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U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP.

Vickers TA, Sabripour M, Crooke ST - Nucleic Acids Res. (2011)

Bottom Line: U1 Adaptors are a recently reported novel approach for targeted reduction of mRNA transcripts.Here, we present data demonstrating U1 adapter-mediated gene silencing can result in significant 'off-target' silencing effects as demonstrated by the reduction of multiple mRNA species that were not intended to be targeted.Our data suggest that a substantial portion of this U1 adaptor-mediated off-target mRNA reduction is the result of sequestration U1 snRNP at levels sufficient to affect splicing and processing of non-target transcripts.

View Article: PubMed Central - PubMed

Affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Inc, 1896 Rutherford Road, Carlsbad, CA 92008, USA. tvickers@isisph.com

ABSTRACT
U1 Adaptors are a recently reported novel approach for targeted reduction of mRNA transcripts. A U1 adaptor oligonucleotide comprising of a target-complimentary hybridization domain and a U1 recruitment domain, directs the U1 snRNP complex to the terminal exon of a targeted gene, subsequently inhibiting poly(A) tail addition and leading to degradation of that RNA species within the nucleus. Here, we present data demonstrating U1 adapter-mediated gene silencing can result in significant 'off-target' silencing effects as demonstrated by the reduction of multiple mRNA species that were not intended to be targeted. Our data suggest that a substantial portion of this U1 adaptor-mediated off-target mRNA reduction is the result of sequestration U1 snRNP at levels sufficient to affect splicing and processing of non-target transcripts.

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Reduction of U1 snRNA with RNAse H ASOs. (a) Chimeric 2′MOE/DNA RNAse H-dependent ASOs were designed to target U1snRNA. The location of 3 RNAse H ASOs is underlined on the U1 snRNA sequence. 469508 (1–20), 469509 (59–78), 469511 (117–138). Northern probe location is highlighted on the U1 snRNA structure. (b) HeLa cells were treated with ASOs at the indicated concentrations (nM). The following day total RNA was purified and U1snRNA reduction analyzed by northern blot with the internal U1 probe (U1-1, upper panel), or with a second probe, U1-2, targeted to the U1 5′-end and the non-target control and U6 snRNA probe (lower panel). Band intensity was quantitated with a Storm 850 Phosphor-Imager and U1 snRNA normalized to U6 snRNA signal for each lane. (c) Effect of U1 adaptor treatment and U1 snRNA reduction on mRNA expression. HeLa cells seeded in 96-well plates were treated with U1A-RAF1, U1A-SMN2, U1A-PCSK9 or anti-U1 ASO 469508, in triplicate at concentrations ranging from 6 to 200 nM. The following day total RNA was purified and mRNA levels of each targeted gene assessed by qRT/PCR. Results are normalized to the total amount of RNA present in each reaction as determined by Ribogreen assay and plotted as percent control compared to mock-treated cells for three to four replicates with the standard error.
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Figure 3: Reduction of U1 snRNA with RNAse H ASOs. (a) Chimeric 2′MOE/DNA RNAse H-dependent ASOs were designed to target U1snRNA. The location of 3 RNAse H ASOs is underlined on the U1 snRNA sequence. 469508 (1–20), 469509 (59–78), 469511 (117–138). Northern probe location is highlighted on the U1 snRNA structure. (b) HeLa cells were treated with ASOs at the indicated concentrations (nM). The following day total RNA was purified and U1snRNA reduction analyzed by northern blot with the internal U1 probe (U1-1, upper panel), or with a second probe, U1-2, targeted to the U1 5′-end and the non-target control and U6 snRNA probe (lower panel). Band intensity was quantitated with a Storm 850 Phosphor-Imager and U1 snRNA normalized to U6 snRNA signal for each lane. (c) Effect of U1 adaptor treatment and U1 snRNA reduction on mRNA expression. HeLa cells seeded in 96-well plates were treated with U1A-RAF1, U1A-SMN2, U1A-PCSK9 or anti-U1 ASO 469508, in triplicate at concentrations ranging from 6 to 200 nM. The following day total RNA was purified and mRNA levels of each targeted gene assessed by qRT/PCR. Results are normalized to the total amount of RNA present in each reaction as determined by Ribogreen assay and plotted as percent control compared to mock-treated cells for three to four replicates with the standard error.

Mentions: To directly evaluate and compare the contribution of U1 snRNP reduction with U1 adaptor off-target activity, we sought to decrease the amount of functional U1 snRNP directly, using RNAse H ASOs designed with complementarity to U1 snRNA (Figure 3a). HeLa cells were transfected with the ASOs at concentrations ranging from 50 nM to 200 nM. The following day, total RNA was purified and U1snRNA reduction analyzed by northern blot. Blots hybridized with the internal U1 northern probe (U1-1) showed a significant shift in the migration of U1snRNA following treatment with 469508 consistent with cleavage of the 5′ U1 snRNA binding sequence, even at the lowest concentration evaluated (Figure 3a and b, upper panel). However, the lack of reduction in signal intensity suggests that there is little degradation of the U1 snRNA following RNaseH cleavage, likely due to the highly structured nature of the RNA target. To confirm this, the same blot was hybridized with a second Northern probe, U1-2, complimentary to the cleaved U1 snRNA 5′-end (Figure 3b, lower panel). As observed with probe U1-1, treatment with ASO 469508 resulted in a shift in mobility consistent with the expected U1 snRNA cleavage product. In addition, a clear reduction in U1 snRNA was observed using probe U1-2, with >75% reduction of U1 snRNA at 50 nM and >90% reduction at 200 nM (normalized to the U6 snRNA loading control). Together, these data confirm that treatment with 469508 efficiently directs RNAse H cleavage of the U1 leader sequence previously shown to be required for U1 splicing activity (8). The other ASOs evaluated, 469509 and 469511, each produced cleavage fragments consistent with their localization on the target, however, the potency of these ASOs was much less than that of 469508.Figure 3.


U1 adaptors result in reduction of multiple pre-mRNA species principally by sequestering U1snRNP.

Vickers TA, Sabripour M, Crooke ST - Nucleic Acids Res. (2011)

Reduction of U1 snRNA with RNAse H ASOs. (a) Chimeric 2′MOE/DNA RNAse H-dependent ASOs were designed to target U1snRNA. The location of 3 RNAse H ASOs is underlined on the U1 snRNA sequence. 469508 (1–20), 469509 (59–78), 469511 (117–138). Northern probe location is highlighted on the U1 snRNA structure. (b) HeLa cells were treated with ASOs at the indicated concentrations (nM). The following day total RNA was purified and U1snRNA reduction analyzed by northern blot with the internal U1 probe (U1-1, upper panel), or with a second probe, U1-2, targeted to the U1 5′-end and the non-target control and U6 snRNA probe (lower panel). Band intensity was quantitated with a Storm 850 Phosphor-Imager and U1 snRNA normalized to U6 snRNA signal for each lane. (c) Effect of U1 adaptor treatment and U1 snRNA reduction on mRNA expression. HeLa cells seeded in 96-well plates were treated with U1A-RAF1, U1A-SMN2, U1A-PCSK9 or anti-U1 ASO 469508, in triplicate at concentrations ranging from 6 to 200 nM. The following day total RNA was purified and mRNA levels of each targeted gene assessed by qRT/PCR. Results are normalized to the total amount of RNA present in each reaction as determined by Ribogreen assay and plotted as percent control compared to mock-treated cells for three to four replicates with the standard error.
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Figure 3: Reduction of U1 snRNA with RNAse H ASOs. (a) Chimeric 2′MOE/DNA RNAse H-dependent ASOs were designed to target U1snRNA. The location of 3 RNAse H ASOs is underlined on the U1 snRNA sequence. 469508 (1–20), 469509 (59–78), 469511 (117–138). Northern probe location is highlighted on the U1 snRNA structure. (b) HeLa cells were treated with ASOs at the indicated concentrations (nM). The following day total RNA was purified and U1snRNA reduction analyzed by northern blot with the internal U1 probe (U1-1, upper panel), or with a second probe, U1-2, targeted to the U1 5′-end and the non-target control and U6 snRNA probe (lower panel). Band intensity was quantitated with a Storm 850 Phosphor-Imager and U1 snRNA normalized to U6 snRNA signal for each lane. (c) Effect of U1 adaptor treatment and U1 snRNA reduction on mRNA expression. HeLa cells seeded in 96-well plates were treated with U1A-RAF1, U1A-SMN2, U1A-PCSK9 or anti-U1 ASO 469508, in triplicate at concentrations ranging from 6 to 200 nM. The following day total RNA was purified and mRNA levels of each targeted gene assessed by qRT/PCR. Results are normalized to the total amount of RNA present in each reaction as determined by Ribogreen assay and plotted as percent control compared to mock-treated cells for three to four replicates with the standard error.
Mentions: To directly evaluate and compare the contribution of U1 snRNP reduction with U1 adaptor off-target activity, we sought to decrease the amount of functional U1 snRNP directly, using RNAse H ASOs designed with complementarity to U1 snRNA (Figure 3a). HeLa cells were transfected with the ASOs at concentrations ranging from 50 nM to 200 nM. The following day, total RNA was purified and U1snRNA reduction analyzed by northern blot. Blots hybridized with the internal U1 northern probe (U1-1) showed a significant shift in the migration of U1snRNA following treatment with 469508 consistent with cleavage of the 5′ U1 snRNA binding sequence, even at the lowest concentration evaluated (Figure 3a and b, upper panel). However, the lack of reduction in signal intensity suggests that there is little degradation of the U1 snRNA following RNaseH cleavage, likely due to the highly structured nature of the RNA target. To confirm this, the same blot was hybridized with a second Northern probe, U1-2, complimentary to the cleaved U1 snRNA 5′-end (Figure 3b, lower panel). As observed with probe U1-1, treatment with ASO 469508 resulted in a shift in mobility consistent with the expected U1 snRNA cleavage product. In addition, a clear reduction in U1 snRNA was observed using probe U1-2, with >75% reduction of U1 snRNA at 50 nM and >90% reduction at 200 nM (normalized to the U6 snRNA loading control). Together, these data confirm that treatment with 469508 efficiently directs RNAse H cleavage of the U1 leader sequence previously shown to be required for U1 splicing activity (8). The other ASOs evaluated, 469509 and 469511, each produced cleavage fragments consistent with their localization on the target, however, the potency of these ASOs was much less than that of 469508.Figure 3.

Bottom Line: U1 Adaptors are a recently reported novel approach for targeted reduction of mRNA transcripts.Here, we present data demonstrating U1 adapter-mediated gene silencing can result in significant 'off-target' silencing effects as demonstrated by the reduction of multiple mRNA species that were not intended to be targeted.Our data suggest that a substantial portion of this U1 adaptor-mediated off-target mRNA reduction is the result of sequestration U1 snRNP at levels sufficient to affect splicing and processing of non-target transcripts.

View Article: PubMed Central - PubMed

Affiliation: Department of Core Antisense Research, ISIS Pharmaceuticals, Inc, 1896 Rutherford Road, Carlsbad, CA 92008, USA. tvickers@isisph.com

ABSTRACT
U1 Adaptors are a recently reported novel approach for targeted reduction of mRNA transcripts. A U1 adaptor oligonucleotide comprising of a target-complimentary hybridization domain and a U1 recruitment domain, directs the U1 snRNP complex to the terminal exon of a targeted gene, subsequently inhibiting poly(A) tail addition and leading to degradation of that RNA species within the nucleus. Here, we present data demonstrating U1 adapter-mediated gene silencing can result in significant 'off-target' silencing effects as demonstrated by the reduction of multiple mRNA species that were not intended to be targeted. Our data suggest that a substantial portion of this U1 adaptor-mediated off-target mRNA reduction is the result of sequestration U1 snRNP at levels sufficient to affect splicing and processing of non-target transcripts.

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