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 (R01GM654321)
- Jef D Boeke
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
Publication 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
Regulation of translation is a fundamental facet of the cellular response to rapidly changing external conditions. Specific RNA-binding proteins (RBPs) co-ordinate the translational regulation of distinct mRNA cohorts during stress. To identify RBPs with previously under-appreciated roles in translational control, we used polysome profiling and mass spectrometry to identify and quantify proteins associated with translating ribosomes in unstressed yeast cells and during oxidative stress and amino acid starvation, which both induce the integrated stress response (ISR). Over 800 proteins were identified across polysome gradient fractions, including ribosomal proteins, translation factors and many others without previously described translation-related roles, including numerous metabolic enzymes. We identified variations in patterns of polysome enrichment in both unstressed and stressed cells and identified proteins enriched in heavy polysomes during stress. Genetic screening of polysome-enriched RBPs identified the cytosolic aspartate aminotransferase, Aat2, as a ribosome-associated protein whose deletion conferred growth sensitivity to oxidative stress. Loss of Aat2 caused aberrantly high activation of the ISR via enhanced eIF2a phosphorylation and GCN4 activation. Importantly, non-catalytic AAT2 mutants retained polysome association and did not show heightened stress sensitivity. Aat2 therefore has a separate ribosome-associated translational regulatory or 'moonlighting' function that modulates the ISR independent of its aspartate aminotransferase activity.
