TY - JOUR
T1 - Affinity proteomics reveals human host factors implicated in discrete stages of LINE-1 retrotransposition
AU - Taylor, Martin S.
AU - LaCava, John
AU - Mita, Paolo
AU - Molloy, Kelly R.
AU - Huang, Cheng Ran Lisa
AU - Li, Donghui
AU - Adney, Emily M.
AU - Jiang, Hua
AU - Burns, Kathleen H.
AU - Chait, Brian T.
AU - Rout, Michael P.
AU - Boeke, Jef D.
AU - Dai, Lixin
PY - 2013/11/21
Y1 - 2013/11/21
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84889600606&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84889600606&partnerID=8YFLogxK
U2 - 10.1016/j.cell.2013.10.021
DO - 10.1016/j.cell.2013.10.021
M3 - Article
C2 - 24267889
AN - SCOPUS:84889600606
VL - 155
SP - 1034
EP - 1048
JO - Cell
JF - Cell
SN - 0092-8674
IS - 5
ER -