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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 alter mini gene splicing and expression. (a) A tetracycline inducible SMN2 mini gene, comprised of the 11-nt long exon 6, a 200-nt shortened intron 6, the 54-nt exon 7, the 444-nt intron 7, the first 75 nt of exon 8, from pCI-SMN2 was subcloned into the vector pcDNA 4/TO using PCR generated HindIII and XbaI sites as detailed in the ‘Materials and Methods’ section. Stable TREX 293 cell transformants from transfected cells were obtained by zeocin selection. (b) Location of qRT/PCR primers and probe used to amplify SMN/TO full-length, exon 7 skipped, and pre-mRNA. (c) Kinetics of SMN/TO tetracycline induction. SMN2/TO-293 cells were treated with TET at 0.25 ug/ml for 30 min to 6 h. Expression of SMN/TO mRNA assessed using the splice-form specific qRT/PCR primer/probe sets shown in Figure 5B and as detailed in ‘Materials and Methods’ section. (d) SMN/TO 293 cells were treated with U1A-RAF, U1A-PCSK9, anti-U1 ASO 469508, or anti-U4 ASO 479333 at 50 nM. The following day SMN/TO expression was induced for 4 h, then expression of SMN/TO and target mRNA assessed. Results are presented as percent mock-treated control (UTC) for each mRNA assayed. Pre-mRNA, filled bars; full-length SMN/TO mRNA, striped bars; exon 7-skipped SMN/TO mRNA, hatched bars; RAF1 mRNA, gray bars; PCSK9 mRNA, open bars.
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Figure 6: U1 adaptors and anti-U1 snRNA ASOs alter mini gene splicing and expression. (a) A tetracycline inducible SMN2 mini gene, comprised of the 11-nt long exon 6, a 200-nt shortened intron 6, the 54-nt exon 7, the 444-nt intron 7, the first 75 nt of exon 8, from pCI-SMN2 was subcloned into the vector pcDNA 4/TO using PCR generated HindIII and XbaI sites as detailed in the ‘Materials and Methods’ section. Stable TREX 293 cell transformants from transfected cells were obtained by zeocin selection. (b) Location of qRT/PCR primers and probe used to amplify SMN/TO full-length, exon 7 skipped, and pre-mRNA. (c) Kinetics of SMN/TO tetracycline induction. SMN2/TO-293 cells were treated with TET at 0.25 ug/ml for 30 min to 6 h. Expression of SMN/TO mRNA assessed using the splice-form specific qRT/PCR primer/probe sets shown in Figure 5B and as detailed in ‘Materials and Methods’ section. (d) SMN/TO 293 cells were treated with U1A-RAF, U1A-PCSK9, anti-U1 ASO 469508, or anti-U4 ASO 479333 at 50 nM. The following day SMN/TO expression was induced for 4 h, then expression of SMN/TO and target mRNA assessed. Results are presented as percent mock-treated control (UTC) for each mRNA assayed. Pre-mRNA, filled bars; full-length SMN/TO mRNA, striped bars; exon 7-skipped SMN/TO mRNA, hatched bars; RAF1 mRNA, gray bars; PCSK9 mRNA, open bars.

Mentions: Since reduction of U1 snRNP has been demonstrated to result in alteration of splicing (7), we utilized an SMN2 minigene system to compare the effects of U1 adaptor treatment and U1 snRNP reduction on alternative splicing. The skipping of exon 7 of the SMN2 gene has been well characterized (14). We subcloned the previously reported SMN2 mini gene (21) into the tetracycline inducible expression plasmid pcDNA 4/TO for stable integration into 293 cells (Figure 6a). SMN2/TO-293 cells were induced with tetracycline at 0.25 ug/ml for 30 min to 6 h. Expression of SMN/TO mRNA assessed using the splice-form specific qRT/PCR primer/probe sets shown in Figure 6b, demonstrated tightly regulated TET induction of the spliced full-length and exon 7 skipped mini gene mRNA (Figure 6c). Reduction of U1 snRNA by ASO 469508, as well as reduction of U4 and U6 snRNAs, was found to result in an increase in SMN2/TO pre-mRNA levels (Supplementary Figure S3A). In addition, reduction of U1, but not U4 or U6 snRNA resulted in an increase in the ratio of skipped relative to full-length spliced mRNA (Supplementary Figure S3B).Figure 6.


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 alter mini gene splicing and expression. (a) A tetracycline inducible SMN2 mini gene, comprised of the 11-nt long exon 6, a 200-nt shortened intron 6, the 54-nt exon 7, the 444-nt intron 7, the first 75 nt of exon 8, from pCI-SMN2 was subcloned into the vector pcDNA 4/TO using PCR generated HindIII and XbaI sites as detailed in the ‘Materials and Methods’ section. Stable TREX 293 cell transformants from transfected cells were obtained by zeocin selection. (b) Location of qRT/PCR primers and probe used to amplify SMN/TO full-length, exon 7 skipped, and pre-mRNA. (c) Kinetics of SMN/TO tetracycline induction. SMN2/TO-293 cells were treated with TET at 0.25 ug/ml for 30 min to 6 h. Expression of SMN/TO mRNA assessed using the splice-form specific qRT/PCR primer/probe sets shown in Figure 5B and as detailed in ‘Materials and Methods’ section. (d) SMN/TO 293 cells were treated with U1A-RAF, U1A-PCSK9, anti-U1 ASO 469508, or anti-U4 ASO 479333 at 50 nM. The following day SMN/TO expression was induced for 4 h, then expression of SMN/TO and target mRNA assessed. Results are presented as percent mock-treated control (UTC) for each mRNA assayed. Pre-mRNA, filled bars; full-length SMN/TO mRNA, striped bars; exon 7-skipped SMN/TO mRNA, hatched bars; RAF1 mRNA, gray bars; PCSK9 mRNA, open bars.
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Figure 6: U1 adaptors and anti-U1 snRNA ASOs alter mini gene splicing and expression. (a) A tetracycline inducible SMN2 mini gene, comprised of the 11-nt long exon 6, a 200-nt shortened intron 6, the 54-nt exon 7, the 444-nt intron 7, the first 75 nt of exon 8, from pCI-SMN2 was subcloned into the vector pcDNA 4/TO using PCR generated HindIII and XbaI sites as detailed in the ‘Materials and Methods’ section. Stable TREX 293 cell transformants from transfected cells were obtained by zeocin selection. (b) Location of qRT/PCR primers and probe used to amplify SMN/TO full-length, exon 7 skipped, and pre-mRNA. (c) Kinetics of SMN/TO tetracycline induction. SMN2/TO-293 cells were treated with TET at 0.25 ug/ml for 30 min to 6 h. Expression of SMN/TO mRNA assessed using the splice-form specific qRT/PCR primer/probe sets shown in Figure 5B and as detailed in ‘Materials and Methods’ section. (d) SMN/TO 293 cells were treated with U1A-RAF, U1A-PCSK9, anti-U1 ASO 469508, or anti-U4 ASO 479333 at 50 nM. The following day SMN/TO expression was induced for 4 h, then expression of SMN/TO and target mRNA assessed. Results are presented as percent mock-treated control (UTC) for each mRNA assayed. Pre-mRNA, filled bars; full-length SMN/TO mRNA, striped bars; exon 7-skipped SMN/TO mRNA, hatched bars; RAF1 mRNA, gray bars; PCSK9 mRNA, open bars.
Mentions: Since reduction of U1 snRNP has been demonstrated to result in alteration of splicing (7), we utilized an SMN2 minigene system to compare the effects of U1 adaptor treatment and U1 snRNP reduction on alternative splicing. The skipping of exon 7 of the SMN2 gene has been well characterized (14). We subcloned the previously reported SMN2 mini gene (21) into the tetracycline inducible expression plasmid pcDNA 4/TO for stable integration into 293 cells (Figure 6a). SMN2/TO-293 cells were induced with tetracycline at 0.25 ug/ml for 30 min to 6 h. Expression of SMN/TO mRNA assessed using the splice-form specific qRT/PCR primer/probe sets shown in Figure 6b, demonstrated tightly regulated TET induction of the spliced full-length and exon 7 skipped mini gene mRNA (Figure 6c). Reduction of U1 snRNA by ASO 469508, as well as reduction of U4 and U6 snRNAs, was found to result in an increase in SMN2/TO pre-mRNA levels (Supplementary Figure S3A). In addition, reduction of U1, but not U4 or U6 snRNA resulted in an increase in the ratio of skipped relative to full-length spliced mRNA (Supplementary Figure S3B).Figure 6.

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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