Uncoupling of RNAi from active translation in mammalian cells - PubMed (original) (raw)

Uncoupling of RNAi from active translation in mammalian cells

Shuo Gu et al. RNA. 2005 Jan.

Abstract

Small inhibitory RNAs (siRNAs) are produced from longer RNA duplexes by the RNAse III family member Dicer. The siRNAs function as sequence-specific guides for RNA cleavage or translational inhibition. The precise mechanism by which siRNAs direct the RNA-induced silencing complex (RISC) to find the complementary target mRNA remains a mystery. Some biochemical evidence connects RNAi with translation making attractive the hypothesis that RISC is coupled with the translational apparatus for scanning mRNAs. Such coupling would facilitate rapid alignment of the siRNA antisense with the complementary target sequence. To test this hypothesis we took advantage of a well-characterized translational switch afforded by the ferritin IRE-IRP to analyze RNAi mediated cleavage of a target mRNA in the presence and absence of translation. Our results demonstrate that neither active translation nor unidirectional scanning is required for siRNA mediated target degradation. Our findings demonstrate that nontranslated mRNAs are highly susceptible to RNAi, and blocking scanning from both the 5' and 3' ends of an mRNA does not impede RNAi. Interestingly, RNAi is about threefold more active in the absence of translation.

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Figures

FIGURE 1.

FIGURE 1.

Iron response element (IRE) and IRP regulate translation in HEK293 cells. (A) sequence and computer-modeled secondary structure of the human ferritin H-chain IRE and synthetic oligonucleotides used in this study. The sequence recognized by the iron regulatory protein (IRP), is highlighted in red. (B) Designs of the two constructs used in this study. (C) Three-hour post-transfection of the constructs in HEK293 cells, Hemin (50 μM final concentration), or deferoxaminemesylate salts (100 μM final concentration) were added to the medium and uptake was allowed to proceed for 21 h. EGFP expression 24 h after transfection is depicted. (D) EGFP mRNA levels analyzed by RT-PCR. GAPDH, served as internal control, and the RT-PCR products are also depicted. U1-GFP plasmid DNA and total RNA from nontransfected HEK293 cells were used as standards and positive controls for EGFP and GAPDH, respectively. To control for DNA contamination, minus RT PCR reactions were carried out for each RNA preparation.

FIGURE 2.

FIGURE 2.

siRNA mediated RNAi is fully functional when translation of the target mRNA is active or blocked. (A) EGFP expression from U1_GFP and U1_IRE_GFP under various conditions. Hemin (iron source) labeled as H or deferoxaminemesylate salts (iron chelator) labeled as D and siRNA targeting the EGFP coding region were added to the culture medium as indicated. (B) Cells were lysed 24 h after treatment, and levels of EGFP and an internal control β-actin were obtained by sequential immunoblotting using anti-eGFP and anti-β-actin antibodies. Lanes (from left to right): U1_GFG plus Hemin; U1_GFP plus deferoxaminemesylate salts; U1_GFP treated with siRNA; U1_GFP treated with both siRNA and deferoxaminemesylate salts; U1_IRE_GFP plus Hemin; U1_IRE_GFP plus deferoxaminemesylate salts; U1_IRE_GFP treated with siRNA; U1_IRE_GFP treated with both siRNA and deferoxaminemesylate salts. (C) Summary of EGFP expression and mRNA levels for each sample. The mean levels of EGFP expression were measured by Dot density assays of the immunoblots (B) after normalization with the internal control β-actin. The EGFP mRNA levels were determined using real time RT-PCR after normalization with GAPDH mRNA levels. EGFP expression and mRNA levels from the U1-GFP plus Hemin in the absence of siRNA were assigned a value of 100 percent. Quantitations of EGFP values and mRNA relative to the U1-GFP values are plotted. The results are derived from averaging values from triplicate experiments. Values for EGFP expression for all of the samples are (from left to right): 100 ± 0%, 93.9 ± 6.6%, 27.3 ± 6.1%, 22.8 ± 5.1%, 108.1 ± 18.8%, 17.7 ± 4.4%, 27.5 ± 8.3%, 3.1 ± 2.1%. Values for mRNA level of all samples are (from left to right): 100 ± 0%, 107.4 ± 1.7%, 18.5 ± 6.4%, 16.6 ± 5.3%, 110.0 ± 19.2%, 107.3 ± 15.3%, 28.9 ± 6.2%, 8.1 ± 2.6%.

FIGURE 3.

FIGURE 3.

Translational elongation mediated by Hygromycin does not protect transcripts from RNAi mediated degradation. (A) Three hours after U1_GFP was transfected into HEK293 cells, Hygromycin (10 nM final concentration) was added to the medium with or without siRNA against EGFP. EGFP expression was monitored 21 h later. (B) EGFP mRNA levels were analyzed by RT-PCR as described in the legend to Figure 2.

FIGURE 4.

FIGURE 4.

RNAs containing IRE duplications at the 5′ and 3′ end are still susceptible to siRNA triggered RNAi. (A) Schematic representation of the Double IRE construct and how it would be protected when the iron concentration is low. (B) Three hours after the U1_Double_IRE_GFP was transfected into HEK293 cells, Hemin (iron source labeled as H) or deferoxaminemesylate salts (iron chelator labeled as D) were added to the medium with or without siRNA against EGFP. EGFP expression was monitored 24 h post-transfection. (C) EGFP mRNA levels are analyzed by RT-PCR as described in the legend to Figure 2.

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