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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 and anti-U1 snRNA ASOs promote reduction of multiple non-targeted mRNAs. HeLa cells were treated with U1A-SMN2, SMN2 ASO 450880, or U1 ASO 469508 at concentrations from 12.5 to 100 nM. The following day cells were harvested and RNA purified. Messenger RNA reduction was analyzed by qRT/PCR. Percent mock-treated control expression is shown for the six transcripts identified by Entrez Gene symbol at the bottom of the figure. (a) Cells treated with U1A-SMN2. (b) Cells treated with anti-U1 RNAse H ASO 469508. (c) Cells treated with SMN2 RNAse H ASO 450880.
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Figure 4: U1 adaptors and anti-U1 snRNA ASOs promote reduction of multiple non-targeted mRNAs. HeLa cells were treated with U1A-SMN2, SMN2 ASO 450880, or U1 ASO 469508 at concentrations from 12.5 to 100 nM. The following day cells were harvested and RNA purified. Messenger RNA reduction was analyzed by qRT/PCR. Percent mock-treated control expression is shown for the six transcripts identified by Entrez Gene symbol at the bottom of the figure. (a) Cells treated with U1A-SMN2. (b) Cells treated with anti-U1 RNAse H ASO 469508. (c) Cells treated with SMN2 RNAse H ASO 450880.

Mentions: To further evaluate off-target effects, HeLa cells were treated with U1A-SMN2, ASO 450880 and ASO 469508 at concentrations from 12.5 to 100 nM. The following day, expression of a panel of mRNAs was evaluated by qRT/PCR. Treatment with U1A-SMN2 resulted in an IC50 for SMN2 mRNA expression of ∼10 nM (Figure 4a). Non-target mRNAs were also effectively reduced by treatment with U1A-SMN2 with IC50's between 20 and 100 nM, and even at the lowest concentration, significant reduction was observed for several of the non-target transcripts. Treatment with anti-U1 snRNA ASO 469508 also resulted in reduction of the same set of transcripts with similar IC50's (Figure 4b). Note that, as in Figure 3c, SMN2 was the target least affected by disabling of U1 snRNA, suggesting that for this mRNA, a significant proportion of the observed activity for the U1 adaptor is specific and mediated by the proposed mechanism. For other targets, such as PCSK9 (Figure 3c) and PIK3CB (compare Figure 4a and b), the similarity of the potency of mRNA reduction between the U1 adaptor and U1 snRNA reduction suggests that the activity is more related to the effects U1snRNP sequestration by the U1 adaptor rather than the proposed specific deadenylation/degradation of the message mediated by the U1 adaptor. SMN2 ASO 450880 had approximately the same IC50 as the corresponding U1 adaptor, but with no significant activity observed at 10 nM for the other mRNA targets and only slight reduction observed for some transcripts at the highest concentration (Figure 4c), again indicating that the observed off-target activity is specific to treatment with U1 adaptors. These results were confirmed and expanded, as an even larger number of overlapping transcripts were identified by PCR array as being reduced by treatment with U1A-RAF1 and the anti-U1 ASO 469508, but not by the corresponding RNAse H-dependent ASO (Supplementary Figure S2).Figure 4.


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 and anti-U1 snRNA ASOs promote reduction of multiple non-targeted mRNAs. HeLa cells were treated with U1A-SMN2, SMN2 ASO 450880, or U1 ASO 469508 at concentrations from 12.5 to 100 nM. The following day cells were harvested and RNA purified. Messenger RNA reduction was analyzed by qRT/PCR. Percent mock-treated control expression is shown for the six transcripts identified by Entrez Gene symbol at the bottom of the figure. (a) Cells treated with U1A-SMN2. (b) Cells treated with anti-U1 RNAse H ASO 469508. (c) Cells treated with SMN2 RNAse H ASO 450880.
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Figure 4: U1 adaptors and anti-U1 snRNA ASOs promote reduction of multiple non-targeted mRNAs. HeLa cells were treated with U1A-SMN2, SMN2 ASO 450880, or U1 ASO 469508 at concentrations from 12.5 to 100 nM. The following day cells were harvested and RNA purified. Messenger RNA reduction was analyzed by qRT/PCR. Percent mock-treated control expression is shown for the six transcripts identified by Entrez Gene symbol at the bottom of the figure. (a) Cells treated with U1A-SMN2. (b) Cells treated with anti-U1 RNAse H ASO 469508. (c) Cells treated with SMN2 RNAse H ASO 450880.
Mentions: To further evaluate off-target effects, HeLa cells were treated with U1A-SMN2, ASO 450880 and ASO 469508 at concentrations from 12.5 to 100 nM. The following day, expression of a panel of mRNAs was evaluated by qRT/PCR. Treatment with U1A-SMN2 resulted in an IC50 for SMN2 mRNA expression of ∼10 nM (Figure 4a). Non-target mRNAs were also effectively reduced by treatment with U1A-SMN2 with IC50's between 20 and 100 nM, and even at the lowest concentration, significant reduction was observed for several of the non-target transcripts. Treatment with anti-U1 snRNA ASO 469508 also resulted in reduction of the same set of transcripts with similar IC50's (Figure 4b). Note that, as in Figure 3c, SMN2 was the target least affected by disabling of U1 snRNA, suggesting that for this mRNA, a significant proportion of the observed activity for the U1 adaptor is specific and mediated by the proposed mechanism. For other targets, such as PCSK9 (Figure 3c) and PIK3CB (compare Figure 4a and b), the similarity of the potency of mRNA reduction between the U1 adaptor and U1 snRNA reduction suggests that the activity is more related to the effects U1snRNP sequestration by the U1 adaptor rather than the proposed specific deadenylation/degradation of the message mediated by the U1 adaptor. SMN2 ASO 450880 had approximately the same IC50 as the corresponding U1 adaptor, but with no significant activity observed at 10 nM for the other mRNA targets and only slight reduction observed for some transcripts at the highest concentration (Figure 4c), again indicating that the observed off-target activity is specific to treatment with U1 adaptors. These results were confirmed and expanded, as an even larger number of overlapping transcripts were identified by PCR array as being reduced by treatment with U1A-RAF1 and the anti-U1 ASO 469508, but not by the corresponding RNAse H-dependent ASO (Supplementary Figure S2).Figure 4.

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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