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).

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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

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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

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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

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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.

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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

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