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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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Global effects of RAF1 U1 adaptor and anti-U1 ASO treatment. HeLa cells were transfected with U1A-RAF, anti-U1 ASO 469508, or ASO 194166 at 50 nM. Total RNA was purified the following day, and mRNA reduction evaluated by qRT/PCR. (a) Affymetrix exon arrays were performed using total RNA. Data were clustered at the transcript level using ANOVA-selected genes (N = 112, treated versus control, P < 0.1). Shown is log2 regulation for U1 adaptor/ASO treated cells versus control. (b) Venn diagrams comparing total signatures for U1 adaptor/ASO versus control. U1A-RAF1, dark green; ASO 469508, light green; ASO 194 166, green.
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Figure 7: Global effects of RAF1 U1 adaptor and anti-U1 ASO treatment. HeLa cells were transfected with U1A-RAF, anti-U1 ASO 469508, or ASO 194166 at 50 nM. Total RNA was purified the following day, and mRNA reduction evaluated by qRT/PCR. (a) Affymetrix exon arrays were performed using total RNA. Data were clustered at the transcript level using ANOVA-selected genes (N = 112, treated versus control, P < 0.1). Shown is log2 regulation for U1 adaptor/ASO treated cells versus control. (b) Venn diagrams comparing total signatures for U1 adaptor/ASO versus control. U1A-RAF1, dark green; ASO 469508, light green; ASO 194 166, green.

Mentions: We next sought to evaluate and compare transcriptome-wide effects on expression and splicing following treatment with U1A-RAF1 or direct inactivation of U1 snRNP with ASO 469508. HeLa cells were transfected with U1A-RAF1, ASO 469508, or ASO 194166, at a concentration of 50 nM. After 16 h, target reduction was confirmed by qRT/PCR (Supplementary Figure S5A) and affymetrix exon arrays were performed using total mRNA purified from the U1 adaptor/ASO-treated cells. ANOVA was used to detect probes showing significant changes in either direction among the groups. P-values were then corrected for multiple testing and probes having adjusted P < 0.01 were selected. Compared to untreated cells, log2-fold expression changes were found to be highly significant for U1A-RAF1 and U1 ASO 469508 treated, but not for cells treated with RAF1 ASO 194166 (Figure 7a). Comparison of total signatures for each treatment group versus mock-treated (P < 1e-3 uncorrected) revealed a highly significant overlap (P = 2.5e-160) between genes regulated by U1A-RAF1 and ASO 469508, with ∼60% of the U1A-regulated genes in common with the anti-U1 regulated genes (Figure 7b). ASO 194166 was found to alter the expression of far fewer genes, which overlap with those transcripts altered by U1A-RAF1 (P = 2.7e-22) or with those regulated by the anti-U1 ASO, 469 508 (P = 1.2e-20).Figure 7.


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

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

Global effects of RAF1 U1 adaptor and anti-U1 ASO treatment. HeLa cells were transfected with U1A-RAF, anti-U1 ASO 469508, or ASO 194166 at 50 nM. Total RNA was purified the following day, and mRNA reduction evaluated by qRT/PCR. (a) Affymetrix exon arrays were performed using total RNA. Data were clustered at the transcript level using ANOVA-selected genes (N = 112, treated versus control, P < 0.1). Shown is log2 regulation for U1 adaptor/ASO treated cells versus control. (b) Venn diagrams comparing total signatures for U1 adaptor/ASO versus control. U1A-RAF1, dark green; ASO 469508, light green; ASO 194 166, green.
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Figure 7: Global effects of RAF1 U1 adaptor and anti-U1 ASO treatment. HeLa cells were transfected with U1A-RAF, anti-U1 ASO 469508, or ASO 194166 at 50 nM. Total RNA was purified the following day, and mRNA reduction evaluated by qRT/PCR. (a) Affymetrix exon arrays were performed using total RNA. Data were clustered at the transcript level using ANOVA-selected genes (N = 112, treated versus control, P < 0.1). Shown is log2 regulation for U1 adaptor/ASO treated cells versus control. (b) Venn diagrams comparing total signatures for U1 adaptor/ASO versus control. U1A-RAF1, dark green; ASO 469508, light green; ASO 194 166, green.
Mentions: We next sought to evaluate and compare transcriptome-wide effects on expression and splicing following treatment with U1A-RAF1 or direct inactivation of U1 snRNP with ASO 469508. HeLa cells were transfected with U1A-RAF1, ASO 469508, or ASO 194166, at a concentration of 50 nM. After 16 h, target reduction was confirmed by qRT/PCR (Supplementary Figure S5A) and affymetrix exon arrays were performed using total mRNA purified from the U1 adaptor/ASO-treated cells. ANOVA was used to detect probes showing significant changes in either direction among the groups. P-values were then corrected for multiple testing and probes having adjusted P < 0.01 were selected. Compared to untreated cells, log2-fold expression changes were found to be highly significant for U1A-RAF1 and U1 ASO 469508 treated, but not for cells treated with RAF1 ASO 194166 (Figure 7a). Comparison of total signatures for each treatment group versus mock-treated (P < 1e-3 uncorrected) revealed a highly significant overlap (P = 2.5e-160) between genes regulated by U1A-RAF1 and ASO 469508, with ∼60% of the U1A-regulated genes in common with the anti-U1 regulated genes (Figure 7b). ASO 194166 was found to alter the expression of far fewer genes, which overlap with those transcripts altered by U1A-RAF1 (P = 2.7e-22) or with those regulated by the anti-U1 ASO, 469 508 (P = 1.2e-20).Figure 7.

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.

Show MeSH