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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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Effect of reducing GW182 paralogs alone or in combination on allele-selective inhibition of HTT expression by duplex RNA P9 in GM04281 fibroblast cells. (A) Inhibiting expression of all three GW182/TNRC6A paralogs reverses allele-selective inhibition. When si6A, si6B and si6C were added together, each was present at 16.6 nM. (B) Inhibiting combinations of any two GW182/TNRC6A paralogs does not reverse allele-selective inhibition. When two siRNAs were used together, their total concentration was 25 nM. (C) Inhibiting expression of all three GW182/TNRC6A paralogs alone or in combination does not block silencing by siRNA siHdh1. When three siRNAs were used together, their total concentration was 25 nM. (D) RIP showing recruitment of GW182 to HTT mRNA after addition of duplex RNA P9 or non-complementary control CM (50 nM). A or si6A: siRNA targeted TNRC6A. B or si6B: siRNA targeted TNRC6B. C or si6C: siRNA targeted TNRC6C.
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gks907-F4: Effect of reducing GW182 paralogs alone or in combination on allele-selective inhibition of HTT expression by duplex RNA P9 in GM04281 fibroblast cells. (A) Inhibiting expression of all three GW182/TNRC6A paralogs reverses allele-selective inhibition. When si6A, si6B and si6C were added together, each was present at 16.6 nM. (B) Inhibiting combinations of any two GW182/TNRC6A paralogs does not reverse allele-selective inhibition. When two siRNAs were used together, their total concentration was 25 nM. (C) Inhibiting expression of all three GW182/TNRC6A paralogs alone or in combination does not block silencing by siRNA siHdh1. When three siRNAs were used together, their total concentration was 25 nM. (D) RIP showing recruitment of GW182 to HTT mRNA after addition of duplex RNA P9 or non-complementary control CM (50 nM). A or si6A: siRNA targeted TNRC6A. B or si6B: siRNA targeted TNRC6B. C or si6C: siRNA targeted TNRC6C.

Mentions: To determine the role of GW182 family proteins in allele-selective inhibition of mutant human HTT, we inhibited expression of TNRC6A, TNRC6B or TNRC6C individually using siRNAs that target their mRNAs (Supplementary Figure S4A and B). Individual reduction of the GW182 paralogs had no effect on inhibition of HTT (Figure 4A). When we simultaneously inhibited all three paralogs, allele-selective inhibition of mutant HTT was reversed, suggesting that GW182 expression was important but that the paralogs had overlapping functions. Subsequent attempts to inhibit any two paralogs in combination also failed to reverse allele-selective inhibition (Figure 4B and Supplementary Figure S4C). While inhibition of all three TNRC6 paralogs blocked allele-selective inhibition of HTT expression, blocking expression of all three paralogs had no effect on non-allele-selective inhibition by a duplex RNA siHdh1 complementary to an mRNA sequence outside of the CAG repeat (Figure 4C and Supplementary Figure S4D). RIP using an anti-TNRC6A antibody demonstrated that addition of anti-CAG duplex RNA caused recruitment of TNRC6A to the HTT mRNA (Figure 4D).Figure 4.


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)

Effect of reducing GW182 paralogs alone or in combination on allele-selective inhibition of HTT expression by duplex RNA P9 in GM04281 fibroblast cells. (A) Inhibiting expression of all three GW182/TNRC6A paralogs reverses allele-selective inhibition. When si6A, si6B and si6C were added together, each was present at 16.6 nM. (B) Inhibiting combinations of any two GW182/TNRC6A paralogs does not reverse allele-selective inhibition. When two siRNAs were used together, their total concentration was 25 nM. (C) Inhibiting expression of all three GW182/TNRC6A paralogs alone or in combination does not block silencing by siRNA siHdh1. When three siRNAs were used together, their total concentration was 25 nM. (D) RIP showing recruitment of GW182 to HTT mRNA after addition of duplex RNA P9 or non-complementary control CM (50 nM). A or si6A: siRNA targeted TNRC6A. B or si6B: siRNA targeted TNRC6B. C or si6C: siRNA targeted TNRC6C.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks907-F4: Effect of reducing GW182 paralogs alone or in combination on allele-selective inhibition of HTT expression by duplex RNA P9 in GM04281 fibroblast cells. (A) Inhibiting expression of all three GW182/TNRC6A paralogs reverses allele-selective inhibition. When si6A, si6B and si6C were added together, each was present at 16.6 nM. (B) Inhibiting combinations of any two GW182/TNRC6A paralogs does not reverse allele-selective inhibition. When two siRNAs were used together, their total concentration was 25 nM. (C) Inhibiting expression of all three GW182/TNRC6A paralogs alone or in combination does not block silencing by siRNA siHdh1. When three siRNAs were used together, their total concentration was 25 nM. (D) RIP showing recruitment of GW182 to HTT mRNA after addition of duplex RNA P9 or non-complementary control CM (50 nM). A or si6A: siRNA targeted TNRC6A. B or si6B: siRNA targeted TNRC6B. C or si6C: siRNA targeted TNRC6C.
Mentions: To determine the role of GW182 family proteins in allele-selective inhibition of mutant human HTT, we inhibited expression of TNRC6A, TNRC6B or TNRC6C individually using siRNAs that target their mRNAs (Supplementary Figure S4A and B). Individual reduction of the GW182 paralogs had no effect on inhibition of HTT (Figure 4A). When we simultaneously inhibited all three paralogs, allele-selective inhibition of mutant HTT was reversed, suggesting that GW182 expression was important but that the paralogs had overlapping functions. Subsequent attempts to inhibit any two paralogs in combination also failed to reverse allele-selective inhibition (Figure 4B and Supplementary Figure S4C). While inhibition of all three TNRC6 paralogs blocked allele-selective inhibition of HTT expression, blocking expression of all three paralogs had no effect on non-allele-selective inhibition by a duplex RNA siHdh1 complementary to an mRNA sequence outside of the CAG repeat (Figure 4C and Supplementary Figure S4D). RIP using an anti-TNRC6A antibody demonstrated that addition of anti-CAG duplex RNA caused recruitment of TNRC6A to the HTT mRNA (Figure 4D).Figure 4.

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