LINE-1 and the cell cycle: protein localization and functional dynamics

Mita, Paolo; Wudzinska, Aleksandra; Sun, Xiaoji; Andrade, Joshua; Nayak, Shruti; Kahler, David J.; Badri, Sana; LaCava, John; Ueberheide, Beatrix; Yun, Chi Young; Fenyö, David; Boeke, Jef D.

doi:10.1101/147587

Cited by 4 publications

(3 citation statements)

References 75 publications

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“…ORF1p-1FLAG did not or only exhibited subtle interactions with the ORF2p-3FLAG-associated nuclear proteins (Figure 1C), suggesting that ORF1p is more abundant in cytoplasmic versus nuclear L1 RNPs or might form unstable complexes with ORF2p-associated nuclear proteins. Thus, in addition to the previously reported associations with PARP1, PCNA, RPA, and HMCES (Mita et al, 2018;Taylor et al, 2013Taylor et al, , 2018, ORF2p interacts with at least seven other nuclear proteins.…”

Section: Identification Of L1 Orf2p Interacting Proteinsmentioning

confidence: 51%

“…Proteins involved in DNA replication and/or DNA repair can facilitate L1 retrotransposition, but their mechanisms of action require elucidation (Benitez-Guijarro et al, 2018;Flasch et al, 2019;Mita et al, 2018;Sultana et al, 2019;Suzuki et al, 2009;Taylor et al, 2013Taylor et al, , 2018. Here, we conducted immunoprecipitation (IP)-coupled mass spectrometry analyses to identify L1 ORF2p interacting proteins.…”

Section: Introductionmentioning

confidence: 99%

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Poly(ADP-Ribose) Polymerase 2 Recruits Replication Protein A to Sites of LINE-1 Integration to Facilitate Retrotransposition

2019

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Long interspersed element-1 (LINE-1 or L1) retrotransposition poses a threat to genome integrity, and cells have evolved mechanisms to restrict retrotransposition. However, how cellular proteins facilitate L1 retrotransposition requires elucidation. Here, we demonstrate that single-strand DNA breaks induced by the L1 endonuclease trigger the recruitment of poly(ADP-ribose) polymerase 2 (PARP2) to L1 integration sites and that PARP2 activation leads to the subsequent recruitment of the replication protein A (RPA) complex to facilitate retrotransposition. We further demonstrate that RPA directly binds activated PARP2 through poly(ADP-ribosyl)ation and can protect single-strand L1 integration intermediates from APOBEC3-mediated cytidine deamination in vitro. Paradoxically, we provide evidence that RPA can guide APOBEC3A, and perhaps other APOBEC3 proteins, to sites of L1 integration. Thus, the interplay of L1-encoded and evolutionarily conserved cellular proteins is required for efficient retrotransposition; however, these interactions also may be exploited to restrict L1 retrotransposition in the human genome.(A) The L1.3 ORF2p-3FLAG expression vector (pTMO2F3). The CMV promoter (black arrow) and 5 0 UTR augment L1 expression. Also indicated are the FLAG epitope tag, SV40 polyadenylation signal, and the EN, Z, RT, and cysteine-rich (C) domains. (B) The ORF2p-3FLAG complex. HEK293T cells were transfected with pAD500 (ORF2p-TAP) or pTMO2F3 (ORF2p-3FLAG). Left: the ORF2p-3FLAG complex was purified using anti-FLAG antibody-conjugated beads, separated on a denaturing polyacrylamide gel, and visualized by silver staining. Right: ORF2p-3FLAG was detected by western blot. Input, the whole-cell lysate used in the IP. Arrows, ORF2p-3FLAG. (C) Validation of proteins in the ORF2p-3FLAG complex. ORF2p-3FLAG complexes were purified (see B) from HEK293T cells transfected with a full-length (pTMF3, lane 2) or monocistronic (pTMO2F3, lane 4) L1 expression constructs. The ORF1p-1FLAG complex was purified from cells transfected with pJM101/L1.3FLAG (lane 5). The pJM101/L1.3 and pAD001 samples (lanes 1 and 3, respectively) served as negative controls. Input, western blot lanes of whole-cell lysate used in the IP. Red triangles, FLAG tags; SSB, single-strand break; NHEJ, non-homologous end-joining; NER, nucleotide excision repair. See also Figure S1 and Table S1.

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Section: Identification Of L1 Orf2p Interacting Proteinsmentioning

confidence: 51%

Section: Introductionmentioning

confidence: 99%

Poly(ADP-Ribose) Polymerase 2 Recruits Replication Protein A to Sites of LINE-1 Integration to Facilitate Retrotransposition

2019

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“…Using a human neuronal model of oxidative stress which displays increased LINE-1 activity as monitored by an increase in ORF1p expression and ORF2p-dependent genomic instability, we observed not only an increase of ORF1p in the cytoplasm, but also a striking translocation of ORF1p to the nucleus. Current consensus postulates that LINE-1 encoded proteins enter the nucleus only during cell cycle 45 which thus would exclude the presence of LINE-1 proteins in the nucleus of non-dividing cells including post-mitotic neurons. However, others have reported the presence of ORF1p in the nucleus in human cells in neurodegenerative context 67 .…”

Section: Discussionmentioning

confidence: 99%

Nuclear translocation of LINE-1 encoded ORF1p alters nuclear envelope integrity and disrupts nucleocytoplasmic transport in human neurons

Znaidi,

Massiani-Beaudoin,

Mailly

et al. 2023

Preprint

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LINE-1 retrotransposons are emerging as possible culprits in neurodegenerative diseases. However, the molecular mechanisms underlying the pathogenic role of LINE-1 and their encoded proteins ORF1p and ORF2p are still not completely understood. While the endonuclease and reverse transcriptase activity of ORF2p has been associated with DNA damage and inflammation, no pathogenic role has yet been assigned to ORF1p. Using a neuronal model of oxidative stress displaying increased LINE-1 expression, we report here that ORF1p stress-dependently translocates into the nucleus, localizes to the nuclear envelope and directly binds to nuclear lamina (Lamin B1), nuclear pore complex (NUP153) components and nuclear import (KPNB1) proteins. Stress-dependent targeting of nuclear envelope components by ORF1p alters nuclear envelope integrity, disrupts nucleo-cytoplasmic transport and induces heterochromatin destructuration, which are key features associated with neurodegenerative diseases and aging. Interestingly, nuclear deformations and heterochromatin destructuration were restored by the small molecule Remodelin which also normalized nuclear ORF1p levels. This study thus reveals a retrotransposition-independent pathogenic action of ORF1p perturbing nuclear envelope barrier function.

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Long interspersed nuclear element-1 expression and retrotransposition in prostate cancer cells

Briggs

Mita

et al. 2018

Mobile DNA

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BackgroundLong Interspersed Nuclear Element-1 (LINE-1) is an autonomous retrotransposon that generates new genomic insertions through the retrotransposition of a RNA intermediate. Expression of LINE-1 is tightly repressed in most somatic tissues to prevent DNA damage and ensure genomic integrity. However, the reactivation of LINE-1 has been documented in cancer and the role of LINE-1 protein expression and retrotransposition has become of interest in the development, progression, and adaptation of many epithelial neoplasms, including prostate cancer.ResultsHere, we examined endogenous LINE-1 protein expression and localization in a panel of prostate cancer cells and observed a diverse range of LINE-1 expression patterns between cell lines. Subcellular localization of LINE-1 proteins, ORF1p and ORF2p, revealed distinct expression patterns. ORF1p, a nucleic acid chaperone that binds LINE-1 mRNA, was predominantly expressed in the cytoplasm, with minor localization in the nucleus. ORF2p, containing endonuclease and reverse transcriptase domains, exhibited punctate foci in the nucleus and also displayed co-localization with PCNA and γH2AX. Using a retrotransposition reporter assay, we found variations in LINE-1 retrotransposition between cell lines.ConclusionsOverall, our findings reveal new insight into the expression and retrotransposition of LINE-1 in prostate cancer. The prostate cancer cells we investigated provide a unique model for investigating endogenous LINE-1 activity and provide a functional model for studying LINE-1 mechanisms in prostate cancer.Electronic supplementary materialThe online version of this article (10.1186/s13100-017-0106-z) contains supplementary material, which is available to authorized users.

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LINE-1 and the cell cycle: protein localization and functional dynamics

Cited by 4 publications

References 75 publications

Poly(ADP-Ribose) Polymerase 2 Recruits Replication Protein A to Sites of LINE-1 Integration to Facilitate Retrotransposition

Poly(ADP-Ribose) Polymerase 2 Recruits Replication Protein A to Sites of LINE-1 Integration to Facilitate Retrotransposition

Nuclear translocation of LINE-1 encoded ORF1p alters nuclear envelope integrity and disrupts nucleocytoplasmic transport in human neurons

Long interspersed nuclear element-1 expression and retrotransposition in prostate cancer cells

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