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Characterization of LINE-1 Ribonucleoprotein Particles


The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility (i.e., retrotransposition). Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1–encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition.


Vyšlo v časopise: Characterization of LINE-1 Ribonucleoprotein Particles. PLoS Genet 6(10): e32767. doi:10.1371/journal.pgen.1001150
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1001150

Souhrn

The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility (i.e., retrotransposition). Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1–encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition.


Zdroje

1. LanderES

LintonLM

BirrenB

NusbaumC

ZodyMC

2001 Initial sequencing and analysis of the human genome. Nature 409 860 921

2. GrimaldiG

SingerMF

1983 Members of the KpnI family of long interspersed repeated sequences join and interrupt alpha-satellite in the monkey genome. Nucleic Acids Res 11 321 338

3. KazazianHHJr

MoranJV

1998 The impact of L1 retrotransposons on the human genome. Nat Genet 19 19 24

4. SassamanDM

DombroskiBA

MoranJV

KimberlandML

NaasTP

1997 Many human L1 elements are capable of retrotransposition. Nat Genet 16 37 43

5. BrouhaB

SchustakJ

BadgeRM

Lutz-PriggeS

FarleyAH

2003 Hot L1s account for the bulk of retrotransposition in the human population. Proc Natl Acad Sci U S A 100 5280 5285

6. BabushokDV

KazazianHHJr

2007 Progress in understanding the biology of the human mutagen LINE-1. Hum Mutat 28 527 539

7. GoodierJL

KazazianHHJr

2008 Retrotransposons revisited: the restraint and rehabilitation of parasites. Cell 135 23 35

8. CordauxR

BatzerMA

2009 The impact of retrotransposons on human genome evolution. Nat Rev Genet 10 691 703

9. SwergoldGD

1990 Identification, characterization, and cell specificity of a human LINE-1 promoter. Mol Cell Biol 10 6718 6729

10. AthanikarJN

BadgeRM

MoranJV

2004 A YY1-binding site is required for accurate human LINE-1 transcription initiation. Nucleic Acids Res 32 3846 3855

11. LavieL

MaldenerE

BrouhaB

MeeseEU

MayerJ

2004 The human L1 promoter: variable transcription initiation sites and a major impact of upstream flanking sequence on promoter activity. Genome Res 14 2253 2260

12. ScottAF

SchmeckpeperBJ

AbdelrazikM

ComeyCT

O'HaraB

1987 Origin of the human L1 elements: proposed progenitor genes deduced from a consensus DNA sequence. Genomics 1 113 125

13. DombroskiBA

MathiasSL

NanthakumarE

ScottAF

KazazianHHJr

1991 Isolation of an active human transposable element. Science 254 1805 1808

14. EsnaultC

MaestreJ

HeidmannT

2000 Human LINE retrotransposons generate processed pseudogenes. Nat Genet 24 363 367

15. HohjohH

SingerMF

1996 Cytoplasmic ribonucleoprotein complexes containing human LINE-1 protein and RNA. Embo J 15 630 639

16. KulpaDA

MoranJV

2005 Ribonucleoprotein particle formation is necessary but not sufficient for LINE-1 retrotransposition. Hum Mol Genet 14 3237 3248

17. KulpaDA

MoranJV

2006 Cis-preferential LINE-1 reverse transcriptase activity in ribonucleoprotein particles. Nat Struct Mol Biol 13 655 660

18. MartinSL

1991 Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells. Mol Cell Biol 11 4804 4807

19. WeiW

GilbertN

OoiSL

LawlerJF

OstertagEM

2001 Human L1 retrotransposition: cis preference versus trans complementation. Mol Cell Biol 21 1429 1439

20. LuanDD

KormanMH

JakubczakJL

EickbushTH

1993 Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72 595 605

21. KuboS

SelemeMC

SoiferHS

PerezJL

MoranJV

2006 L1 retrotransposition in nondividing and primary human somatic cells. Proc Natl Acad Sci U S A 103 8036 8041

22. FengQ

MoranJV

KazazianHHJr

BoekeJD

1996 Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell 87 905 916

23. CostGJ

BoekeJD

1998 Targeting of human retrotransposon integration is directed by the specificity of the L1 endonuclease for regions of unusual DNA structure. Biochemistry 37 18081 18093

24. FuranoAV

2000 The biological properties and evolutionary dynamics of mammalian LINE-1 retrotransposons. Prog Nucleic Acid Res Mol Biol 64 255 294

25. KoloshaVO

MartinSL

1997 In vitro properties of the first ORF protein from mouse LINE-1 support its role in ribonucleoprotein particle formation during retrotransposition. Proc Natl Acad Sci U S A 94 10155 10160

26. MartinSL

LiJ

WeiszJA

2000 Deletion analysis defines distinct functional domains for protein-protein and nucleic acid interactions in the ORF1 protein of mouse LINE-1. J Mol Biol 304 11 20

27. MoranJV

GilbertN

2002 Mammalian LINE-1 retrotransposons and related elements.

CraigN

CraggieR

GellertM

LambowitzA

Mobile DNA II Washington, DC ASM Press 836 869

28. DuvernellDD

TurnerBJ

1998 Swimmer 1, a new low-copy-number LINE family in teleost genomes with sequence similarity to mammalian L1. Mol Biol Evol 15 1791 1793

29. FuranoAV

DuvernellDD

BoissinotS

2004 L1 (LINE-1) retrotransposon diversity differs dramatically between mammals and fish. Trends Genet 20 9 14

30. KhazinaE

WeichenriederO

2009 Non-LTR retrotransposons encode noncanonical RRM domains in their first open reading frame. Proc Natl Acad Sci U S A 106 731 736

31. GoodierJL

ZhangL

VetterMR

KazazianHHJr

2007 LINE-1 ORF1 protein localizes in stress granules with other RNA-binding proteins, including components of RNA interference RNA-induced silencing complex. Mol Cell Biol 27 6469 6483

32. MoranJV

HolmesSE

NaasTP

DeBerardinisRJ

BoekeJD

1996 High frequency retrotransposition in cultured mammalian cells. Cell 87 917 927

33. BasameS

Wai-Lun LiP

HowardG

BranciforteD

KellerD

2006 Spatial Assembly and RNA Binding Stoichiometry of a LINE-1 Protein Essential for Retrotransposition. J Mol Biol 357 351 357

34. MartinSL

BranciforteD

KellerD

BainDL

2003 Trimeric structure for an essential protein in L1 retrotransposition. Proc Natl Acad Sci U S A 100 13815 13820

35. MartinSL

CruceanuM

BranciforteD

Wai-Lun LiP

KwokSC

2005 LINE-1 retrotransposition requires the nucleic acid chaperone activity of the ORF1 protein. J Mol Biol 348 549 561

36. McMillanJP

SingerMF

1993 Translation of the human LINE-1 element, L1Hs. Proc Natl Acad Sci U S A 90 11533 11537

37. AlischRS

Garcia-PerezJL

MuotriAR

GageFH

MoranJV

2006 Unconventional translation of mammalian LINE-1 retrotransposons. Genes Dev 20 210 224

38. LiPW

LiJ

TimmermanSL

KrushelLA

MartinSL

2006 The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition. Nucleic Acids Res 34 853 864

39. DmitrievSE

AndreevDE

TereninIM

OlovnikovIA

PrassolovVS

2007 Efficient translation initiation directed by the 900-nucleotide-long and GC-rich 5′ untranslated region of the human retrotransposon LINE-1 mRNA is strictly cap dependent rather than internal ribosome entry site mediated. Mol Cell Biol 27 4685 4697

40. MathiasSL

ScottAF

KazazianHHJr

BoekeJD

GabrielA

1991 Reverse transcriptase encoded by a human transposable element. Science 254 1808 1810

41. FanningT

SingerM

1987 The LINE-1 DNA sequences in four mammalian orders predict proteins that conserve homologies to retrovirus proteins. Nucleic Acids Res 15 2251 2260

42. GoodierJL

OstertagEM

EnglekaKA

SelemeMC

KazazianHHJr

2004 A potential role for the nucleolus in L1 retrotransposition. Hum Mol Genet 13 1041 1048

43. RigautG

ShevchenkoA

RutzB

WilmM

MannM

1999 A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17 1030 1032

44. NakataniY

OgryzkoV

2003 Immunoaffinity purification of mammalian protein complexes. Methods Enzymol 370 430 444

45. FoutsDE

TrueHL

CelanderDW

1997 Functional recognition of fragmented operator sites by R17/MS2 coat protein, a translational repressor. Nucleic Acids Res 25 4464 4473

46. ValegardK

MurrayJB

StonehouseNJ

van den WormS

StockleyPG

1997 The three-dimensional structures of two complexes between recombinant MS2 capsids and RNA operator fragments reveal sequence-specific protein-RNA interactions. J Mol Biol 270 724 738

47. FreemanJD

GoodchildNL

MagerDL

1994 A modified indicator gene for selection of retrotransposition events in mammalian cells. Biotechniques 17 46, 48 49, 52

48. WeiW

MorrishTA

AlischRS

MoranJV

2000 A transient assay reveals that cultured human cells can accommodate multiple LINE-1 retrotransposition events. Anal Biochem 284 435 438

49. HolmesSE

SingerMF

SwergoldGD

1992 Studies on p40, the leucine zipper motif-containing protein encoded by the first open reading frame of an active human LINE-1 transposable element. J Biol Chem 267 19765 19768

50. LeiboldDM

SwergoldGD

SingerMF

ThayerRE

DombroskiBA

1990 Translation of LINE-1 DNA elements in vitro and in human cells. Proc Natl Acad Sci U S A 87 6990 6994

51. RangwalaSH

KazazianHHJr

2009 The L1 retrotransposition assay: a retrospective and toolkit. Methods 49 219 226

52. KedershaN

StoecklinG

AyodeleM

YaconoP

Lykke-AndersenJ

2005 Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. J Cell Biol 169 871 884

53. TourriereH

ChebliK

ZekriL

CourselaudB

BlanchardJM

2003 The RasGAP-associated endoribonuclease G3BP assembles stress granules. J Cell Biol 160 823 831

54. LiuJ

Valencia-SanchezMA

HannonGJ

ParkerR

2005 MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies. Nat Cell Biol 7 719 723

55. KedershaN

ChenS

GilksN

LiW

MillerIJ

2002 Evidence that ternary complex (eIF2-GTP-tRNA(i)(Met))-deficient preinitiation complexes are core constituents of mammalian stress granules. Mol Biol Cell 13 195 210

56. Garcia-PerezJL

DoucetAJ

BuchetonA

MoranJV

GilbertN

2007 Distinct mechanisms for trans-mediated mobilization of cellular RNAs by the LINE-1 reverse transcriptase. Genome Res

57. ErgunS

BuschmannC

HeukeshovenJ

DammannK

SchniedersF

2004 Cell type-specific expression of LINE-1 open reading frames 1 and 2 in fetal and adult human tissues. J Biol Chem 279 27753 27763

58. MorrishTA

GilbertN

MyersJS

VincentBJ

StamatoTD

2002 DNA repair mediated by endonuclease-independent LINE-1 retrotransposition. Nat Genet 31 159 165

59. GoodierJL

MandalPK

ZhangL

KazazianHHJr

2010 Discrete subcellular partitioning of human retrotransposon RNAs despite a common mechanism of genome insertion. Hum Mol Genet 19 1712 1725

60. Beliakova-BethellN

BeckhamC

GiddingsTHJr

WineyM

ParkerR

2006 Virus-like particles of the Ty3 retrotransposon assemble in association with P-body components. RNA 12 94 101

61. CheckleyMA

NagashimaK

LockettSJ

NyswanerKM

GarfinkelDJ

2010 P-body components are required for Ty1 retrotransposition during assembly of retrotransposition-competent virus-like particles. Mol Cell Biol 30 382 398

62. DutkoJA

KennyAE

GamacheER

CurcioMJ

2010 5′ to 3′ mRNA decay factors colocalize with Ty1 gag and human APOBEC3G and promote Ty1 retrotransposition. J Virol 84 5052 5066

63. DombroskiBA

ScottAF

KazazianHHJr

1993 Two additional potential retrotransposons isolated from a human L1 subfamily that contains an active retrotransposable element. Proc Natl Acad Sci U S A 90 6513 6517

64. BoireauS

MaiuriP

BasyukE

de la MataM

KnezevichA

2007 The transcriptional cycle of HIV-1 in real-time and live cells. J Cell Biol 179 291 304

65. BertrandE

ChartrandP

SchaeferM

ShenoySM

SingerRH

1998 Localization of ASH1 mRNA particles in living yeast. Mol Cell 2 437 445

66. FuscoD

AccorneroN

LavoieB

ShenoySM

BlanchardJM

2003 Single mRNA molecules demonstrate probabilistic movement in living mammalian cells. Curr Biol 13 161 167

67. BradfordMM

1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 248 254

68. QiHH

OngusahaPP

MyllyharjuJ

ChengD

PakkanenO

2008 Prolyl 4-hydroxylation regulates Argonaute 2 stability. Nature 455 421 424

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