A Dicer-2-dependent 80S complex cleaves targeted mRNAs during RNAi in Drosophila

John W. Pham; Janice L. Pellino; Young Sik Lee; Richard W. Carthew; Erik J. Sontheimer

doi:10.1016/S0092-8674(04)00258-2

A Dicer-2-dependent 80S complex cleaves targeted mRNAs during RNAi in Drosophila

John W. Pham, Janice L. Pellino, Young Sik Lee, Richard W. Carthew, Erik J. Sontheimer^*

^*Corresponding author for this work

Molecular Biosciences

Research output: Contribution to journal › Article › peer-review

351 Scopus citations

Abstract

We use native gel electrophoresis to characterize complexes that mediate RNA interference (RNAi) in Drosophila. Our data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex. R1 serves as a precursor to both the R2 and R3 complexes. R3 is a large (80S), ATP-enhanced complex that contains unwound siRNAs, cofractionates with known RNAi factors, and binds and cleaves targeted mRNAs in a cognate-siRNA-dependent manner. Our results establish an ordered biochemical pathway for RISC assembly and indicate that siRNAs must first interact with Dcr-2 to reach the R3 "holo-RISC" complex. Dcr-2 does not simply transfer siRNAs to a distinct effector complex, but rather assembles into RISC along with the siRNAs, indicating that its role extends beyond the initiation phase of RNAi.

Original language	English (US)
Pages (from-to)	83-94
Number of pages	12
Journal	Cell
Volume	117
Issue number	1
DOIs	https://doi.org/10.1016/S0092-8674(04)00258-2
State	Published - Apr 2 2004

Funding

We are very grateful to P. Zamore for the guanylyl transferase that we used in our early experiments; A. Caudy, G. Hannon, and E. Izaurralde for antibodies; and Q. Liu and X. Wang for antibodies and for communicating results before publication. We thank J. Gorra and Z. He for help with fly maintenance and lysate preparation, R. Lieberman for assistance with column chromatography, T. Karginov and R. Fahlman for help with sedimentation analysis, and P. Bellare, A. Lamond, J. Staley, and O. Uhlenbeck for comments on the manuscript. J.W.P. and J.L.P. were supported by NIH Training Grants in Biophysics and the Molecular and Cellular Basis of Disease, respectively. Y.S.L. was supported by a FRAXA postdoctoral fellowship. This work was supported by a Burroughs Wellcome Fund New Investigator Award to E.J.S. and by a grant from the National Institutes of Health (GM068743-01) to R.W.C. and E.J.S.

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology

Access to Document

10.1016/S0092-8674(04)00258-2

Cite this

@article{2695843092a34e45bec680a71e4b1e98,

title = "A Dicer-2-dependent 80S complex cleaves targeted mRNAs during RNAi in Drosophila",

abstract = "We use native gel electrophoresis to characterize complexes that mediate RNA interference (RNAi) in Drosophila. Our data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex. R1 serves as a precursor to both the R2 and R3 complexes. R3 is a large (80S), ATP-enhanced complex that contains unwound siRNAs, cofractionates with known RNAi factors, and binds and cleaves targeted mRNAs in a cognate-siRNA-dependent manner. Our results establish an ordered biochemical pathway for RISC assembly and indicate that siRNAs must first interact with Dcr-2 to reach the R3 {"}holo-RISC{"} complex. Dcr-2 does not simply transfer siRNAs to a distinct effector complex, but rather assembles into RISC along with the siRNAs, indicating that its role extends beyond the initiation phase of RNAi.",

author = "Pham, {John W.} and Pellino, {Janice L.} and Lee, {Young Sik} and Carthew, {Richard W.} and Sontheimer, {Erik J.}",

note = "Funding Information: We are very grateful to P. Zamore for the guanylyl transferase that we used in our early experiments; A. Caudy, G. Hannon, and E. Izaurralde for antibodies; and Q. Liu and X. Wang for antibodies and for communicating results before publication. We thank J. Gorra and Z. He for help with fly maintenance and lysate preparation, R. Lieberman for assistance with column chromatography, T. Karginov and R. Fahlman for help with sedimentation analysis, and P. Bellare, A. Lamond, J. Staley, and O. Uhlenbeck for comments on the manuscript. J.W.P. and J.L.P. were supported by NIH Training Grants in Biophysics and the Molecular and Cellular Basis of Disease, respectively. Y.S.L. was supported by a FRAXA postdoctoral fellowship. This work was supported by a Burroughs Wellcome Fund New Investigator Award to E.J.S. and by a grant from the National Institutes of Health (GM068743-01) to R.W.C. and E.J.S. ",

year = "2004",

month = apr,

day = "2",

doi = "10.1016/S0092-8674(04)00258-2",

language = "English (US)",

volume = "117",

pages = "83--94",

journal = "Cell",

issn = "0092-8674",

publisher = "Cell Press",

number = "1",

}

TY - JOUR

T1 - A Dicer-2-dependent 80S complex cleaves targeted mRNAs during RNAi in Drosophila

AU - Pham, John W.

AU - Pellino, Janice L.

AU - Lee, Young Sik

AU - Carthew, Richard W.

AU - Sontheimer, Erik J.

N1 - Funding Information: We are very grateful to P. Zamore for the guanylyl transferase that we used in our early experiments; A. Caudy, G. Hannon, and E. Izaurralde for antibodies; and Q. Liu and X. Wang for antibodies and for communicating results before publication. We thank J. Gorra and Z. He for help with fly maintenance and lysate preparation, R. Lieberman for assistance with column chromatography, T. Karginov and R. Fahlman for help with sedimentation analysis, and P. Bellare, A. Lamond, J. Staley, and O. Uhlenbeck for comments on the manuscript. J.W.P. and J.L.P. were supported by NIH Training Grants in Biophysics and the Molecular and Cellular Basis of Disease, respectively. Y.S.L. was supported by a FRAXA postdoctoral fellowship. This work was supported by a Burroughs Wellcome Fund New Investigator Award to E.J.S. and by a grant from the National Institutes of Health (GM068743-01) to R.W.C. and E.J.S.

PY - 2004/4/2

Y1 - 2004/4/2

N2 - We use native gel electrophoresis to characterize complexes that mediate RNA interference (RNAi) in Drosophila. Our data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex. R1 serves as a precursor to both the R2 and R3 complexes. R3 is a large (80S), ATP-enhanced complex that contains unwound siRNAs, cofractionates with known RNAi factors, and binds and cleaves targeted mRNAs in a cognate-siRNA-dependent manner. Our results establish an ordered biochemical pathway for RISC assembly and indicate that siRNAs must first interact with Dcr-2 to reach the R3 "holo-RISC" complex. Dcr-2 does not simply transfer siRNAs to a distinct effector complex, but rather assembles into RISC along with the siRNAs, indicating that its role extends beyond the initiation phase of RNAi.

AB - We use native gel electrophoresis to characterize complexes that mediate RNA interference (RNAi) in Drosophila. Our data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex. R1 serves as a precursor to both the R2 and R3 complexes. R3 is a large (80S), ATP-enhanced complex that contains unwound siRNAs, cofractionates with known RNAi factors, and binds and cleaves targeted mRNAs in a cognate-siRNA-dependent manner. Our results establish an ordered biochemical pathway for RISC assembly and indicate that siRNAs must first interact with Dcr-2 to reach the R3 "holo-RISC" complex. Dcr-2 does not simply transfer siRNAs to a distinct effector complex, but rather assembles into RISC along with the siRNAs, indicating that its role extends beyond the initiation phase of RNAi.

UR - http://www.scopus.com/inward/record.url?scp=1842766252&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=1842766252&partnerID=8YFLogxK

U2 - 10.1016/S0092-8674(04)00258-2

DO - 10.1016/S0092-8674(04)00258-2

M3 - Article

C2 - 15066284

AN - SCOPUS:1842766252

SN - 0092-8674

VL - 117

SP - 83

EP - 94

JO - Cell

JF - Cell

IS - 1

ER -

A Dicer-2-dependent 80S complex cleaves targeted mRNAs during RNAi in Drosophila

Abstract

Funding

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this