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. 2008 Nov;36(20):6511-22.
doi: 10.1093/nar/gkn687. Epub 2008 Oct 15.

A role for the Dicer helicase domain in the processing of thermodynamically unstable hairpin RNAs

Harris S Soifer  1 , Masayuki Sano, Kumi Sakurai, Pritsana Chomchan, Pål Saetrom, Mark A Sherman, Michael A Collingwood, Mark A Behlke, John J Rossi
Affiliations

A role for the Dicer helicase domain in the processing of thermodynamically unstable hairpin RNAs

Harris S Soifer et al. Nucleic Acids Res. 2008 Nov.

Abstract

In humans a single species of the RNAseIII enzyme Dicer processes both microRNA precursors into miRNAs and long double-stranded RNAs into small interfering RNAs (siRNAs). An interesting but poorly understood domain of the mammalian Dicer protein is the N-terminal helicase-like domain that possesses a signature DExH motif. Cummins et al. created a human Dicer mutant cell line by inserting an AAV targeting cassette into the helicase domain of both Dicer alleles in HCT116 cells generating an in-frame 43-amino-acid insertion immediately adjacent to the DExH box. This insertion creates a Dicer mutant protein with defects in the processing of most, but not all, endogenous pre-miRNAs into mature miRNA. Using both biochemical and computational approaches, we provide evidence that the Dicer helicase mutant is sensitive to the thermodynamic properties of the stems in microRNAs and short-hairpin RNAs, with thermodynamically unstable stems resulting in poor processing and a reduction in the levels of functional mi/siRNAs. Paradoxically, this mutant exhibits enhanced processing efficiency and concomitant RNA interference when thermodynamically stable, long-hairpin RNAs are used. These results suggest an important function for the Dicer helicase domain in the processing of thermodynamically unstable hairpin structures.

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Figures

Figure 1.
Figure 1.
Processing and efficacy of microRNAs in vivo. (A) Northern blot analyses of miRNA expression in wild-type and mHelicase cells. For miR-21, small RNAs were harvested from wild-type and Dicer mHelicase cells. The unprocessed pre-miR-21 (arrowhead) and mature miR-21 species (arrow) are indicated. The asterisk denotes a nonspecific signal that runs slightly >70 nt, which likely represents cross hybridization of the 2′-O-methyl-modified oligonucleotide to an abundant tRNA, serves as an internal loading control. The illustrated size marker is based on the migration of the Ambion Decade ladder. For miR-23a and miR-27a, total RNA was harvested from wild-type and mHelicase cells transfected with either the control U6 vector (Control) or vectors expressing U6-driven shRNA (SII-c21) or lhRNA (SII-c50). Arrows denote the migration of the mature miR-23a and miR-27a. For miR-27a, the ∼63 nt pre-miR-27a is indicated by the arrowhead. The illustrated size marker is based on the migration of the Ambion Decade ladder. U6 snRNA expression was also detected as a loading standard. (B) Inhibition of miRNA gene expression by an ectopic miRNA expression construct. Twenty-five nanograms of the psiCHECK-miR-93 reporter vector and 100 ng of the MCM7 miRNA expression vector were co-transfected with an empty vector or a vector expressing the wild-type Dicer cDNA into wild-type or Dicer mHelicase cells. Luciferase values were determined 24 h later and the Renilla target was normalized to Firefly luciferase, which is contained on the same reporter construct but is not subject to downregulation by the miRNA. Luciferase values in response to the irrelevant HIV miRNA expression vector (HIV) were set at 100%. The mean and standard deviations from three experiments are presented. *P < 0.02, **P < 0.05.
Figure 2.
Figure 2.
Expression and efficacy of shRNA or lhRNAs in vivo. (A) Schematic representation of short and long hairpin constructs. Predicted structures of shRNA and lhRNAs, which target the HIV-1 rev exon (site II or SII) and the relative positioning of the oligonucleotide probes for detection of processed siRNAs are shown. The bases shown in the top strand indicate altered nucleotides in the passenger stand of the duplex, which create G:U wobble base parings within the sh- or lhRNA but do not affect the perfect complementarity of the guide strand with its target. (B) Northern analyses of siRNA production in wild-type or mHelicase cells transfected with each hairpin RNA expression vector. Total RNA extracted from cells transfected with the empty vector was used as a negative control for cross-hybridization of each probe. The mature siRNA is indicated with the arrowhead and the respective precursor molecules are indicated by the vertical bars. The membrane was subsequently stripped and probed for miR-21. U6 snRNA expression was also detected as a loading standard. The same membrane was also stripped and probed for siRNAs using Probe 2 and Probe 3. The illustrated size marker is based on the migration of the Ambion Decade ladder. (C) The processing efficiency for each hairpin RNA (Probe 1, Probe 2, Probe 3) was determined by autoradiography and dosimetry as follows: processing efficiency = matureIDV/(matureIDV + precursorIDV). IDV, Integrated Density Value. The mean and standard deviation of three experiments is represented.
Figure 3.
Figure 3.
In vitro processing of hairpin RNA substrates. (A) In vitro processing assays were performed using cell extracts from wild-type or Dicer mHelicase HCT116 cells. Cell extracts (25 μg each) from either wild-type or mHelicase cells were incubated with 5′-end-labeled synthetic pre-miR-21 at 37°C for 1 h, run on a 15% denaturing polyacrylamide gel and visualized by autoradiography. The Ambion Decade marker and mature miR-21 (arrowhead) are illustrated. (B) Uniformly radiolabeled RNAs corresponding to the SII-21 shRNA and SII-50 lhRNA were incubated with either the wild-type (25 μg) or mHelicase (25 μg) cell extract at 37°C for 2 h and then electrophoresed in a 10% denaturing polyacrylamide gel and visualized by phosphorimager analysis. The control lane represents substrate RNA that was incubated with reaction buffer but no cell extract. The Ambion Decade marker is illustrated and the arrowheads mark the migration of the processed siRNAs. In the SII-50 gel, lanes 3–6 marked ‘Irrelevant samples’ were performed for a different experiment and are not included in our analysis. The blank lane has no RNA. (C) The processing efficiency for each radiolabeled substrate RNA was determined by phosphorimager analysis as follows: processing efficiency = matureIDV/(matureIDV + precursorIDV). IDV, Integrated Density Value. The mean and standard deviation of three experiments is represented.
Figure 4.
Figure 4.
Expression and efficacy of perfectly complementary shRNA and lhRNAs. (A) Northern analyses of siRNA production in wild-type or mHelicase cells following transfection with perfectly complementary hairpin RNA constructs. Total RNA extracted from cells transfected with the empty vector was used as a negative control for cross-hybridization of each probe. The mature siRNA is indicated with the arrowhead and the precursor molecules are indicated by the vertical bars. The membrane was subsequently stripped and probed for miR-21. U6 snRNA expression was also detected as a loading standard. The same membrane was also stripped and probed for siRNA using Probe 2. The illustrated size marker is based on the migration of the Ambion Decade ladder. (B, C) The processing efficiency for each siRNA [Probe 1 (B) and Probe 2 (C)] was determined by autoradiography and dosimetry as follows: processing efficiency = matureIDV/(matureIDV + precursorIDV) IDV, Integrated Density Value. The mean and standard deviation of three experiments is represented. (D) Inhibition of reporter gene expression by the shRNA or lhRNA constructs. The sequence corresponding to either target 1 or 2 was inserted into the cloning site in the 3′ UTR of the Renilla luciferase gene in the psiCHECK-2 plasmid. One hundred nanograms of each reporter construct and 1 ng of either SII-c21 or SII-50 hairpin RNA expression vector were co-transfected into wild-type or mHelicase cells. Luciferase values were determined 24 h later and the Renilla target was normalized to Firefly luciferase, which is contained on the same reporter construct but is not subject to downregulation by the shRNA. Luciferase values in response to the empty vector (control) were set at 100%. P-values compare mHelicase versus wild type: target 1: SII-c21:P < 0.6; SII-c50:P < 0.001; target 2: SII-c50: P < 0.001.
Figure 5.
Figure 5.
Structural properties of short hairpin precursors affect their processing in Dicer mHelicase cells. (A) Northern blot detection of siRNAs derived from various shRNAs in the wild type or mHelicase cells. Northern blotting analyses were performed as described in Figure 3. The mature siRNA is indicated with the arrowhead and the precursor molecules are indicated by the vertical bar. The membrane was subsequently stripped and probed for miR-21. U6 snRNA expression was also detected as a loading standard. The illustrated size marker is based on the migration of the Ambion Decade ladder. (B) The processing efficiency for each shRNA was determined by autoradiography and dosimetry as follows: processing efficiency = matureIDV/(matureIDV + precursorIDV). IDV, Integrated Density Value. The mean and standard deviation of three experiments is represented.
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