Functional polarity is introduced by Dicer processing of short substrate RNAs - PubMed (original) (raw)

Functional polarity is introduced by Dicer processing of short substrate RNAs

Scott D Rose et al. Nucleic Acids Res. 2005.

Abstract

Synthetic RNA duplexes that are substrates for Dicer are potent triggers of RNA interference (RNAi). Blunt 27mer duplexes can be up to 100-fold more potent than traditional 21mer duplexes. Not all 27mer duplexes show increased potency. Evaluation of the products of in vitro dicing reactions using electrospray ionization mass spectrometry reveals that a variety of products can be produced by Dicer cleavage. Use of asymmetric duplexes having a single 2-base 3'-overhang restricts the heterogeneity that results from dicing. Inclusion of DNA residues at the ends of blunt duplexes also limits heterogeneity. Combination of asymmetric 2-base 3'-overhang with 3'-DNA residues on the blunt end result in a duplex form which directs dicing to predictably yield a single primary cleavage product. It is therefore possible to design a 27mer duplex which is processed by Dicer to yield a specific, desired 21mer species. Using this strategy, two different 27mers can be designed that result in the same 21mer after dicing, one where the 3'-overhang resides on the antisense (AS) strand and dicing proceeds to the 'right' ('R') and one where the 3'-overhang resides on the sense (S) strand and dicing proceeds to the 'left' ('L'). Interestingly, the 'R' version of the asymmetric 27mer is generally more potent in reducing target gene levels than the 'L' version 27mer. Strand targeting experiments show asymmetric strand utilization between the two different 27mer forms, with the 'R' form favoring S strand and the 'L' form favoring AS strand silencing. Thus, Dicer processing confers functional polarity within the RNAi pathway.

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Figures

Figure 1

Figure 1

ESI-MS analysis of in vitro dicing reactions. Mass spectra of duplexes are shown before (top) and after (middle) digestion with recombinant human Dicer. Sequence of the substrate RNA duplex is provided with detected cleavage products along with calculated molecular weight and strand length (bottom). RNA bases are upper case, DNA bases are lower case bold and ‘p’ represents 5′-phosphate. (A) Blunt duplex EGFPS2 R 27/27; (B) asymmetric duplex EGFPS2 R 25/27; (C) asymmetric duplex EGFPS2 R 27/25; (D) asymmetric duplex EGFPS2 R 27/25(3′D); (E) asymmetric duplex EGFPS2 R 25D/27; (F) asymmetric duplex EGFPS2 L 27/25D; (G) asymmetric duplex EGFPS1 R 25D/27; (H) asymmetric duplex EGFPS1 L 27/25D.

Figure 1

Figure 1

ESI-MS analysis of in vitro dicing reactions. Mass spectra of duplexes are shown before (top) and after (middle) digestion with recombinant human Dicer. Sequence of the substrate RNA duplex is provided with detected cleavage products along with calculated molecular weight and strand length (bottom). RNA bases are upper case, DNA bases are lower case bold and ‘p’ represents 5′-phosphate. (A) Blunt duplex EGFPS2 R 27/27; (B) asymmetric duplex EGFPS2 R 25/27; (C) asymmetric duplex EGFPS2 R 27/25; (D) asymmetric duplex EGFPS2 R 27/25(3′D); (E) asymmetric duplex EGFPS2 R 25D/27; (F) asymmetric duplex EGFPS2 L 27/25D; (G) asymmetric duplex EGFPS1 R 25D/27; (H) asymmetric duplex EGFPS1 L 27/25D.

Figure 1

Figure 1

ESI-MS analysis of in vitro dicing reactions. Mass spectra of duplexes are shown before (top) and after (middle) digestion with recombinant human Dicer. Sequence of the substrate RNA duplex is provided with detected cleavage products along with calculated molecular weight and strand length (bottom). RNA bases are upper case, DNA bases are lower case bold and ‘p’ represents 5′-phosphate. (A) Blunt duplex EGFPS2 R 27/27; (B) asymmetric duplex EGFPS2 R 25/27; (C) asymmetric duplex EGFPS2 R 27/25; (D) asymmetric duplex EGFPS2 R 27/25(3′D); (E) asymmetric duplex EGFPS2 R 25D/27; (F) asymmetric duplex EGFPS2 L 27/25D; (G) asymmetric duplex EGFPS1 R 25D/27; (H) asymmetric duplex EGFPS1 L 27/25D.

Figure 1

Figure 1

ESI-MS analysis of in vitro dicing reactions. Mass spectra of duplexes are shown before (top) and after (middle) digestion with recombinant human Dicer. Sequence of the substrate RNA duplex is provided with detected cleavage products along with calculated molecular weight and strand length (bottom). RNA bases are upper case, DNA bases are lower case bold and ‘p’ represents 5′-phosphate. (A) Blunt duplex EGFPS2 R 27/27; (B) asymmetric duplex EGFPS2 R 25/27; (C) asymmetric duplex EGFPS2 R 27/25; (D) asymmetric duplex EGFPS2 R 27/25(3′D); (E) asymmetric duplex EGFPS2 R 25D/27; (F) asymmetric duplex EGFPS2 L 27/25D; (G) asymmetric duplex EGFPS1 R 25D/27; (H) asymmetric duplex EGFPS1 L 27/25D.

Figure 2

Figure 2

‘R’ form duplexes are more potent than ‘L’ form duplexes. Top: relative expression data are shown. Bottom: target sequences (S strand) are shown and transfected RNAi duplexes are aligned beneath in duplex form with sense strand top (5′→3′) and antisense strand bottom (3′→5′). RNA bases are upper case, DNA bases are lower case bold, and ‘p’ represents 5′-phosphate. (A) Relative EGFP fluorescence was measured following co-transfection of an EGFP expression plasmid and EGFPS2 RNA duplexes; (B) relative EGFP fluorescence was measured following co-transfection of an EGFP expression plasmid and EGFPS1 RNA duplexes; (C) relative EGFP fluorescence was measured following co-transfection of an EGFP-hnRNPH fusion vector and hnRNPH RNA duplexes; (D) relative light emission from firefly luciferase was measured following co-transfection of a luciferase expression vector and luciferase RNA duplexes; (E) RNA duplexes specific for La antigen mRNA were transfected into cells at 2.5 nM concentration and protein extracts were prepared 72 h post-transfection. Western blots were done using anti-La antibodies and anti-enolase (control) antibodies. RNA duplexes were transfected into cells at 1, 2.5 and 10 nM concentrations, and RNA was prepared 24 h post-transfection. Quantitative real-time RT–PCR was used to measure relative La mRNA levels, using RPLP0 as an internal normalization standard.

Figure 2

Figure 2

‘R’ form duplexes are more potent than ‘L’ form duplexes. Top: relative expression data are shown. Bottom: target sequences (S strand) are shown and transfected RNAi duplexes are aligned beneath in duplex form with sense strand top (5′→3′) and antisense strand bottom (3′→5′). RNA bases are upper case, DNA bases are lower case bold, and ‘p’ represents 5′-phosphate. (A) Relative EGFP fluorescence was measured following co-transfection of an EGFP expression plasmid and EGFPS2 RNA duplexes; (B) relative EGFP fluorescence was measured following co-transfection of an EGFP expression plasmid and EGFPS1 RNA duplexes; (C) relative EGFP fluorescence was measured following co-transfection of an EGFP-hnRNPH fusion vector and hnRNPH RNA duplexes; (D) relative light emission from firefly luciferase was measured following co-transfection of a luciferase expression vector and luciferase RNA duplexes; (E) RNA duplexes specific for La antigen mRNA were transfected into cells at 2.5 nM concentration and protein extracts were prepared 72 h post-transfection. Western blots were done using anti-La antibodies and anti-enolase (control) antibodies. RNA duplexes were transfected into cells at 1, 2.5 and 10 nM concentrations, and RNA was prepared 24 h post-transfection. Quantitative real-time RT–PCR was used to measure relative La mRNA levels, using RPLP0 as an internal normalization standard.

Figure 3

Figure 3

Strand bias is introduced by the direction of Dicer processing. Reporter constructs were made in the psiCheck-2™ vector that contained a fragment of EGFP or hnRNPH coding sequence cloned into the 3′-UTR of the Renilla luciferase gene in both ‘S’ and ‘AS’ orientations. Reporter plasmids were co-transfected into cells without (control) or with the indicated RNA duplexes and luciferase activity was measured 24 h post-transfection. Results are reported as the ratio of Renilla luciferase (target) to firefly luciferase (internal control); (A) EGFP strand targeting; (B) hnRNPH strand targeting. Schematic diagrams of the four synthetic reporter constructs employed with orientation of target sequences relative to Renilla luciferase are shown.

Figure 4

Figure 4

Structure–activity features of Dicer–substrate RNA duplexes. (A) Comparison of activity of ‘R’ form 27mer duplexes with RNA versus DNA bases in the single 2-base 3′-overhang. (B) Comparison of activity of ‘R’ form 27mer duplexes with 1, 2, 3 or 4-base RNA 3′-overhangs. (C) Comparison of activity of ‘R’ form 27mer duplexes with every possible RNA 2-base 3′-overhang. Top: For A and B, an EGFP-hnRNPH fusion protein expression plasmid was transfected into cells without (control) or with the indicated RNA duplexes and EGFP fluorescence was measured at 48 h post-transfection. For C, a luciferase expression vector containing hnRNPH sequence in the 3′-UTR was transfected into cells without (control) or with the indicated RNA duplexes and luciferase activity was assayed 48 h post-transfection. Bottom: hnRNPH target sequence (S strand) is shown and duplexes employed in transfection are aligned beneath in duplex form with sense strand top (5′→3′) and antisense strand bottom (3′→5′). RNA bases are upper case, DNA bases are lower case bold, and ‘p’ represents 5′-phosphate. Two neighboring sites were targeted, H-1 and H-3 shifted by two bases along the hnRNPH sequence.

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