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Human LINE-1 ORF1p complex LC-MS/MS

ABSTRACT: Host proteins regulate LINE-1 (L1) retrotransposition. Here, we use immunoprecipitation followed by liquid chromatography-tandem mass spectrometry to identify proteins that associate with the L1 ORF1-encoded protein (ORF1p) in ribonucleoprotein particles.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Cervix Carcinoma

SUBMITTER: Tomoichiro Miyoshi

LAB HEAD: Tomoichiro Miyoshi

PROVIDER: PXD038851 | Pride | 2023-03-11

REPOSITORIES: Pride

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			Action	DRS
	F022536.dat	Other
	F022536.mgf	Mgf
	F022536.mzid.gz	Mzid
	F022536.raw	Raw
	F022537.dat	Other

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Publications

The interferon stimulated gene-encoded protein HELZ2 inhibits human LINE-1 retrotransposition and LINE-1 RNA-mediated type I interferon induction.

Luqman-Fatah Ahmad A Watanabe Yuzo Y Uno Kazuko K Ishikawa Fuyuki F Moran John V JV Miyoshi Tomoichiro T

Nature communications 20230113 1

Some interferon stimulated genes (ISGs) encode proteins that inhibit LINE-1 (L1) retrotransposition. Here, we use immunoprecipitation followed by liquid chromatography-tandem mass spectrometry to identify proteins that associate with the L1 ORF1-encoded protein (ORF1p) in ribonucleoprotein particles. Three ISG proteins that interact with ORF1p inhibit retrotransposition: HECT and RLD domain containing E3 ubiquitin-protein ligase 5 (HERC5); 2'-5'-oligoadenylate synthetase-like (OASL); and helicas ...[more]

PMID: 36639706

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Dissection of affinity captured LINE-1 macromolecular complexes

Project description:Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications, quantitative mass spectrometry, and next generation RNA sequencing, we have characterized the proteins and nucleic acids associated with distinctive, enzymatically active L1 macromolecular complexes. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1-RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in target-primed reverse transcription.

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Characterization of L1 ORF1p macromolecular complexes from Mael-/- mouse testes

Project description:piRNA-deficient Maelstrom (Mael) null mice are characterized by a strong upregulation of retrotransposon LINE-1 (L1) in meiotic spermatocytes. This defect turns out in the accumulation of L1 RNA and ORF1p in their cytoplasm and the formation of prominent ribonucleoprotein aggregates. We used 3-months-old Mael-/- male mice to characterize the protein composition of those ORF1p aggregates.

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Enrichment of processed pseudogene transcripts in L1-ribonucleoprotein particles

Project description:We explored the RNA binding properties of LINE-1 ORF1p, both free and in the L1 RNP, using a recently developed photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation technique (PAR-CLIP) to comprehensively identify ORF1p binding sites in the transcriptome of human cells (HEK293T). Our results show that ORF1p binds to a wide range of cellular mRNAs, with an enrichment for binding at the 3’ UTR. Our data also show that ORF1p binds very strongly with retrotransposable RNA, i.e., L1, Alu and SVA. PAR-CLIP analysis of L1 RNPs and free ORF1p RNA binding profiles, comparison to HuR RNA binding profile

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Dissolution of RNA condensates by the embryonic stem cell protein L1TD1 [CLIP-seq]

Project description:L1TD1 is a cytoplasmic RNA-binding protein that is specifically expressed in pluripotent stem cells and, unlike its mouse paralogue, is essential for the maintenance of stemness in human cells. Although L1TD1 is the only known domesticated gene from a LINE-1 (L1) retroelement, the functional legacy from its ancestral protein, ORF1p of L1, and the novelty are so far unknown. Here, we determined RNAs associated with L1TD1 and found that, like ORF1p, L1TD1 binds L1 RNAs, localizes to high-density ribonucleoprotein (RNP) condensates, and increases L1 retrotransposition. Unexpectedly, L1TD1 enhanced the translation of a subset of mRNAs enriched in the condensates. In HeLa cells, ectopically expressed L1TD1 facilitated the dissolution of stress granules and granules formed by pathological mutations of TDP-43 and FUS. The glutamate-rich domain and the ORF1-homology domain facilitated dispersal of the RNPs and induced autophagy, respectively. These results provide insights into how L1TD1 regulates gene expression in pluripotent stem cells. We propose that the ability of L1TD1 to dissolve stress granules may provide novel opportunities for treatments of neurodegenerative diseases caused by disturbed stress granule dynamics.

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Dissolution of RNA condensates by the embryonic stem cell protein L1TD1 [RNA-seq]

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ORF1p interactors in the mouse brain

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2024-09-10 | PXD047160 | Pride

Characterization of the RNA present in L1 ORF1p macromolecular complexes from Mael-/- mouse testes

Project description:piRNA-deficient Maelstrom (Mael) null mice are characterized by a strong upregulation of LINE-1 (L1) retrotransposon in meiotic spermatocytes. This defect turns out in the accumulation of L1 RNA and ORF1p in their cytoplasm and the formation of prominent ribonucleoprotein aggregates. We used 3-months-old Mael-/- male mice to characterize the RNA present in those ORF1p aggregates. To favor the isolation of complexed versus free ORF1p protein, we first fractionated Mael-/- testis extracts (that we refer to as TOTAL) by sucrose gradient ultracentrifugation, in the presence of EDTA. We then pooled the sucrose fractions where ORF1p macromolecular complexes sediment (fractions 5-8) and used this pool as the INPUT for an anti-ORF1p co-immunoprecipitation (IP) followed by RNA-seq.

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Colorectal cancer anti-ORF1p LFQ IP-MS

Project description:Here, three colorectal tumors were subjected to anti-ORF1p LFQ, IP-MS: (A) Krukenberg Carcinoma, Ovary; (B) Metastatic Rectal Adenocarcinoma, Liver; (C) Adenocarcinoma, Colon. These were accompanied by matched normal IP controls and/or mouse IgG IP controls. The objective is to map LINE-1 RNP interactions in cancer.

2019-08-23 | PXD013743 | Pride

RNAseq analysis of Huh7 cells upon stable-knockdown of L1 elements

Project description:L1 elements are repeat elements known to get activated during hepatocarcinogenesis. We have knocked-down expression of L1 elements using shRNA targeting L1-ORF1 in Huh7 cell line and a control non-targeting cell line was also generated. Influence of L1 knockdown on overall cellular transcritome was then analysed by RNAseq followed by pathway analysis. Overall, a crosstalk between L1 elements and TGFbeta signaling pathway is observed.

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