Shintaro Iwasaki, Tomoko Kawamata, Yukihide Tomari  Molecular Cell 

Slides:



Advertisements
Similar presentations
Cotranscriptional Recruitment of the mRNA Export Factor Yra1 by Direct Interaction with the 3′ End Processing Factor Pcf11  Sara Ann Johnson, Gabrielle.
Advertisements

Volume 35, Issue 4, Pages (August 2009)
Volume 28, Issue 4, Pages (November 2007)
Purusharth Rajyaguru, Meipei She, Roy Parker  Molecular Cell 
Volume 3, Issue 1, Pages (January 1999)
Angiogenin-Induced tRNA Fragments Inhibit Translation Initiation
Reconstitution of an Argonaute-Dependent Small RNA Biogenesis Pathway Reveals a Handover Mechanism Involving the RNA Exosome and the Exonuclease QIP 
Volume 38, Issue 5, Pages (June 2010)
Volume 36, Issue 2, Pages (October 2009)
HnRNP L and HnRNP A1 Induce Extended U1 snRNA Interactions with an Exon to Repress Spliceosome Assembly  Ni-Ting Chiou, Ganesh Shankarling, Kristen W.
Transcriptional Activators Enhance Polyadenylation of mRNA Precursors
Volume 59, Issue 1, Pages (July 2015)
Fátima Gebauer, Marica Grskovic, Matthias W Hentze  Molecular Cell 
Monica C. Rodrigo-Brenni, Erik Gutierrez, Ramanujan S. Hegde 
Human mRNA Export Machinery Recruited to the 5′ End of mRNA
Volume 64, Issue 3, Pages (November 2016)
Volume 62, Issue 2, Pages (April 2016)
Volume 55, Issue 2, Pages (July 2014)
Adenylation of Maternally Inherited MicroRNAs by Wispy
Communication with the Exon-Junction Complex and Activation of Nonsense-Mediated Decay by Human Upf Proteins Occur in the Cytoplasm  Guramrit Singh, Steffen.
Gad Asher, Zippi Bercovich, Peter Tsvetkov, Yosef Shaul, Chaim Kahana 
Volume 25, Issue 1, Pages (January 2007)
Volume 56, Issue 1, Pages (October 2014)
Volume 38, Issue 5, Pages (June 2010)
Volume 22, Issue 4, Pages (May 2006)
Hiro-oki Iwakawa, Yukihide Tomari  Molecular Cell 
Volume 50, Issue 3, Pages (May 2013)
Volume 39, Issue 5, Pages (September 2010)
Antti Nykänen, Benjamin Haley, Phillip D. Zamore  Cell 
PARP1 Represses PAP and Inhibits Polyadenylation during Heat Shock
Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain
Volume 29, Issue 2, Pages (February 2008)
Volume 15, Issue 6, Pages (September 2004)
Volume 29, Issue 4, Pages (February 2008)
Gracjan Michlewski, Jeremy R. Sanford, Javier F. Cáceres 
Takashi Fukaya, Hiro-oki Iwakawa, Yukihide Tomari  Molecular Cell 
Volume 66, Issue 5, Pages e4 (June 2017)
Sorting of Drosophila Small Silencing RNAs
The Mammalian RNA Polymerase II C-Terminal Domain Interacts with RNA to Suppress Transcription-Coupled 3′ End Formation  Syuzo Kaneko, James L. Manley 
Jens Herold, Raul Andino  Molecular Cell 
Volume 35, Issue 6, Pages (September 2009)
A Critical Role for Noncoding 5S rRNA in Regulating Mdmx Stability
Inhibitor Mediated Protein Degradation
Volume 5, Issue 3, Pages (November 2013)
Volume 39, Issue 2, Pages (July 2010)
The DEAD-Box Protein Ded1 Modulates Translation by the Formation and Resolution of an eIF4F-mRNA Complex  Angela Hilliker, Zhaofeng Gao, Eckhard Jankowsky,
Autoantigen La Promotes Efficient RNAi, Antiviral Response, and Transposon Silencing by Facilitating Multiple-Turnover RISC Catalysis  Ying Liu, Huiling.
Cotranscriptional Recruitment of the mRNA Export Factor Yra1 by Direct Interaction with the 3′ End Processing Factor Pcf11  Sara Ann Johnson, Gabrielle.
Volume 26, Issue 6, Pages (June 2007)
Molecular Basis for Target RNA Recognition and Cleavage by Human RISC
tRNA Binds to Cytochrome c and Inhibits Caspase Activation
C-Raf Inhibits MAPK Activation and Transformation by B-RafV600E
Richard W. Deibler, Marc W. Kirschner  Molecular Cell 
RISC Assembly Defects in the Drosophila RNAi Mutant armitage
Hua Gao, Yue Sun, Yalan Wu, Bing Luan, Yaya Wang, Bin Qu, Gang Pei 
Posttranscriptional Derepression of GADD45α by Genotoxic Stress
Volume 129, Issue 6, Pages (June 2007)
Lin28 Mediates the Terminal Uridylation of let-7 Precursor MicroRNA
Dianne S Schwarz, György Hutvágner, Benjamin Haley, Phillip D Zamore 
USP15 Negatively Regulates Nrf2 through Deubiquitination of Keap1
The Innate Immune Sensor LGP2 Activates Antiviral Signaling by Regulating MDA5- RNA Interaction and Filament Assembly  Annie M. Bruns, George P. Leser,
Regulation of Yeast mRNA 3′ End Processing by Phosphorylation
Takashi Fukaya, Yukihide Tomari  Molecular Cell 
Volume 36, Issue 6, Pages (December 2009)
Volume 35, Issue 6, Pages (September 2009)
Volume 42, Issue 4, Pages (May 2011)
CDK Phosphorylation of Translation Initiation Factors Couples Protein Translation with Cell-Cycle Transition  Tai An, Yi Liu, Stéphane Gourguechon, Ching.
c-IAP1 Cooperates with Myc by Acting as a Ubiquitin Ligase for Mad1
Volume 3, Issue 1, Pages (January 1999)
Volume 31, Issue 5, Pages (September 2008)
Presentation transcript:

Drosophila Argonaute1 and Argonaute2 Employ Distinct Mechanisms for Translational Repression  Shintaro Iwasaki, Tomoko Kawamata, Yukihide Tomari  Molecular Cell  Volume 34, Issue 1, Pages 58-67 (April 2009) DOI: 10.1016/j.molcel.2009.02.010 Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 1 Both Ago1 and Ago2 Can Repress Translation of Their Target mRNAs (A) Ago1-RISC or Ago2-RISC was programmed in embryo lysate for 30 min, and Renilla luciferase (RL) reporter mRNAs bearing zero, two, four, and eight let-7 target sites were translated in vitro for 1 hr together with firefly luciferase (FL) control mRNA. let-7/let-7∗ entered Ago1-RISC, whereas let-7, RL, and CXCR4 siRNAs entered Ago2-RISC (Figure S1A). The RL/FL luminescence was normalized to the value of no RISC programming. RL siRNA had a target site within the ORF of RL (positive control), while CXCR4 siRNA had no target site (negative control). The graph shows means and standard deviations from three independent trials. (B) Stability of the reporter RL mRNAs bearing zero and eight target sites divided by that of the control FL mRNA normalized to the value of no RISC programming. Except for the RL siRNA, which cleaves a site within the ORF of RL, no significant change of mRNA levels was detected. The graph shows means and standard deviations quantified from three independent northern blot analyses, and representative data are shown in Figure S3A. (C) Schematic representation of the RNase H assay used in (D). (D) Ago1, but not Ago2, induces deadenylation of the target. Asterisks designate nonspecific signals. The positions of poly(A)+ and poly(A)− markers run on the same gel are indicated. Note that the length of the RNase H product differs depending on the number of the target sites. Molecular Cell 2009 34, 58-67DOI: (10.1016/j.molcel.2009.02.010) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 2 Ago1 and Ago2 Employ Distinct Mechanisms for Translational Regulation (A) Schematic representation of the RL target mRNAs used in (B) and (C). (B) Ago1 inhibits translation primarily by deadenylation but can secondarily block a step after cap recognition. (C) Ago2 represses translation by blocking the function of the cap structure. For (B) and (C), the RL/FL luminescence was normalized to the value of no RISC programming. The graph shows means and standard deviations from three independent trials. Black, m7G-capped RL mRNA bearing 8x target sites; red, A-capped reaper-IRES-containing RL mRNA bearing 8x target sites. Molecular Cell 2009 34, 58-67DOI: (10.1016/j.molcel.2009.02.010) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 3 Repression by Ago2 Is Independent of GW182, While Deadenylation by Ago1 Requires GW182 and ATP (A) Translational repression of poly(A)+ target mRNA by Ago1 (solid circles), but not by Ago2 (open circles), was inhibited by the addition of GST-GW182(ΔC). (B) Deadenylation by Ago1 requires GW182. GST-GW182(ΔC) blocked the Ago1-mediated deadenylation of the target mRNAs. GST or GST-GW182(ΔC) (12 μM) was present throughout the reaction. (C) Deadenylation by Ago1 and GW182 requires ATP. Northern blot analysis after RNase H treatment is shown. CHX, 0.5 mM cycloheximide was added before the addition of target. −ATP, Ago1-RISC was programmed in the presence of ATP, and then ATP was depleted before the target was added. Molecular Cell 2009 34, 58-67DOI: (10.1016/j.molcel.2009.02.010) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 4 Ago2-RISC Binds to eIF4E Only When Bound to a Cognate Target mRNA (A) Ago1-RISC or Ago2-RISC was programmed in S2 lysate. m7G-capped RL mRNA bearing eight target sites (m7G-cap RL 8x) was added, and anti-eIF4E immunoprecipitation was then performed. Both Ago1 and Ago2 were coimmunoprecipitated with eIF4E, but Ago2 coimmunoprecipitation was significantly enhanced only when the cognate target for Ago2-RISC was present. (B) Same as (A), but 100–800 mM KCl was supplemented in the washing buffer (which by itself contains 100 mM KOAc). Ago1 coimmunoprecipitation was eliminated, but the RNA-enhanced Ago2 coimmunoprecipitation was refractory to this treatment. (C) RNase A treatment abolished Ago2 coimmunoprecipitation. (D) Same as (A), but A-capped RL RNA bearing eight target sites (A-cap RL 8x) was used. A-cap RL 8x enhanced the Ago2 coimmunoprecipitation, to the same degree as m7G-cap RL 8x, indicating that the eIF4E-Ago2 interaction is not simply due to tethering by the target RNA. (E) Same as (A), but m7G-capped or A-capped short RNA lacking the entire RL ORF and containing two target sites (m7G- or A-cap 2x) was used. The short RNAs, in which the region complementary to the let-7 seed sequence was mutated (mm), did not enhance the Ago2 coimmunoprecipitation. Molecular Cell 2009 34, 58-67DOI: (10.1016/j.molcel.2009.02.010) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 5 Ago2-RISC Competes with eIF4G for eIF4E (A) Trp-117 of eIF4E is essential for its association with Ago2. eif4e[RNAi] S2 lysate was supplemented with 200 nM GST-eIF4E(WT) or GST-eIF4E(W117A) and immunoprecipitated with anti-eIF4E antibody. Ago2 was efficiently coimmunoprecipitated only when GST-eIF4E(WT) was present. (B) Ago2 binds to eIF4E independently of eIF4G or Cup. Addition of an increasing concentration of recombinant 4E-BP inhibited the coimmunoprecipitation of Ago2, Cup, and eIF4G by eIF4E antibody. The western blot signals were reasonably within a quantitative linear range (Figure S9). (C) Quantification of (B). The data were fitted to the Hill equation. The Ago2-RISC-eIF4E interaction was more tolerant to 4E-BP than Cup-eIF4E and eIF4G-eIF4E interactions. (D) Cap-radiolabeled target mRNA remained associated with eIF4E, but a significant amount dissociated from eIF4G when it was targeted by Ago2-RISC. In contrast, Ago1-RISC did not affect the association of the target with eIF4E and eIF4G. The amount of eIF4G-bound target divided by that of eIF4E-bound target was normalized to the value of CXCR4 siRNA, and means and standard deviations from four independent trials are shown by the graph in the inset. The p values were calculated using Student's two-tailed t test. Molecular Cell 2009 34, 58-67DOI: (10.1016/j.molcel.2009.02.010) Copyright © 2009 Elsevier Inc. Terms and Conditions

Figure 6 A Model for Distinct Translational Repression Mechanisms by Drosophila Ago Proteins (A) Ago1-RISC represses translation primarily by ATP-dependent shortening of the poly(A) tail of its mRNA targets. Secondarily, Ago1-RISC can also block a step after cap recognition. These two processes are independent, but both require the P body component GW182. (B) Ago2-RISC competitively inhibits translational initiation by blocking the interaction of eIF4E with eIF4G. This occurs only when Ago2-RISC binds to a cognate target mRNA. Molecular Cell 2009 34, 58-67DOI: (10.1016/j.molcel.2009.02.010) Copyright © 2009 Elsevier Inc. Terms and Conditions