MCB.21.6.1973-1985.2001

    • PubMed
    • Google Scholar
92. 1. Swergold GD

    (1990) Identification, characterization, and cell specificity of a human LINE-1 promoter

    Molecular and Cellular Biology 10:6718–6729.

    https://doi.org/10.1128/mcb.10.12.6718-6729.1990

    • PubMed
    • Google Scholar
93. 1. Tauber D
    2. Tauber G
    3. Khong A
    4. Van Treeck B
    5. Pelletier J
    6. Parker R

    (2020) Modulation of RNA condensation by the DEAD-box protein eIF4A

    Cell 180:411–426.

    https://doi.org/10.1016/j.cell.2019.12.031

    • PubMed
    • Google Scholar
94. 1. Taylor MS
    2. LaCava J
    3. Mita P
    4. Molloy KR
    5. Huang CRL
    6. Li D
    7. Adney EM
    8. Jiang H
    9. Burns KH
    10. Chait BT
    11. Rout MP
    12. Boeke JD
    13. Dai L

    (2013) Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition

    Cell 155:1034–1048.

    https://doi.org/10.1016/j.cell.2013.10.021

    • PubMed
    • Google Scholar
95. 1. Taylor MS
    2. Altukhov I
    3. Molloy KR
    4. Mita P
    5. Jiang H
    6. Adney EM
    7. Wudzinska A
    8. Badri S
    9. Ischenko D
    10. Eng G
    11. Burns KH
    12. Fenyö D
    13. Chait BT
    14. Alexeev D
    15. Rout MP
    16. Boeke JD
    17. LaCava J

    (2018) Dissection of affinity captured LINE-1 macromolecular complexes

    eLife 7:e30094.

    https://doi.org/10.7554/eLife.30094

    • PubMed
    • Google Scholar
96. 1. Tóth-Petróczy A
    2. Simon I
    3. Fuxreiter M
    4. Levy Y

    (2009) Disordered tails of homeodomains facilitate DNA recognition by providing a trade-off between folding and specific binding

    Journal of the American Chemical Society 131:15084–15085.

    https://doi.org/10.1021/ja9052784

    • PubMed
    • Google Scholar
97. 1. Wei W
    2. Gilbert N
    3. Ooi SL
    4. Lawler JF
    5. Ostertag EM
    6. Kazazian HH
    7. Boeke JD
    8. Moran JV

    (2001) Human L1 retrotransposition: cis preference versus trans complementation

    Molecular and Cellular Biology 21:1429–1439.

    https://doi.org/10.1128/MCB.21.4.1429-1439.2001

    • PubMed
    • Google Scholar
98. 1. Weidenfeld I
    2. Gossen M
    3. Löw R
    4. Kentner D
    5. Berger S
    6. Görlich D
    7. Bartsch D
    8. Bujard H
    9. Schönig K

    (2009) Inducible expression of coding and inhibitory RNAs from retargetable genomic loci

    Nucleic Acids Research 37:e50.

    https://doi.org/10.1093/nar/gkp108

    • PubMed
    • Google Scholar
99. 1. Wheeler JR
    2. Matheny T
    3. Jain S
    4. Abrisch R
    5. Parker R

    (2016) Distinct stages in stress granule assembly and disassembly

    eLife 5:e18413.

    https://doi.org/10.7554/eLife.18413

    • PubMed
    • Google Scholar
100. 1. Woods-Samuels P
     2. Wong C
     3. Mathias SL
     4. Scott AF
     5. Kazazian HH
     6. Antonarakis SE

     (1989) Characterization of a nondeleterious L1 insertion in an intron of the human factor VIII gene and further evidence of open reading frames in functional L1 elements

     Genomics 4:290–296.

     https://doi.org/10.1016/0888-7543(89)90332-7

     • PubMed
     • Google Scholar
101. 1. Yang P
     2. Mathieu C
     3. Kolaitis R-M
     4. Zhang P
     5. Messing J
     6. Yurtsever U
     7. Yang Z
     8. Wu J
     9. Li Y
     10. Pan Q
     11. Yu J
     12. Martin EW
     13. Mittag T
     14. Kim HJ
     15. Taylor JP

     (2020) G3Bp1 is a tunable switch that triggers phase separation to assemble stress granules

     Cell 181:325–345.

     https://doi.org/10.1016/j.cell.2020.03.046

     • PubMed
     • Google Scholar
102. 1. Zhang H
     2. Elbaum-Garfinkle S
     3. Langdon EM
     4. Taylor N
     5. Occhipinti P
     6. Bridges AA
     7. Brangwynne CP
     8. Gladfelter AS

     (2015) Rna controls polyQ protein phase transitions

     Molecular Cell 60:220–230.

     https://doi.org/10.1016/j.molcel.2015.09.017

     • PubMed
     • Google Scholar

Author details

1. Srinjoy Sil

   Institute for Systems Genetics, New York University Langone Medical Center, New York, United States

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing - original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6722-4311

2. Sarah Keegan

   Institute for Systems Genetics, New York University Langone Medical Center, New York, United States

   Contribution

   Data curation, Software, Formal analysis, Visualization, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

3. Farida Ettefa

   Institute for Systems Genetics, New York University Langone Medical Center, New York, United States

   Contribution

   Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

4. Lance T Denes

   Institute for Systems Genetics, New York University Langone Medical Center, New York, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

5. Jef D Boeke

   Institute for Systems Genetics, New York University Langone Medical Center, New York, United States

   Contribution

   Resources, Writing – review and editing

   Competing interests

   is a Founder and Director of CDI Labs, Inc, a Founder of and consultant to Neochromosome, Inc, a Founder, SAB member of and consultant to ReOpen Diagnostics, LLC and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc, Modern Meadow, Inc, Rome Therapeutics, Inc, Sample6, Inc, Tessera Therapeutics, Inc and the Wyss Institute

   "This ORCID iD identifies the author of this article:" 0000-0001-5322-4946

6. Liam J Holt

   Institute for Systems Genetics, New York University Langone Medical Center, New York, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing – review and editing

   For correspondence

   Liam.Holt@nyulangone.org

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4002-0861

• 3,427

  views

• 435

  downloads

• 15

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.