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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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U1 adaptors bind the 5′ sequence of U1 snRNA and inhibit antisense hybridization. (a) 293-T cells were transfected with 100 nM U1 adaptor/ASO for 6 h. RNAse H protection assays were performed with whole-cell extracts ± 2U E. coli RNAse H and 2 µM DNA oligonucleotide antisense to the 5′ sequence of U1. Cleavage of U1 snRNA was detected by northern blot using internal U1 probe (U1-1), and normalized using a U3 probe (U3). (b) Cellular RNAse protection assay. Cells were treated with U1A_SMN2 or ASO at concentrations ranging from 10 to 200 nM as shown. Cells were then transfected with anti U1 RNAse H ASO 469508 at 50 nM and U1 snRNA cleavage assessed by northern blot using internal U1 probe (U1-1). Band intensity was quantitated with a Storm 850 Phosphor-Imager and U1 snRNA signal normalized to U3 for each lane.
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Figure 5: U1 adaptors bind the 5′ sequence of U1 snRNA and inhibit antisense hybridization. (a) 293-T cells were transfected with 100 nM U1 adaptor/ASO for 6 h. RNAse H protection assays were performed with whole-cell extracts ± 2U E. coli RNAse H and 2 µM DNA oligonucleotide antisense to the 5′ sequence of U1. Cleavage of U1 snRNA was detected by northern blot using internal U1 probe (U1-1), and normalized using a U3 probe (U3). (b) Cellular RNAse protection assay. Cells were treated with U1A_SMN2 or ASO at concentrations ranging from 10 to 200 nM as shown. Cells were then transfected with anti U1 RNAse H ASO 469508 at 50 nM and U1 snRNA cleavage assessed by northern blot using internal U1 probe (U1-1). Band intensity was quantitated with a Storm 850 Phosphor-Imager and U1 snRNA signal normalized to U3 for each lane.

Mentions: To verify that U1 adaptors sequester U1 snRNP, we performed RNAse H protection assays (Figure 5a). Extracts from cells transfected with U1A-RAF1 and U1A-SMN2 or with the corresponding RNAse H ASOs were incubated with E. coli RNAse H and a DNA ASO probe complimentary to the U1 snRNA 5′ leader sequence. In extracts prepared from untreated cells or cells treated with ASOs, a fragment corresponding to the size expected for RNAse H cleavage was observed. However, in extracts from cells treated with either U1 adaptor, no cleavage product was observed, indicating that the U1 adaptor prevented binding of the DNA ASO probe and subsequent RNAse H digestion.Figure 5.


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

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

U1 adaptors bind the 5′ sequence of U1 snRNA and inhibit antisense hybridization. (a) 293-T cells were transfected with 100 nM U1 adaptor/ASO for 6 h. RNAse H protection assays were performed with whole-cell extracts ± 2U E. coli RNAse H and 2 µM DNA oligonucleotide antisense to the 5′ sequence of U1. Cleavage of U1 snRNA was detected by northern blot using internal U1 probe (U1-1), and normalized using a U3 probe (U3). (b) Cellular RNAse protection assay. Cells were treated with U1A_SMN2 or ASO at concentrations ranging from 10 to 200 nM as shown. Cells were then transfected with anti U1 RNAse H ASO 469508 at 50 nM and U1 snRNA cleavage assessed by northern blot using internal U1 probe (U1-1). Band intensity was quantitated with a Storm 850 Phosphor-Imager and U1 snRNA signal normalized to U3 for each lane.
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Figure 5: U1 adaptors bind the 5′ sequence of U1 snRNA and inhibit antisense hybridization. (a) 293-T cells were transfected with 100 nM U1 adaptor/ASO for 6 h. RNAse H protection assays were performed with whole-cell extracts ± 2U E. coli RNAse H and 2 µM DNA oligonucleotide antisense to the 5′ sequence of U1. Cleavage of U1 snRNA was detected by northern blot using internal U1 probe (U1-1), and normalized using a U3 probe (U3). (b) Cellular RNAse protection assay. Cells were treated with U1A_SMN2 or ASO at concentrations ranging from 10 to 200 nM as shown. Cells were then transfected with anti U1 RNAse H ASO 469508 at 50 nM and U1 snRNA cleavage assessed by northern blot using internal U1 probe (U1-1). Band intensity was quantitated with a Storm 850 Phosphor-Imager and U1 snRNA signal normalized to U3 for each lane.
Mentions: To verify that U1 adaptors sequester U1 snRNP, we performed RNAse H protection assays (Figure 5a). Extracts from cells transfected with U1A-RAF1 and U1A-SMN2 or with the corresponding RNAse H ASOs were incubated with E. coli RNAse H and a DNA ASO probe complimentary to the U1 snRNA 5′ leader sequence. In extracts prepared from untreated cells or cells treated with ASOs, a fragment corresponding to the size expected for RNAse H cleavage was observed. However, in extracts from cells treated with either U1 adaptor, no cleavage product was observed, indicating that the U1 adaptor prevented binding of the DNA ASO probe and subsequent RNAse H digestion.Figure 5.

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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