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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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Potency and specificity of U1 adaptors and RNAse H ASOs. A second U1 adaptor was designed to target SMN2 (U1A-SMN2). HeLa cells were treated at concentrations of 3 nM, 10 nM, 30 nM, or 100 nM with U1A-SMN2, U1A-RAF1, ASO 194166 (RAF1) or ASO 450880 (SMN2). After 24 h, cells were harvested and RNA purified. Messenger RNA reduction was analyzed by qRT/PCR. The percent mean mock-treated control (UTC) for three to four replicates and the standard error is shown for each concentration. (a) Percent control RAF1 mRNA expression. (b) Percent control SMN2 mRNA expression. (c) Percent control PTEN mRNA expression.
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Figure 2: Potency and specificity of U1 adaptors and RNAse H ASOs. A second U1 adaptor was designed to target SMN2 (U1A-SMN2). HeLa cells were treated at concentrations of 3 nM, 10 nM, 30 nM, or 100 nM with U1A-SMN2, U1A-RAF1, ASO 194166 (RAF1) or ASO 450880 (SMN2). After 24 h, cells were harvested and RNA purified. Messenger RNA reduction was analyzed by qRT/PCR. The percent mean mock-treated control (UTC) for three to four replicates and the standard error is shown for each concentration. (a) Percent control RAF1 mRNA expression. (b) Percent control SMN2 mRNA expression. (c) Percent control PTEN mRNA expression.

Mentions: Since it has been reported that IC50's of <5 nM can be achieved with U1 adaptors (1), we wanted to determine if a window of specific target reduction could be observed at lower treatment concentrations. HeLa cells were transfected with U1A-RAF1, a second U1 adaptor targeted to SMN2 (U1A-SMN2) or the corresponding RNAse H-dependent ASOs at multiple concentrations. Expression of RAF1 mRNA was reduced by U1A-RAF1 with an IC50 of ∼20 nM (Figure 2a). U1A-SMN2 also effectively reduced RAF1 mRNA with approximately the same IC50. Reduction of SMN2 mRNA was also evaluated (Figure 2b). While U1A-SMN2 reduced expression of the targeted RNA with an IC50 of ∼3 nM, off-target reduction of SMN2 by U1A-RAF1 was less robust with an IC50 of ∼75 nM. Both U1 adaptors also effectively reduced expression of PTEN, an unrelated, non-target mRNA, with an IC50 of ∼25 nM for U1A-RAF1 and ∼10 nM for U1A-SMN (Figure 2c). ASO 194166 (RAF1) was ∼7-fold more potent than the corresponding U1 adaptor, while ASO 450880 (SMN2) and the corresponding U1 adaptor were equipotent. No significant reduction in non-targeted messages was observed for either ASO. Similar levels of reduction in non-targeted transcripts were observed in primary human umbilical vein endothelial cells (HUVEC) and in 293T cells treated with U1 adaptors (Supplementary Figure S1). Together, these data suggest that transcripts may be differentially sensitive to U1 adaptor-mediated off-target activity and that off-target activity can vary by site and target.Figure 2.


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

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

Potency and specificity of U1 adaptors and RNAse H ASOs. A second U1 adaptor was designed to target SMN2 (U1A-SMN2). HeLa cells were treated at concentrations of 3 nM, 10 nM, 30 nM, or 100 nM with U1A-SMN2, U1A-RAF1, ASO 194166 (RAF1) or ASO 450880 (SMN2). After 24 h, cells were harvested and RNA purified. Messenger RNA reduction was analyzed by qRT/PCR. The percent mean mock-treated control (UTC) for three to four replicates and the standard error is shown for each concentration. (a) Percent control RAF1 mRNA expression. (b) Percent control SMN2 mRNA expression. (c) Percent control PTEN mRNA expression.
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Figure 2: Potency and specificity of U1 adaptors and RNAse H ASOs. A second U1 adaptor was designed to target SMN2 (U1A-SMN2). HeLa cells were treated at concentrations of 3 nM, 10 nM, 30 nM, or 100 nM with U1A-SMN2, U1A-RAF1, ASO 194166 (RAF1) or ASO 450880 (SMN2). After 24 h, cells were harvested and RNA purified. Messenger RNA reduction was analyzed by qRT/PCR. The percent mean mock-treated control (UTC) for three to four replicates and the standard error is shown for each concentration. (a) Percent control RAF1 mRNA expression. (b) Percent control SMN2 mRNA expression. (c) Percent control PTEN mRNA expression.
Mentions: Since it has been reported that IC50's of <5 nM can be achieved with U1 adaptors (1), we wanted to determine if a window of specific target reduction could be observed at lower treatment concentrations. HeLa cells were transfected with U1A-RAF1, a second U1 adaptor targeted to SMN2 (U1A-SMN2) or the corresponding RNAse H-dependent ASOs at multiple concentrations. Expression of RAF1 mRNA was reduced by U1A-RAF1 with an IC50 of ∼20 nM (Figure 2a). U1A-SMN2 also effectively reduced RAF1 mRNA with approximately the same IC50. Reduction of SMN2 mRNA was also evaluated (Figure 2b). While U1A-SMN2 reduced expression of the targeted RNA with an IC50 of ∼3 nM, off-target reduction of SMN2 by U1A-RAF1 was less robust with an IC50 of ∼75 nM. Both U1 adaptors also effectively reduced expression of PTEN, an unrelated, non-target mRNA, with an IC50 of ∼25 nM for U1A-RAF1 and ∼10 nM for U1A-SMN (Figure 2c). ASO 194166 (RAF1) was ∼7-fold more potent than the corresponding U1 adaptor, while ASO 450880 (SMN2) and the corresponding U1 adaptor were equipotent. No significant reduction in non-targeted messages was observed for either ASO. Similar levels of reduction in non-targeted transcripts were observed in primary human umbilical vein endothelial cells (HUVEC) and in 293T cells treated with U1 adaptors (Supplementary Figure S1). Together, these data suggest that transcripts may be differentially sensitive to U1 adaptor-mediated off-target activity and that off-target activity can vary by site and target.Figure 2.

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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