Journal: PLoS Biology

Article Title: Translation Repression in Human Cells by MicroRNA-Induced Gene Silencing Requires RCK/p54

doi: 10.1371/journal.pbio.0040210

Figure Lengend Snippet: (A) Immunoprecipitation and immunoblot analyses. TCEs from HeLa cells co-expressing Myc-Ago2 and YFP-Ago1, YFP-Dcp2, YFP-RCK/p54, YFP-eIF4E, YFP-Lsm1, or YFP were treated with +/− RNase A followed by Myc-Ago2 immunoprecipitation. TCE and anti-Myc IPs were analyzed by immunoblot using anti-GFP and anti-Myc antibodies. (B) In vivo localization of RCK/p54 and Ago2 to P-bodies. HeLa cells expressing YFP-Lsm1 and CFP-Ago2 (a, b, and c), YFP-RCK/p54 and CFP-Ago2 (d, e, and f) were visualized by confocal microscopy at 24 h post-transfection. (C) Visualization of interactions between RCK/p54 and Ago2 in P-bodies by FRET. HeLa cells expressing YFP-RCK/p54 and CFP-Ago2 were fixed at 24 h post-transfection. FRET was measured by an acceptor photobleaching method. Fluorescence images of donor (CFP-Ago2) and acceptor (YFP-RCK/p54) molecules were taken before and after photobleaching YFP. FRET efficiencies were calculated as described [ , , ], and data were analyzed by Leica confocal software. Arrows point to P-bodies, which are enlarged in insets. (D) FRET efficiencies between different P-body protein donor: acceptor pairs. HeLa cells co-expressing YFP-RCK/p54 and CFP-Ago2, YFP-Lsm1 and CFP-Ago2, YFP-RCK/p54 and CFP, YFP-Ago1 and CFP-Ago2, YFP-Ago2 and CFP-Ago1, YFP-RCK/p54 and CFP-Ago-1, as well as YFP-Ago1 and CFP, were fixed and FRET efficiencies were measured.

Article Snippet: Ago1 and Ago2 expression vectors with N-terminal YFP- or CFP-epitope tags were generated by PCR amplification of Ago 1 and Ago 2 coding sequences from pMyc-Ago1 and pMyc-Ago2 followed by cloning into the Xba I and EcoR I sites of pEYFP-C1 and pECFP-C1 (BD Biosciences, Palo Alto, California, United States).

Techniques: Immunoprecipitation, Western Blot, Expressing, In Vivo, Confocal Microscopy, Transfection, Fluorescence, Software

Journal: PLoS Biology

Article Title: Translation Repression in Human Cells by MicroRNA-Induced Gene Silencing Requires RCK/p54

doi: 10.1371/journal.pbio.0040210

Figure Lengend Snippet: (A) Experimental outline to purify active human RISC. The guide strands of siRNA complexes targeting GFP (si-GFP) were conjugated with 3′ biotin (si-GFP-Bi; blue strands) and transfected into HeLa cells. RISCs were captured by incubating cell extracts with streptavidin-magnetic beads. (B) Target mRNA is cleaved by biotin-captured RISC. Bead (B) and supernatant (S) phases of captured RISC were incubated with 124-nt 32 P-cap-labeled GFP target mRNA. The reactions were stopped after 120 min, and products were resolved on 6% denaturing polyacrylamide gels. (C) Biotin-captured RISC contains proteins associated with mRNA processing. Active human RISC from HeLa cells expressing Flag-Ago1 was captured by biotin-siRNA and its protein composition was analyzed by immunoblot using anti-Flag, anti-Ago2, anti-RCK/p54, anti-Lsm1, and anti-eIF4E antibodies.

Article Snippet: Ago1 and Ago2 expression vectors with N-terminal YFP- or CFP-epitope tags were generated by PCR amplification of Ago 1 and Ago 2 coding sequences from pMyc-Ago1 and pMyc-Ago2 followed by cloning into the Xba I and EcoR I sites of pEYFP-C1 and pECFP-C1 (BD Biosciences, Palo Alto, California, United States).

Techniques: Transfection, Magnetic Beads, Incubation, Labeling, Expressing, Western Blot

Journal: PLoS Biology

Article Title: Translation Repression in Human Cells by MicroRNA-Induced Gene Silencing Requires RCK/p54

doi: 10.1371/journal.pbio.0040210

Figure Lengend Snippet: (A) Affinity-purified miRISCs associated with PCK/p54 retain cleavage activity. To purify miRISC associated with RCK/p54, magnetic protein A beads coupled with rabbit IgG, rabbit anti-Ago2, or rabbit anti-RCK/p54 antibodies were incubated with HeLa cytoplasmic extracts. After immunoprecipitation, RISC activities were analyzed by incubating the supernatant (S) or bead (B) phases with 182-nt 32 P-cap-labeled let -7 substrate mRNAs having a perfectly complementary or mismatched sequence to the let- 7 miRNA. Cleavage products were resolved on 6% denaturing polyacrylamide gels. CE, cytoplasmic extract; PM, perfect match; MM, mismatch. (B) Affinity-purified miRISCs retain cleavage activity. let -7 miRISC cleavage of a perfectly matched RNA target was inhibited by 2′- O -Me oligonucleotides complementary to let -7 miRNA ( let -7–2′- O- Me or let -7–2′- O- Me-biotin). A 182-nt 32 P-cap-labeled let -7 substrate mRNA was incubated with the supernatant (S) or bead (B) phases of captured miRISC. The reactions were stopped after 120 min, and products were resolved on 6% denaturing polyacrylamide gels. (C) miRISCs contain proteins associated with mRNA processing. Cytoplasmic extracts of HeLa cells expressing Flag-Ago2 and Myc-Ago1 were incubated with 2′- O -Me oligonucleotides complementary to let -7 miRNA ( let -7–2′- O- Me or let -7–2′- O- Me-biotin), affinity-purified by streptavidin-magnetic beads to capture let -7 miRISC. Supernatant (S) and beads (B) after biotin capture were analyzed by immunoblot using anti-Myc, anti-Flag, anti-RCK/p54, and anti-eIF4E antibodies.

Article Snippet: Ago1 and Ago2 expression vectors with N-terminal YFP- or CFP-epitope tags were generated by PCR amplification of Ago 1 and Ago 2 coding sequences from pMyc-Ago1 and pMyc-Ago2 followed by cloning into the Xba I and EcoR I sites of pEYFP-C1 and pECFP-C1 (BD Biosciences, Palo Alto, California, United States).

Techniques: Affinity Purification, Activity Assay, Incubation, Immunoprecipitation, Labeling, Sequencing, Expressing, Magnetic Beads, Western Blot

Journal: PLoS Biology

Article Title: Translation Repression in Human Cells by MicroRNA-Induced Gene Silencing Requires RCK/p54

doi: 10.1371/journal.pbio.0040210

Figure Lengend Snippet: RISC contains Ago2 (red), Ago1 (green), RCK/p54 (blue, labeled p54), and other known (e.g., Dicer and TRBP) and unidentified proteins (pink) and is distributed throughout the cytoplasm. siRISC binds to its target mRNA by perfectly matching base pairs, cleaves the target mRNA for degradation, recycles the complex, and does not require P-body structures for its function. Multiple numbers (n) of miRISC bind to target mRNA by forming a bulge sequence in the middle that is not suitable for RNA cleavage, accumulate in P-bodies, and repress translation by exploiting global translational suppressors such as RCK/p54. The translationally repressed mRNA is either stored in P-bodies or enters the mRNA decay pathway for destruction. Depending upon cellular conditions and stimuli, stored mRNA can either re-enter the translation or mRNA decay pathways.

Article Snippet: Ago1 and Ago2 expression vectors with N-terminal YFP- or CFP-epitope tags were generated by PCR amplification of Ago 1 and Ago 2 coding sequences from pMyc-Ago1 and pMyc-Ago2 followed by cloning into the Xba I and EcoR I sites of pEYFP-C1 and pECFP-C1 (BD Biosciences, Palo Alto, California, United States).

Techniques: Labeling, Sequencing