Dissection of affinity captured LINE-1 macromolecular complexes
- Massachusetts General Hospital, United States
- Moscow Institute of Physics and Technology, Russian Federation
- The Rockefeller University, United States
- NYU Langone Medical Center, United States
- Johns Hopkins University School of Medicine, United States
- Novosibirsk State University, Russian Federation
Abstract
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.
Data availability
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RNA-seq FASTAQ filesPublicly available at ProteomeXchange (accession no: PXD008542).
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RNAs associated with affinity captured LINE-1 ribonucleoproteinsPublicly available at the NCBI Gene Expression Omnibus (accession no: GSE108270).
Article and author information
Author details
Funding
National Institutes of Health (P41GM109824)
- Michael P Rout
National Institutes of Health (P41GM103314)
- Brian T Chait
National Institutes of Health (P50GM107632)
- Jef D Boeke
National Institutes of Health (R01GM126170)
- John LaCava
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Stephen P Goff, Howard Hughes Medical Institute, Columbia University, United States
Version history
- Received: July 1, 2017
- Accepted: December 18, 2017
- Accepted Manuscript published: January 8, 2018 (version 1)
- Accepted Manuscript updated: January 10, 2018 (version 2)
- Version of Record published: February 21, 2018 (version 3)
- Version of Record updated: May 15, 2018 (version 4)
- Version of Record updated: September 11, 2018 (version 5)
- Version of Record updated: January 8, 2019 (version 6)
Copyright
© 2018, Taylor et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Dissection of affinity captured LINE-1 macromolecular complexes
eLife 7:e30094.
https://doi.org/10.7554/eLife.30094
Further reading
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- Cell Biology
- Genetics and Genomics
The mechanisms by which a retrotransposon called LINE-1 duplicates itself and spreads through the human genome are becoming clearer.
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- Biochemistry and Chemical Biology
- Cell Biology
Mediator of ERBB2-driven Cell Motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.
