Limits...
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.

Show MeSH
U1 adaptor downregulates multiple mRNAs. A U1 adaptor targeted to RAF1 (U1A-RAF1) was administered to Hela cells at a single-dose of 100 nM. A fully modified 2′MOE ASO (13649) and a chimeric 2′MOE/DNA RNAse H-dependent ASO (194166) complementary to the same target sequence were also administered at the same concentration. After 24 h, cells were harvested, RNA was purified and mRNA reduction assayed by qRT/PCR using primers specific for 3 mRNAs; RAF1, PTEN and SMN2. The bars represent per cent mean untreated control for three replicates and the standard errors. UTC = mock-treated control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3105408&req=5

Figure 1: U1 adaptor downregulates multiple mRNAs. A U1 adaptor targeted to RAF1 (U1A-RAF1) was administered to Hela cells at a single-dose of 100 nM. A fully modified 2′MOE ASO (13649) and a chimeric 2′MOE/DNA RNAse H-dependent ASO (194166) complementary to the same target sequence were also administered at the same concentration. After 24 h, cells were harvested, RNA was purified and mRNA reduction assayed by qRT/PCR using primers specific for 3 mRNAs; RAF1, PTEN and SMN2. The bars represent per cent mean untreated control for three replicates and the standard errors. UTC = mock-treated control.

Mentions: To discriminate potential off-target effects due to sequestration of U1 snRNP by U1 adaptors from those resulting from binding to the mRNA target alone, we evaluated the activity of several U1 adaptors along with ASOs targeted to the same site on the mRNA. An ASO would be expected to bind the targeted mRNA transcript with an affinity similar to that of the U1 adaptor target domain without recruitment of the U1 snRNP, allowing a direct comparison with the alternate degradation mechanism proposed for U1 adaptors. HeLa cells were transfected at 100 nM with a U1 adaptor, a fully 2′-O-methoxyethyl ribose (2′MOE) ASO, or a chimeric 2′MOE/DNA ASO targeted to the same site on RAF1 mRNA. Target mRNA reduction was evaluated by qRT/PCR the following day (Figure 1). The RNAse H-dependent ASO (194166) reduced RAF1 mRNA (solid bars) by >85%, with little reduction of levels of SMN2 (striped bars) or PTEN (hatched bars) non-target messages observed. Treatment with the RAF1 U1 adaptor (U1A-RAF1) at the same concentration resulted in a ∼90% reduction in RAF1 mRNA. In addition, the level of PTEN mRNA was reduced by approximately the same amount, while SMN2 mRNA was reduced by ∼75%. Treatment with ASO 13 496, which does not support cleavage by RNAse H (20), resulted in no significant reduction of either RAF1 or non-target mRNAs. The lack of activity observed with ASO 13496 suggests that binding alone cannot be responsible for specific or non-specific mRNA reduction resulting from treatment with the U1 adaptor or RNase H-dependent ASO.Figure 1.


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 adaptor downregulates multiple mRNAs. A U1 adaptor targeted to RAF1 (U1A-RAF1) was administered to Hela cells at a single-dose of 100 nM. A fully modified 2′MOE ASO (13649) and a chimeric 2′MOE/DNA RNAse H-dependent ASO (194166) complementary to the same target sequence were also administered at the same concentration. After 24 h, cells were harvested, RNA was purified and mRNA reduction assayed by qRT/PCR using primers specific for 3 mRNAs; RAF1, PTEN and SMN2. The bars represent per cent mean untreated control for three replicates and the standard errors. UTC = mock-treated control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105408&req=5

Figure 1: U1 adaptor downregulates multiple mRNAs. A U1 adaptor targeted to RAF1 (U1A-RAF1) was administered to Hela cells at a single-dose of 100 nM. A fully modified 2′MOE ASO (13649) and a chimeric 2′MOE/DNA RNAse H-dependent ASO (194166) complementary to the same target sequence were also administered at the same concentration. After 24 h, cells were harvested, RNA was purified and mRNA reduction assayed by qRT/PCR using primers specific for 3 mRNAs; RAF1, PTEN and SMN2. The bars represent per cent mean untreated control for three replicates and the standard errors. UTC = mock-treated control.
Mentions: To discriminate potential off-target effects due to sequestration of U1 snRNP by U1 adaptors from those resulting from binding to the mRNA target alone, we evaluated the activity of several U1 adaptors along with ASOs targeted to the same site on the mRNA. An ASO would be expected to bind the targeted mRNA transcript with an affinity similar to that of the U1 adaptor target domain without recruitment of the U1 snRNP, allowing a direct comparison with the alternate degradation mechanism proposed for U1 adaptors. HeLa cells were transfected at 100 nM with a U1 adaptor, a fully 2′-O-methoxyethyl ribose (2′MOE) ASO, or a chimeric 2′MOE/DNA ASO targeted to the same site on RAF1 mRNA. Target mRNA reduction was evaluated by qRT/PCR the following day (Figure 1). The RNAse H-dependent ASO (194166) reduced RAF1 mRNA (solid bars) by >85%, with little reduction of levels of SMN2 (striped bars) or PTEN (hatched bars) non-target messages observed. Treatment with the RAF1 U1 adaptor (U1A-RAF1) at the same concentration resulted in a ∼90% reduction in RAF1 mRNA. In addition, the level of PTEN mRNA was reduced by approximately the same amount, while SMN2 mRNA was reduced by ∼75%. Treatment with ASO 13 496, which does not support cleavage by RNAse H (20), resulted in no significant reduction of either RAF1 or non-target mRNAs. The lack of activity observed with ASO 13496 suggests that binding alone cannot be responsible for specific or non-specific mRNA reduction resulting from treatment with the U1 adaptor or RNase H-dependent ASO.Figure 1.

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