title: Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition.
abstract: LINE-1s are active human DNA parasites that are agents of genome dynamics in evolution and disease. These streamlined elements require host factors to complete their life cycles, whereas hosts have developed mechanisms to combat retrotransposition's mutagenic effects. As such, endogenous L1 expression levels are extremely low, creating a roadblock for detailed interactomic analyses. Here, we describe a system to express and purify highly active L1 RNP complexes from human suspension cell culture and characterize the copurified proteome, identifying 37 high-confidence candidate interactors. These data sets include known interactors PABPC1 and MOV10 and, with in-cell imaging studies, suggest existence of at least three types of compositionally and functionally distinct L1 RNPs. Among the findings, UPF1, a key nonsense-mediated decay factor, and PCNA, the polymerase-delta-associated sliding DNA clamp, were identified and validated. PCNA interacts with ORF2p via a PIP box motif; mechanistic studies suggest that this occurs during or immediately after target-primed reverse transcription.
authors.affiliation: High Throughput Biology Center and Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Martin S
authors.initials: MS
authors.lastname: Taylor
authors.forename: John
authors.initials: J
authors.lastname: LaCava
authors.forename: Paolo
authors.initials: P
authors.lastname: Mita
authors.forename: Kelly R
authors.initials: KR
authors.lastname: Molloy
authors.forename: Cheng Ran Lisa
authors.initials: CR
authors.lastname: Huang
authors.forename: Donghui
authors.initials: D
authors.lastname: Li
authors.forename: Emily M
authors.initials: EM
authors.lastname: Adney
authors.forename: Hua
authors.initials: H
authors.lastname: Jiang
authors.forename: Kathleen H
authors.initials: KH
authors.lastname: Burns
authors.forename: Brian T
authors.initials: BT
authors.lastname: Chait
authors.forename: Michael P
authors.initials: MP
authors.lastname: Rout
authors.forename: Jef D
authors.initials: JD
authors.lastname: Boeke
authors.forename: Lixin
authors.initials: L
authors.lastname: Dai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015536:Down-Regulation; D015894:Genome, Human; D006801:Humans; D020084:Long Interspersed Nucleotide Elements; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D020365:RNA Helicases; D012261:Ribonucleoproteins / Q000032:analysis* / Q000302:isolation & purification; D016415:Sequence Alignment; D015534:Trans-Activators / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D018809:Proliferating Cell Nuclear Antigen; D020543:Proteome; D012261:Ribonucleoproteins; D015534:Trans-Activators; D020365:RNA Helicases; C101828:UPF1 protein, human