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

Newton, JC; Gy, Li; Naik, MT; Fawzi, NL; Jm, Sedivy; Jogl, G.

doi:10.1101/2020.10.29.360719

Cited by 9 publications

(11 citation statements)

References 61 publications

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“…A recent study also described phase separation behavior of ORF1p in biochemical reconstitution experiments (Newton et al 2021). This study characterized the formation of an ORF1p condensed phase that is mediated by electrostatic interactions.…”

Section: Discussionmentioning

confidence: 99%

“…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 phase separation (Newton et al 2021). Here we demonstrate that ORF1p expressed from a full-length active L1 element rapidly forms cytoplasmic condensates in cells.…”

Section: Introductionmentioning

confidence: 97%

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Condensation of LINE-1 is required for retrotransposition

Sil

Boeke

Holt

2022

Preprint

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LINE-1 (L1) is the only autonomously active retrotransposon in the human genome, and accounts for 17% of the human genome. The L1 mRNA encodes two proteins, ORF1p and ORF2p. ORF1p is a homotrimeric RNA-binding protein that plays a critical role in assembling functional L1 ribonucleoprotein (RNP) complexes. Here we show that condensation of ORF1p is required for L1 retrotransposition. Using a combination of biochemical reconstitution and live-cell imaging, we demonstrate that RNA binding, electrostatic interactions, and trimer conformational dynamics together tune the properties of ORF1p assemblies to allow for efficient L1 condensate formation in cells. Furthermore, we directly relate the dynamics of ORF1p assembly to the ability to complete the entire retrotransposon life-cycle. Mutations that prevented ORF1 condensation led to loss of retrotransposition activity, while orthogonal restoration of coiled-coil conformational flexibility rescued both condensation and retrotransposition. Based on these observations, we propose that ORF1p oligomerization on L1 RNA drives the formation of a dynamic L1 condensate that is essential for retrotransposition.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 97%

Condensation of LINE-1 is required for retrotransposition

Sil

Boeke

Holt

2022

Preprint

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“…RNA and RNA binding proteins (RBPs) are key components of these cytoplasmic condensates 30 . Recently, by microscopy and NMR spectroscopy, human L1 ORF1 protein was shown to form liquid droplets in vitro in a salt dependent manner 31 . To test whether L1 ORF1 foci in mESCs undergo similar LLPS, we treated Dicer_KO mESCs with 3% 1,6 Hexanediol for 15 minutes, a concentration at which proteins undergoing LLPS have been previously observed to change solubility from being in foci to becoming diffused in mESCs 32 .…”

Section: Resultsmentioning

confidence: 99%

Sequestration of LINE-1 in novel cytosolic bodies by MOV10 restricts retrotransposition

Arora

Bodak

Penouty

et al. 2021

Preprint

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LINE-1 (L1) are autonomous retroelements that have retained their ability to mobilize. Mechanisms regulating L1 mobility include DNA methylation in somatic cells and the Piwi-interacting RNA pathway in the germline. During pre-implantation stages of mouse embryonic development, however, both pathways are inactivated leading to a critical window necessitating alternate means of L1 regulation. We previously reported an increase in L1 levels in Dicer_KO mouse embryonic stem cells (mESCs). Intriguingly this was accompanied by only a marginal increase in retrotransposition, suggestive of additional mechanisms suppressing L1 mobility. Here, we demonstrate that L1 Ribonucleoprotein complexes (L1 RNP) accumulate as aggregates in Dicer_KO cytoplasm along with the RNA helicase MOV10. The combined overexpression of L1 RNAs and MOV10 is sufficient to create L1 RNP aggregates in stem cells. In Dicer_KO mESCs, MOV10 is upregulated due to the loss of its direct regulation by miRNAs. The newly discovered post-transcriptional regulation of Mov10 expression, and its role in preventing L1 retrotransposition by driving novel cytosolic aggregation affords alternate routes to explore for therapy and disease progression.

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“…Constituent subunits of condensates, including SC proteins, can enter and exit condensates and move within them. Coiled-coils can facilitate phase-separation (47, 48), potentially by promoting multivalent interactions (49, 50). This is consistent with the poor per-site conservation of SC proteins, since multivalent interactions can rely on molecular features exhibited by groups of amino acids (e.g., charge or hydrophobicity) rather than tight, ‘lock-and-key’ interfaces formed by specific tertiary structures.…”

Section: Discussionmentioning

confidence: 99%

Unconventional conservation reveals structure-function relationships in the synaptonemal complex

Kursel

Cope

Rog

2021

Preprint

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Functional requirements constrain protein evolution, commonly manifesting in conserved primary amino acid sequence. Here, we extend this idea to secondary structural features by tracking their conservation in essential meiotic proteins with highly diverged sequences. The synaptonemal complex (SC) aligns parental chromosome pairs and regulates exchanges between them. In electron micrographs of meiocytes from all eukaryotic clades, the SC appears as a ~100 nm-wide ladder-like structure with regular striations. Despite the conserved ultrastructure and functions, the proteins that make up the SC are highly divergent in sequence. Here we found that, within the Caenorhabditis genus, SC proteins are significantly more diverged than other proteins. However, SC proteins have highly conserved protein length and coiled-coil domain structure. The same unconventional conservation signature holds true for SC proteins in Drosophila and mammals, suggesting it could be a universal feature of SC proteins. We used this evolutionary signature to identify a novel SC protein in the nematode Pristionchus pacificus, Ppa-SYP-1, which has no significant homology to any protein outside of Pristionchus. Our work suggests that the length and relative arrangement of coiled-coils play a key role in the structure and function of the SC. Furthermore, our analysis implies that expanding sequence analysis beyond measures of per-site identity or similarity can enhance our understanding of protein evolution and function.

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

Cited by 9 publications

References 61 publications

Condensation of LINE-1 is required for retrotransposition

Condensation of LINE-1 is required for retrotransposition

Sequestration of LINE-1 in novel cytosolic bodies by MOV10 restricts retrotransposition

Unconventional conservation reveals structure-function relationships in the synaptonemal complex

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