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Mechanism of allele-selective inhibition of huntingtin expression by duplex RNAs that target CAG repeats: function through the RNAi pathway.

Hu J, Liu J, Yu D, Chu Y, Corey DR - Nucleic Acids Res. (2012)

Bottom Line: In contrast, inhibiting the expression of mutant HTT protein is highly sensitive to reduced expression of GW182 (TNRC6A) and its two paralogs, a protein family associated with miRNA action.Allele-selective inhibition may involve cooperative binding of multiple protein-RNA complexes to the expanded repeat.These data suggest that allele-selective inhibition proceeds through an RNA interference pathway similar to that used by miRNAs and that discrimination between mutant and wild-type alleles of HTT mRNA is highly sensitive to the pool of AGO2 and GW182 family proteins inside cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pharmacology and Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX 75390-9041, USA.

ABSTRACT
Huntington's disease is an incurable neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat within one allele of the huntingtin (HTT) gene. Agents that block expression of mutant HTT and preserve expression of wild-type HTT target the cause of the disease and are an alternative for therapy. We have previously demonstrated that mismatch-containing duplex RNAs complementary to the expanded trinucleotide repeat are potent and allele-selective inhibitors of mutant HTT expression, but the mechanism of allele selectivity was not explored. We now report that anti-CAG duplex RNA preferentially recruits argonaute 2 (AGO2) to mutant rather than wild-type HTT mRNA. Efficient inhibition of mutant HTT protein expression requires less AGO2 than needed for inhibiting wild-type expression. In contrast, inhibiting the expression of mutant HTT protein is highly sensitive to reduced expression of GW182 (TNRC6A) and its two paralogs, a protein family associated with miRNA action. Allele-selective inhibition may involve cooperative binding of multiple protein-RNA complexes to the expanded repeat. These data suggest that allele-selective inhibition proceeds through an RNA interference pathway similar to that used by miRNAs and that discrimination between mutant and wild-type alleles of HTT mRNA is highly sensitive to the pool of AGO2 and GW182 family proteins inside cells.

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Reducing AGO2 expression converts duplex RNA REP and other previously non-allele-selective duplexes into allele-selective silencing agents. Experiments were performed in GM04281 fibroblasts. In an initial transfection levels of cellular AGO2 were reduced by transfection with 50 nM siAGO2 designed to target AGO2 mRNA. For comparison transfection was also done with 50 nM non-complementary duplex CM. (A) Sequences of duplex RNAs. (B) Effect of adding a non-complementary RNA duplex (CM, 50 nM) during the initial transfection on silencing by duplex RNA REP. (C) Effect of reducing AGO2 using an anti-AGO2 duplex RNA (50 nM) of duplex RNA REP. (D) Effect of adding a non-complementary RNA duplex (CM) during the initial transfection on silencing by several different non-allele-selective RNAs (12 nM). (E) Effect of reducing AGO2 expression, using anti-siAGO2 duplex RNA (50 nM), on the action of previously non-allele-selective RNAs.
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gks907-F3: Reducing AGO2 expression converts duplex RNA REP and other previously non-allele-selective duplexes into allele-selective silencing agents. Experiments were performed in GM04281 fibroblasts. In an initial transfection levels of cellular AGO2 were reduced by transfection with 50 nM siAGO2 designed to target AGO2 mRNA. For comparison transfection was also done with 50 nM non-complementary duplex CM. (A) Sequences of duplex RNAs. (B) Effect of adding a non-complementary RNA duplex (CM, 50 nM) during the initial transfection on silencing by duplex RNA REP. (C) Effect of reducing AGO2 using an anti-AGO2 duplex RNA (50 nM) of duplex RNA REP. (D) Effect of adding a non-complementary RNA duplex (CM) during the initial transfection on silencing by several different non-allele-selective RNAs (12 nM). (E) Effect of reducing AGO2 expression, using anti-siAGO2 duplex RNA (50 nM), on the action of previously non-allele-selective RNAs.

Mentions: We next examined the impact of reducing levels of AGO2 on inhibition of HTT expression by duplex RNA REP and other non-allele-selective duplex RNAs (Figure 3A). In contrast to the narrow window for achieving detectable effects of AGO2 depletion on allele-selective inhibition of mutant HTT, reduced expression of AGO2 over a wide range of concentrations caused REP to change from a non-allele-selective inhibitor (Figure 3B and Supplementary Figure S3A) to an efficient allele-selective agent (Figure 3C and Supplementary Figure S3A). We tested several other non-allele-selective inhibitors (Figure 3D and Supplementary Figure S3B) with complementarity to the CAG repeat and found that they all became selective when levels of AGO2 were reduced (Figure 3E and Supplementary Figure S3C).Figure 3.


Mechanism of allele-selective inhibition of huntingtin expression by duplex RNAs that target CAG repeats: function through the RNAi pathway.

Hu J, Liu J, Yu D, Chu Y, Corey DR - Nucleic Acids Res. (2012)

Reducing AGO2 expression converts duplex RNA REP and other previously non-allele-selective duplexes into allele-selective silencing agents. Experiments were performed in GM04281 fibroblasts. In an initial transfection levels of cellular AGO2 were reduced by transfection with 50 nM siAGO2 designed to target AGO2 mRNA. For comparison transfection was also done with 50 nM non-complementary duplex CM. (A) Sequences of duplex RNAs. (B) Effect of adding a non-complementary RNA duplex (CM, 50 nM) during the initial transfection on silencing by duplex RNA REP. (C) Effect of reducing AGO2 using an anti-AGO2 duplex RNA (50 nM) of duplex RNA REP. (D) Effect of adding a non-complementary RNA duplex (CM) during the initial transfection on silencing by several different non-allele-selective RNAs (12 nM). (E) Effect of reducing AGO2 expression, using anti-siAGO2 duplex RNA (50 nM), on the action of previously non-allele-selective RNAs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526262&req=5

gks907-F3: Reducing AGO2 expression converts duplex RNA REP and other previously non-allele-selective duplexes into allele-selective silencing agents. Experiments were performed in GM04281 fibroblasts. In an initial transfection levels of cellular AGO2 were reduced by transfection with 50 nM siAGO2 designed to target AGO2 mRNA. For comparison transfection was also done with 50 nM non-complementary duplex CM. (A) Sequences of duplex RNAs. (B) Effect of adding a non-complementary RNA duplex (CM, 50 nM) during the initial transfection on silencing by duplex RNA REP. (C) Effect of reducing AGO2 using an anti-AGO2 duplex RNA (50 nM) of duplex RNA REP. (D) Effect of adding a non-complementary RNA duplex (CM) during the initial transfection on silencing by several different non-allele-selective RNAs (12 nM). (E) Effect of reducing AGO2 expression, using anti-siAGO2 duplex RNA (50 nM), on the action of previously non-allele-selective RNAs.
Mentions: We next examined the impact of reducing levels of AGO2 on inhibition of HTT expression by duplex RNA REP and other non-allele-selective duplex RNAs (Figure 3A). In contrast to the narrow window for achieving detectable effects of AGO2 depletion on allele-selective inhibition of mutant HTT, reduced expression of AGO2 over a wide range of concentrations caused REP to change from a non-allele-selective inhibitor (Figure 3B and Supplementary Figure S3A) to an efficient allele-selective agent (Figure 3C and Supplementary Figure S3A). We tested several other non-allele-selective inhibitors (Figure 3D and Supplementary Figure S3B) with complementarity to the CAG repeat and found that they all became selective when levels of AGO2 were reduced (Figure 3E and Supplementary Figure S3C).Figure 3.

Bottom Line: In contrast, inhibiting the expression of mutant HTT protein is highly sensitive to reduced expression of GW182 (TNRC6A) and its two paralogs, a protein family associated with miRNA action.Allele-selective inhibition may involve cooperative binding of multiple protein-RNA complexes to the expanded repeat.These data suggest that allele-selective inhibition proceeds through an RNA interference pathway similar to that used by miRNAs and that discrimination between mutant and wild-type alleles of HTT mRNA is highly sensitive to the pool of AGO2 and GW182 family proteins inside cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pharmacology and Biochemistry, UT Southwestern Medical Center at Dallas, Dallas, TX 75390-9041, USA.

ABSTRACT
Huntington's disease is an incurable neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat within one allele of the huntingtin (HTT) gene. Agents that block expression of mutant HTT and preserve expression of wild-type HTT target the cause of the disease and are an alternative for therapy. We have previously demonstrated that mismatch-containing duplex RNAs complementary to the expanded trinucleotide repeat are potent and allele-selective inhibitors of mutant HTT expression, but the mechanism of allele selectivity was not explored. We now report that anti-CAG duplex RNA preferentially recruits argonaute 2 (AGO2) to mutant rather than wild-type HTT mRNA. Efficient inhibition of mutant HTT protein expression requires less AGO2 than needed for inhibiting wild-type expression. In contrast, inhibiting the expression of mutant HTT protein is highly sensitive to reduced expression of GW182 (TNRC6A) and its two paralogs, a protein family associated with miRNA action. Allele-selective inhibition may involve cooperative binding of multiple protein-RNA complexes to the expanded repeat. These data suggest that allele-selective inhibition proceeds through an RNA interference pathway similar to that used by miRNAs and that discrimination between mutant and wild-type alleles of HTT mRNA is highly sensitive to the pool of AGO2 and GW182 family proteins inside cells.

Show MeSH
Related in: MedlinePlus