1. Volume 22, Issue 4, Pages 553-560 (May 2006)
Recapitulation of Short RNA-Directed Translational Gene Silencing In Vitro 
Bingbing Wang, Tara M. Love, Matthew E. Call, John G. Doench, Carl D. Novina 
Molecular Cell 
Volume 22, Issue 4, Pages (May 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Comparison of Silencing by Translational Repression and mRNA Cleavage
(A) Reporters used in these studies. Reporters contain no sites, imperfectly complementary binding sites (“X”), or perfectly complementary binding sites (“P”) for the antisense strand of the CXCR4 siRNA.
(B) Ethidium bromide-stained agarose gel of capped (cap) and polyadenylated (polyA) FL0, FL2X, FL4X, FL6X, and FL1P.
(C) Silencing reactions using the reporters in (B) without (−) and with (+) CXCR4 siRNA (siCXCR4; top) or control siRNA (siGFP; bottom). Gene silencing (percentage reduction) is indicated.
(D) Northern blot of the reactions in (C). A firefly probe recognizes FL0, FL2X, FL4X, FL6X, and FL1P, and a Renilla probe recognizes the RL0 (input; left). mRNA stability is measured for FL0, FL2X, FL4X, FL6X, and FL1P in the absence (−) and in the presence (+) of CXCR4 siRNA (percentage reduction; right).
(E) Reactions with [α32P]GTP cap-labeled FL6X and FL1P RL0 mRNAs (input, lanes 1–3) and unlabeled amino acid cocktails in the absence (−, lanes 4 and 6) and in the presence (+, lanes 5 and 7) of CXCR4 siRNA. mRNA stability (percentage reduction) is quantitated.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

3. Figure 2
Translational Silencing In Vitro Demonstrates Molecular Hallmarks of miRNA Activity
(A) Translational repression requires perfect 5′ seed region complementarity between the antisense strand of the CXCR4 siRNA and its binding site. Three nucleotide mutations in the 5′ seed region or 3′ end of the CXCR4 siRNA binding site are shown. The 5′ seed region mutant, the 3′ end mutant, or the wild-type FL6X reporter was added to reactions in the absence (−) and presence (+) of CXCR4 siRNAs (left). The 5′ seed region mutant FL6X reporter was added to reactions in the absence (−) and presence of wild-type CXCR4 siRNA (siCXCR4), a CXCR4 siRNA with compensatory mutations to the FL6X with 5′ seed region mutations (2-4-6 comp siCXCR4) or a control siRNA (siGFP, right).
(B) Translational repression requires 5′ phosphorylated short RNAs. FL6X and RL0 were added to reactions in the absence (−) or in the presence (+) of CXCR4 siRNA, a 5′ phosphorylated antisense strand of the CXCR4 siRNA, or an unphosphorylated antisense strand of the CXCR4 siRNA.
(C) Short RNAs do not repress translation by an antisense mechanism. Capped and polyadenylated FL6X and RL0 were added to reactions in the absence (−) or in the presence of CXCR4 siRNA (siCXCR4), a 31-mer CXCR4 RNA, a 2′O-methylated RNA (2′O-Me CXCR4 RNA), or a control siRNA (siGFP). Gene silencing (percentage reduction) is indicated.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

4. Figure 3
Short RNAs Can Associate with the mRNA prior to miRNP Recruitment, In Vitro
FL6X and RL0 mRNAs polyadenylated with adenosine:biotinyl-adenosine (100:1) contained an average of two biotins per 200 nucleotide polyA tail (FL6X-bi, RL0-bi). These reporters were translated in the absence (−, lanes 1 and 4) or in the presence (+) of CXCR4 siRNA alone (lane 2) or in the presence of streptavidin beads (lane 3). Alternately, CXCR4 siRNAs were annealed to these reporters, bound to streptavidin beads, and subjected to pull-down and washing before translation (top) or modified Northern blotting with a probe specific for the antisense strand of the CXCR4 siRNA (as-siCXCR4, middle panel). Isoleucine tRNA served as a loading control (Ile tRNA, bottom). Gene silencing of FL6X-bi in the presence of streptavidin beads (lane 5) is approximately the same as the FL6X-bi-streptavidin bead precipitate after precipitation and washing (lane 6). The supernatant from FL6X-bi-streptavidin bead precipitate (lane 7) demonstrated little gene silencing and CXCR4 siRNA determined by modified Northern blotting. Conversely, the control Isoleucine tRNA was not strongly associated with the precipitated mRNA (lane 6) but was identified in the supernatant (lane 7). Gene silencing (percentage reduction) is indicated.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

5. Figure 4
Translational Gene Silencing Is 7-Methyl G Cap and PolyA Tail Dependent
(A) Silencing reactions using capped (+/− cap) and/or polyadenylated (+/− polyA; 0.2 kb) FL1P and RL0 mRNAs.
(B) Silencing reactions using FL6X and RL0 mRNA prepared as in (A). The right panels are the same lanes as FL6X (0.2 kb) 0 and 6:1 in (C).
(C) Silencing reactions using siRNA:mRNA ratios of 0:1, 1:1, and 6:1; FL6X and RL0 mRNAs were not capped (−cap) or capped (+cap) and polyadenylated to the lengths indicated. Gene silencing (percentage reduction) is indicated.
(D) An ethidium bromide-stained agarose gel demonstrates mRNA polyadenylation (polyA; kb).
(E) mRNA looping model of miRNA function. The 7-methyl G cap (green ball) is brought into proximity of the polyA tail through interactions between the cap binding protein eIF4E (4E), the polyA tail binding protein (PABP), and a cofactor eIF4G (4G, left). At nonphysiological polyA tail lengths, enough PABP binds to the polyA tail to recruit eIF4E without a cap binding. Thus, cap-independent translational gene silencing may occur with long polyA tails (right).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions