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.
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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Birnaviruses are a group of double-stranded RNA (dsRNA) viruses infecting birds, fish, and insects. Early endosomes (EE) constitute the platform for viral replication. Here, we study the mechanism of birnaviral targeting of EE membranes. Using the Infectious Bursal Disease Virus (IBDV) as a model, we validate that the viral protein 3 (VP3) binds to phosphatidylinositol-3-phosphate (PI3P) present in EE membranes. We identify the domain of VP3 involved in PI3P-binding, named P2 and localized in the core of VP3, and establish the critical role of the arginine at position 200 (R200), conserved among all known birnaviruses. Mutating R200 abolishes viral replication. Moreover, we propose a two-stage modular mechanism for VP3 association with EE. Firstly, the carboxy-terminal region of VP3 adsorbs on the membrane, and then the VP3 core reinforces the membrane engagement by specifically binding PI3P through its P2 domain, additionally promoting PI3P accumulation.
