RNase H1-Dependent Antisense Oligonucleotides Are Robustly Active in Directing RNA Cleavage in Both the Cytoplasm and the Nucleus

Abstract

RNase H1-dependent antisense oligonucleotides (ASOs) are active in reducing levels of both cytoplasmic mRNAs and nuclear retained RNAs. Although ASO activity in the nucleus has been well demonstrated, the cytoplasmic activity of ASOs is less clear. Using kinetic and subcellular fractionation studies, we evaluated ASO activity in the cytoplasm. Upon transfection, ASOs targeting exonic regions rapidly reduced cytoplasmically enriched mRNAs, whereas an intron-targeting ASO that only degrades the nuclear pre-mRNA reduced mRNA levels at a slower rate, similar to normal mRNA decay. Importantly, some exon-targeting ASOs can rapidly and vigorously reduce mRNA levels without decreasing pre-mRNA levels, suggesting that pre-existing cytoplasmic mRNAs can be cleaved by RNase H1-ASO treatment. In addition, we expressed a cytoplasm-localized mutant 7SL RNA that contains a partial U16 small nucleolar RNA (snoRNA) sequence. Treatment with an ASO simultaneously reduced both the nuclear U16 snoRNA and the cytoplasmic 7SL mutant RNA as early as 30 min after transfection in an RNase H1-dependent manner. Both the 5' and 3' cleavage products of the 7SL mutant RNA were accumulated in the cytoplasm. Together, these results demonstrate that RNase H1-dependent ASOs are robustly active in both the cytoplasm and nucleus.

Keywords: RNA; RNase H1; antisense; cytoplasm; nucleus; oligonucleotide.

Figure 1

ASOs Can Degrade mRNAs at a Rate Faster Than Normal mRNA Decay (A) qRT-PCR for U16 and MALAT1 RNA levels in HeLa cells transfected with either 50 nM ASO462026 or 5 nM ASO395254, respectively, for different times. (B) qRT-PCR for NCL1 and Drosha mRNA levels in HeLa cells transfected with 50 nM ASO110074 (for NCL1) or 25690 (for Drosha), respectively, for different times. (C) qRT-PCR for NCL1 and MALAT1 RNA levels in HEK293 cells transfected with 50 nM ASO110074 or 5 nM ASO395254, respectively, for different times. (D) qRT-PCR for SRB and MALAT1 RNA levels in mouse MHT cells transfected with 30 nM ASO 205382 or 5 nM ASO 399479, respectively, for different times. (E) qRT-PCR for different RNA levels in HeLa cells treated with 100 μg/mL DRB for different times. (F) qRT-PCR for NCL1 pre-mRNA levels in HeLa cells treated with 100 μg/mL DRB for different times. The gray dashed lines indicate a 50% reduction of the RNAs. Error bars are SDs from three independent experiments.

Figure 2

Some ASOs Can Rapidly Reduce Mature mRNAs without Reducing Pre-mRNA Levels (A) qRT-PCR for Drosha mRNA levels in HeLa cells transfected with different ASOs at 40 nM for different times. (B) qRT-PCR for Drosha pre-mRNA levels in the same samples as in (A). (C) qRT-PCR for Drosha mRNA levels in HeLa cells transfected with either 40 nM ASO25690 or 3 nM Drosha siRNA for different times. (D) qRT-PCR for Ago2 mRNA levels in HeLa cells transfected with different ASOs at 50 nM for different times. (E) qRT-PCR for pre-mRNA levels of Ago2 in the same samples as in (D). (F) qRT-PCR for Ago2 mRNA levels in cells treated with 100 μg/mL DRB for different times. Error bars are SDs from three independent experiments.

Figure 3

Pre-mRNA and Processed Mature RNA Can Have Different Accessibility to ASOs (A) Schematic representation of partial NCL1 pre-mRNA and processing of the intronic snoRNA U23. U23 snoRNA and its flanking introns are marked with white boxes or lines, respectively. (B) qRT-PCR for mature and pre-mRNA levels of NCL1 in HeLa cells transfected for 4 hr with different concentrations of ASO110128. (C) Northern hybridization for U23 snoRNA in HeLa cells transfected for 4 hr with two different ASOs. U16 snoRNA was detected and served as a loading control. (D) qRT-PCR for mature or pre-mRNA levels of NCL1 in HeLa cells transfected with 100 nM ASOs for 20 hr. Two different primer probe sets were used to detect the pre-mRNA of NCL1. The two ASOs used, as in (C), are underlined. (E) Western blot analysis of XRN2 in HeLa cells transfected with a control luciferase siRNA or an siRNA specific to XRN2 mRNA for 72 hr. La protein was detected and served as the loading control. (F) Northern hybridization for U23 snoRNA in luciferase siRNA- or XRN2 siRNA-treated HeLa cells subsequently transfected with or without 50 nM ASO483788 for 4 hr. Ethidium bromide staining of the PAGE gel is shown in the left panel, and the sizes of known RNA bands are given. The northern blot was hybridized with probe XL099, and images with shorter or longer (middle and right panels, respectively) exposure time are shown. The identities of the hybridized bands are indicated. (G) Sequence of the 5′-RACE product for the U23 intermediate. The 5′ end of the product is highlighted. (H) Northern hybridization of U4 snRNA in cells treated for 24 hr with control ASO (129700) and U4-specific ASO (479333) at 30 nM. U3 snoRNA was detected and served as a loading control. (I) qRT-PCR for mature and pre-mRNA levels of NCL1 in control cells (con) or U4 reduced cells. (J) qRT-PCR for NCL1 pre-mRNA levels in different cells transfected with the U23 snoRNA ASO at different doses for 4 hr. (K) qRT-PCR for SOD1 pre-mRNA levels in different cells transfected with the SOD1 intron-targeting ASO at different doses for 4 hr. Error bars in different panels are SDs from three experiments.

Figure 4

mRNA Reduction Can Occur in the Cytoplasm (A) qRT-PCR for Drosha mature and pre-mRNA levels in different subcellular fractions prepared from HeLa cells treated with 5 nM ASO25690 for 2 hr. Cy and Nu indicate cytoplasmic and nuclear fractions, respectively. +ASO and −ASO indicate cells treated with or without ASO25690, respectively. (B) qRT-PCR for NCL1 mature and pre-mRNA levels in the same samples as used in (A). (C) qRT-PCR for cytoplasmic 7SL RNA and nuclear Malat1 RNA levels in the same samples as in (A). (D) Western blot analyses of nuclear protein PSF and cytoplasmic protein GAPDH in cytoplasmic and nuclear fractions prepared from HeLa cells treated with [(+)ASO] or without [(−)ASO] ASOs. (E) qRT-PCR for 28S rRNA, 7SL RNA, and U16 snoRNA levels in cytoplasmic or nuclear RNAs prepared from aliquots of cytoplasmic and nuclear samples as in (D). The same set of RNAs was also subjected to qRT-PCR analyses for mature and pre-mRNA levels of SOD1 (F), NCL1 (G), Ago2 (H), and MALAT1 (I) RNA. Error bars are SDs from three experiments.

Figure 5

RNase H1 Protein Is Present in the Cytosol and the Reduction of RNase H1 Dramatically Decreases ASO Activity (A) Cytosolic, mitochondrial, and nuclear fractions were prepared, and proteins of each fraction from an equal number of cells were analyzed by SDS-PAGE and western blot assay. The membrane was probed for the following, using specific antibodies: RNase H1; nuclear proteins PSF, P54nrb, and Lamin A; mitochondrial proteins P32, GRSF1, and Hsp60; and cytoplasmic protein GAPDH. The RNase H1 protein level was quantified using ImageJ software (NIH). The mean levels and SDs from three independent experiments are shown below the lanes. (B) qRT-PCR (left) and western blot (right) analyses for the levels of RNase H1 and RNase H2 mRNAs and proteins in HeLa cells treated with specific siRNAs or a control luciferase siRNA for 60 hr. GAPDH was probed in western blot analysis and served as a loading control. (C–H) Control, RNase H1-, and RNase H2-siRNA-treated cells, as used in (A), were treated for 4 hr with different ASOs, and the levels of the targeted MALAT1 (C), SOD1 pre-mRNA (D), Ago2 (E and F), NCL1 (G), and Drosha (H) RNAs were determined by qRT-PCR. Error bars are SDs from three experiments. p values were calculated using the F test (curve comparison between control and H1-reduced cells).

Figure 6

An Intron-Targeting ASO that Degrades Nuclear Pre-mRNAs Causes a Slow Reduction of mRNA Levels (A) qRT-PCR for mature and pre-mRNA levels of SOD1 in HeLa cells transfected for different times with 60 nM intron-targeting ASO480774. (B) qRT-PCR for mature and pre-mRNA levels of SOD1 in HeLa cells transfected for different times with 60 nM exon-targeting ASO333632. (C) qRT-PCR for mature SOD1 mRNA levels in HeLa cells treated with 100 μg/mL DRB for the indicated times. (D) qRT-PCR for mature and pre-mRNA levels of SOD1 in HeLa cells transfected for different times with 120 nM ASO480774. The gray dashed lines indicate a 50% reduction of the RNAs. Error bars indicate SDs from three experiments.

Figure 7

A Mutant 7SLm RNA Could Be Rapidly Degraded by ASOs in HeLa Cells (A) Schematic depiction of human 7SL RNA. The position of the insertion site, the insert sequences, and the ASO target site are shown. (B) Northern hybridization analysis of 7SLm RNA expression in HeLa cells transfected with the expression plasmid (+7SLm) or in mock-transfected cells (con). The northern membrane was hybridized with a 5′-end labeled probe XL272 that recognizes both U16 and the mutant 7SLm RNAs. (C) Northern hybridization analysis of 7SLm RNA and U16 snoRNA levels in HeLa cells transiently expressing 7SLm RNA and transfected with different concentrations of ASO462026 for 4 hr. The upper panel indicates the hybridization result using probe XL272. The membrane was re-hybridized with a probe specific to U4 snRNA, which served as a loading control. The hybridization signals for 7SLm and U16 RNAs were quantified using ImageJ, and the relative levels are plotted in the right panel. (D) Northern hybridization of different RNAs in HeLa cells transiently expressing 7SLm RNA and transfected with 15 nM ASO462026 for different times. The PAGE gel was stained with ethidium bromide and the identities of the abundant RNAs are indicated in the upper panel. The membrane was either probed for the wild-type 7SL RNA or hybridized with probe XL272, which recognizes both U16 snoRNA and 7SLm RNA (middle panel). An image with a shorter exposure time for U16 is shown in the lower panel. The 30-min time point is marked with an arrow. (E) 7SLm RNA, the ASO target site, and the expected sizes of the cleaved fragments are depicted in the upper panel. The positions of hybridization probes used in subsequent studies are indicated. The lower panel shows northern hybridization analysis of the same blot as in (D) using a 3′ probe (XL297) that detects both the full-length and 3′ cleaved fragment of the 7SLm RNA. The identities of detected bands are indicated. (F) Northern hybridization analysis of 7SLm RNA using a 5′ probe (XL299). HeLa cells expressing 7SLm RNA were transfected with 20 nM ASO462026 for different times and total RNA was prepared and subjected to northern hybridization analyses. The 5.8S and 5S rRNA visualized by ethidium bromide staining served as loading controls. (G) Northern hybridization analysis of U16 and 7SLm RNA in HeLa cells treated [(+)ActD] or not treated [(−)ActD] with 5 μg/mL Actinomycin D for 8 hr. The ethidium bromide staining of 5.8S and 5S rRNAs was used as a loading control. (H) qRT-PCR for NCL1 mRNA levels in the RNA samples as used in (G). Error bars indicate SDs from three experiments.

Figure 8

The Full-Length and Cleaved Fragments of 7SLm RNA Are Accumulated in the Cytoplasm (A) Northern hybridization analysis of RNAs prepared from cytoplasmic or nuclear fractions of cells treated with [(+)ASO] or without (UTC) 20 nM ASO462026 for 2 hr. The upper panel shows ethidium bromide staining of the PAGE gel. The identities and the sizes of known abundant RNAs are indicated, which served as size markers. The lower panels are northern hybridization for wild-type 7SL RNA and tRNA^tyr, which served as markers for cytoplasmic RNAs, and U1 snRNA and U3 snoRNA, which served as markers for nuclear RNAs, respectively. (B) Northern hybridization of the membrane transferred from the gel in (A) using probe XL297, which detects the full-length and the 3′ cleaved fragments of 7SLm RNA. The size ladder was generated from the known RNAs as shown in (A). The cleaved 3′ fragment and its expected size are indicated. (C) Northern hybridization of the same membrane as in (B) using the 5′ probe XL299. The cleaved fragment and the expected size are indicated. (D) Western analyses of RNase H1 and RNase H2 in HeLa cells treated with either a control luciferase siRNA (Luci-siRNA) or specific siRNAs for 60 hr. GAPDH was detected and served as a loading control. (E) Northern hybridization of 7SLm RNA and U16 snoRNA in 7SLm RNA-expressing HeLa cells transfected with siRNAs for 60h, followed by transfection of 15 nM ASO462026 for 5 hr. U3 snoRNA was detected and served as a loading control. The relative levels of 7SLm and U16 were quantified and normalized to U3 RNA and are shown below the lanes.