Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 May;19(5):639-48.
doi: 10.1261/rna.037424.112. Epub 2013 Mar 26.

Multiple sensors ensure guide strand selection in human RNAi pathways

Cameron L Noland  1 , Jennifer A Doudna
Affiliations

Multiple sensors ensure guide strand selection in human RNAi pathways

Cameron L Noland et al. RNA. 2013 May.

Abstract

Small RNAs guide RNA-induced silencing complexes (RISCs) to bind to cognate mRNA transcripts and trigger silencing of protein expression during RNA interference (RNAi) in eukaryotes. A fundamental aspect of this process is the asymmetric loading of one strand of a short interfering RNA (siRNA) or microRNA (miRNA) duplex onto RISCs for correct target recognition. Here, we use a reconstituted system to determine the extent to which the core components of the human RNAi machinery contribute to RNA guide strand selection. We show that Argonaute2 (Ago2), the endonuclease that binds directly to siRNAs and miRNAs within RISC, has intrinsic but substrate-dependent RNA strand selection capability. This activity can be enhanced substantially when Ago2 is in complex with the endonuclease Dicer and the double-stranded RNA-binding proteins (dsRBPs)-trans-activation response (TAR) RNA-binding protein (TRBP) or protein activator of PKR (PACT). The extent to which human Dicer/dsRBP complexes contribute to strand selection is dictated by specific duplex parameters such as thermodynamics, 5' nucleotide identity, and structure. Surprisingly, our results also suggest that strand selection for some miRNAs is enhanced by PACT-containing complexes but not by those containing TRBP. Furthermore, overall mRNA targeting by miRNAs is disfavored for complexes containing TRBP but not PACT. These findings demonstrate that multiple proteins collaborate to ensure optimal strand selection in humans and reveal the possibility of delineating RNAi pathways based on the presence of TRBP or PACT.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Human Ago2 efficiently cleaves target RNAs in vitro using duplex siRNAs and miRNAs. (A) Coomassie Blue-stained SDS-PAGE gel of recombinantly purified Ago2. (B) Sequences and thermodynamic analysis of siRNA-1 and miRNA-1. Thermodynamics were calculated for the four terminal base pairs and 2-nt 3′ overhangs at each end of the duplex. (C) Recombinant human Ago2 is active for target cleavage using either a duplex siRNA (siRNA-1, left panel) or duplex miRNA (miRNA-1, right panel) as a guide. Neither DMSO nor the Hsp90 inhibitor 17-AAG inhibit the activity of human Ago2, whereas EDTA does inhibit activity. (D) siRNA-1 and miRNA-1 remain double-stranded at 37°C, as demonstrated by 15% native PAGE analysis.
FIGURE 2.
FIGURE 2.
Dicer and dsRBPs are required for efficient strand selection with some siRNAs. (A) Superose 6 elution profile for reconstituted Ago2/Dicer/PACT. Peak 1 contains the full complex and Peak 2 contains free Ago2 and free PACT. (B) Superose 6 elution profile for reconstituted Ago2/Dicer/TRBP. Peak 1 contains the full complex and Peak 2 contains free Ago2 and free TRBP. (C) Coomassie Blue-stained SDS-PAGE gel showing purified Ago2/Dicer/PACT and Ago2/Dicer/TRBP. (D) Ago2 alone exhibits minimal levels of strand selection when loaded with siRNA-1. Complexes containing Dicer and either TRBP or PACT enhance strand selection. Lanes labeled “G” contain a target corresponding to the guide strand, and lanes labeled “P” contain a target that is complementary to the passenger strand. (E) Quantification of the ratio of target cleavage mediated by the guide strand compared to that mediated by the passenger strand. Data shown are means (±SD) from three separate experimental replicates.
FIGURE 3.
FIGURE 3.
Ago2/Dicer/dsRBP complexes are sensitive to changes in duplex thermodynamics. (A) Sequence and thermodynamic analysis of siRNA-2. Thermodynamics were calculated for the four terminal base pairs and 2-nt 3′ overhangs at each end of the duplex. (B) Ago2 alone exhibits minimal levels of strand selection when loaded with siRNA-2. Complexes containing Dicer and either TRBP or PACT enhance strand selection. Lanes labeled “G” contain a target corresponding to the guide strand, and lanes labeled “P” contain a target that is complementary to the passenger strand. (C) Quantification of the ratio of target cleavage mediated by the guide strand compared to that mediated by the passenger strand. Data shown are means (±SD) from three separate experimental replicates.
FIGURE 4.
FIGURE 4.
5′ nucleotide identity affects strand selection by Ago2. (A) Sequence and thermodynamic analysis of siRNA-3. Thermodynamics were calculated for the four terminal base pairs and 2-nt 3′ overhangs at each end of the duplex. (B) Ago2 alone cleaves target RNAs strand-selectively using siRNA-3. This level of strand selection is slightly attenuated by complexes containing Dicer and either TRBP or PACT. Lanes labeled “G” contain a target corresponding to the guide strand, and lanes labeled “P” contain a target that is complementary to the passenger strand. (C) Quantification of the ratio of target cleavage mediated by the guide strand compared to that mediated by the passenger strand. Data shown are means (±SD) from three separate experimental replicates.
FIGURE 5.
FIGURE 5.
RNA duplex terminal base-pairing affects strand selection by Ago2. (A) Sequence and thermodynamic analysis of siRNA-4. Thermodynamics were calculated for the four terminal base pairs and 2-nt 3′ overhangs at each end of the duplex. (B) Ago2 alone cleaves targets strand-selectively using siRNA-4. Complexes containing Dicer and either TRBP or PACT do not enhance this strand selection. Lanes labeled “G” contain a target corresponding to the guide strand, and lanes labeled “P” contain a target that is complementary to the passenger strand. (C) Quantification of the ratio of target cleavage mediated by the guide strand compared to that mediated by the passenger strand. Data shown are means (±SD) from three separate experimental replicates.
FIGURE 6.
FIGURE 6.
Complexes containing PACT but not TRBP enhance miRNA strand selection. (A) Sequence and thermodynamic analysis of miRNA-1, miRNA-2, and miRNA-3. Thermodynamics were calculated for the four terminal base pairs and 2-nt 3′ overhangs at each end of the duplex. (B) Ago2 alone cleaves target RNAs strand-selectively using miRNA-1. This strand selection is not enhanced by the presence of Dicer and TRBP but is strongly enhanced by the presence of Dicer and PACT. Lanes labeled “G” contain a target corresponding to the guide strand, and lanes labeled “P” contain a target that is complementary to the passenger strand. (C) Quantification of the ratio of target cleavage mediated by the guide strand compared to that mediated by the passenger strand. Data shown are means (±SD) from three separate experimental replicates. (D) Ago2 alone exhibits low levels of strand-selectivity using miRNA-2. Complexes containing Dicer and either TRBP or PACT enhance this strand-selectivity. (E) Quantification of the ratio of target cleavage mediated by the guide strand compared to that mediated by the passenger strand. Data shown are means (±SD) from three separate experimental replicates. (F) Ago2 alone exhibits strong strand-selectivity using miRNA-3. This strand selection is not enhanced by the presence of Dicer and TRBP but is strongly enhanced by the presence of Dicer and PACT. (G) Quantification of the ratio of target cleavage mediated by the guide strand compared to that mediated by the passenger strand. Data shown are means (±SD) from three separate experimental replicates.
FIGURE 7.
FIGURE 7.
Several duplex parameters dictate the relative contributions of human RNAi components to guide strand selection. Left: Only Ago2/Dicer/dsRBP complexes are able to efficiently direct strand selection with perfectly matched siRNAs containing identical 5′ nucleotides on both strands or a disfavored 5′ nucleotide on the guide strand. Center: Ago2 alone as well as Dicer/dsRBP-containing complexes are capable of strand selection with duplex RNAs harboring a disfavored 5′ nucleotide on the passenger strand, terminal mismatches, or central mismatches. In certain cases (as with siRNA-3), strand selection by Ago2 may be enhanced above that of Dicer/dsRBP-containing complexes. Right: Ago2 and an Ago2/Dicer/TRBP complexes exhibit robust strand selection with duplex RNAs containing seed mismatches or combined seed and central mismatches, although overall activity is diminished with a TRBP-containing complex (denoted by gray arrow). Strand selection with these RNAs is enhanced by Ago2/Dicer/PACT complexes (denoted by large arrow). Red strand denotes the guide strand. Green highlights the less stable duplex ends.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bernstein E, Caudy AA, Hammond SM, Hannon GJ 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409: 363–366 - PubMed
    1. Betancur JG, Tomari Y 2012. Dicer is dispensable for asymmetric RISC loading in mammals. RNA 18: 24–30 - PMC - PubMed
    1. Borer PN, Dengler B, Tinoco I, Uhlenbeck OC 1974. Stability of ribonucleic acid double-stranded helices. J Mol Biol 86: 843–853 - PubMed
    1. Chakravarthy S, Sternberg SH, Kellenberger CA, Doudna JA 2010. Substrate-specific kinetics of Dicer-catalyzed RNA processing. J Mol Biol 404: 392–402 - PMC - PubMed
    1. Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K, Shiekhattar R 2005. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436: 740–744 - PMC - PubMed

Publication types

MeSH terms