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
N1 - Funding Information:
We thank Jeffrey Han for gifts of antibodies and helpful discussions; Phil Cole and Carolyn Machamer for helpful discussion and critical reading of the manuscript; Jennifer Wang, Chih-Yung Lee, and Geraldine Seydoux for assistance with confocal microscopy and helpful discussions; and Yana Li, Dan Leahy, Jennifer Kavran, and Jacqueline McCabe for help with suspension cell culture. The DNA template for artwork in Figure 7 was adapted from http://www.dragonartz.net with permission. This work was supported in part by NIH grant U54 GM103511 to M.P.R. and B.T.C., grants R01 GM36481 and U54 GM103520 to J.D.B., and grant P41 GM103314 to B.T.C.
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.
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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 -