Published online 03 February 2016 Nucleic Acids Research, 2016, Vol. 44, No.

7 3351–3363
doi: 10.1093/nar/gkw065

RNA cleavage products generated by antisense

oligonucleotides and siRNAs are processed by the
RNA surveillance machinery
Walt F. Lima, Cheryl L. De Hoyos, Xue-hai Liang and Stanley T. Crooke*

Ionis Pharmaceuticals Inc., Carlsbad, CA, USA

Received November 02, 2015; Revised January 15, 2016; Accepted January 25, 2016

ABSTRACT of improperly processed transcripts both before and after

export into the cytoplasm.
DNA-based antisense oligonucleotides (ASOs) elicit Eukaryotic mRNAs contain stabilizing elements, a
cleavage of the targeted RNA by the endoribonu- 5 7-methylguanosine cap and a 3 poly(A) tail that
clease RNase H1, whereas siRNAs mediate cleav- bind to the protein complex containing the 5 -cap bind-
age through the RNAi pathway. To determine the ing protein eIF4E and to the poly(A) binding protein
fates of the cleaved RNA in cells, we lowered the (PABP), respectively, which protect the mRNA from
levels of the factors involved in RNA surveillance exonuclease degradation. Degradation of mRNAs
prior to treating cells with ASOs or siRNA and an- may be deadenylation-dependent or deadenylation-
alyzed cleavage products by RACE. The cytoplasmic independent or may involve endonucleolytic mechanisms.
5 to 3 exoribonuclease XRN1 was responsible for The deadenylation-independent mechanism is associated
the degradation of the downstream cleavage prod- with nonsense mediated decay (NMD), which involves
decapping followed by 5 to 3 degradation by the XRN
ucts generated by ASOs or siRNA targeting mRNAs.
exoribonucleases (1,2). The deadenylation-dependent
In contrast, downstream cleavage products gener- mechanism begins with the shortening of the 3 -poly(A)
ated by ASOs targeting nuclear long non-coding tail by either the CCR4-NOT or PARN deadenylases
RNA Malat 1 and pre-mRNA were degraded by nu- (3–5). Removal of the poly(A) tail results in the release
clear XRN2. The downstream cleavage products did of PABP and the 5 -cap binding complex (6–11). Once
not appear to be degraded in the 3 to 5 direction deadenylation is complete, the LSM1–7 complex binds
as the majority of these products contained intact and recruits the decapping enzymes DCP1 and 2 (12–14).
poly(A) tails and were bound by the poly(A) binding Once deadenylated and decapped, the mRNA is degraded
protein. The upstream cleavage products of Malat1 in the 5 to 3 direction by either the cytoplasmic XRN1
were degraded in the 3 to 5 direction by the exo- exoribonuclease or by the nuclear XRN2 exoribonuclease
some complex containing the nuclear exoribonucle- and in the 3 to 5 direction by the multi-protein exosome
complex (4,15–18).
ase Dis3. The exosome complex containing Dis3 or
The exosome is a multi-protein complex containing the
cytoplasmic Dis3L1 degraded mRNA upstream cleav- six structural subunits Mtr3, Rrp41, rRp42, Rrp43, Rrp45
age products, which were not bound by the 5 -cap and Rrp46/EXOSC5 arranged in a ring configuration (18–
binding complex and, consequently, were suscepti- 21). The structural core is capped with the Csl4, Rrp4 and
ble to degradation in the 5 to 3 direction by the XRN Rrp40 proteins (22). Depending on its subcellular localiza-
exoribonucleases. tion, the structural core recruits the various catalytically ac-
tive exonuclease subunits Rrp6/Exosc10, the nuclear exonu-
clease Rrp44/Dis3 or cytoplasmic exonuclease Dis3L1 (18–
INTRODUCTION 21). These exonuclease subunits bind to the core ring struc-
mRNA surveillance mechanisms are utilized by organisms ture at the surface opposite the exosome cap structure (22).
to ensure fidelity and quality of RNA molecules. A num- A highly efficient means of destroying mRNA begins with
ber of surveillance mechanisms function at various steps endonucleolytic cleavage of the mRNA. Several endoge-
of RNA biogenesis to mark aberrant RNA molecules for nous endoribonucleases have been shown to target mR-
degradation. In eukaryotes, these mechanisms are known to NAs including PMR1, IRE1 and the RNase MRP (23–28).
function in both the nucleus and cytoplasm. Fidelity checks These endoribonucleases are both highly specific and tightly
of mRNA molecules in the nucleus result in the degradation regulated. RNase MRP, for example, is restricted to the nu-

* To whom correspondence should be addressed. Tel: +1 760 603 2301; Fax: +1 760 603 4561; Email: [email protected]

C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact
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3352 Nucleic Acids Research, 2016, Vol. 44, No. 7

cleolus and mitochondria (24,25,29). Following endonucle- were grown to ≈70–80% confluency in 10-cm dishes and
olytic cleavage of the mRNA, the 5 fragment (upstream transfected with a final concentration of 10 nM siRNA
cleavage product) and 3 fragment (downstream cleavage using RNAiMAX (Life Technologies) for 4 h using the
product) is presumably degraded by the RNA surveillance protocol supplied by the manufacturer. After 48 h, cells
machinery, although the specific mechanism is unclear. were transfected with either a final concentration of 50 nM
Short interfering RNAs (siRNAs) may initiate endonu- target ASO or 10 nM siRNA using Lipofectamine 2000
cleolytic cleavage of mRNAs mediated by endoribonucle- (Life Technologies) or RNAiMAX for 4 h, respectively.
ase Argonaute-2 (Ago2), which is followed by degradation As negative controls, cells were mock-transfected using the
of the downstream cleavage products by either XRN1 or same transfection reagent but without ASO or siRNA. The
XRN4 (30–33). This finding is consistent with the cyto- mock-transfected cells are referred to as untreated controls
plasmic localization of XRN1 and Ago2. In addition, some (UTC). For shRNA treated cells, the shRNAs (Vigene Bio-
studies have claimed that siRNAs function in the nucleus sciences) are delivered via lentivirus transduction to cells
(34,35). However, the activity of siRNAs in the nucleus has overlayed with DMEM at a multiplicity of infection (MOI)
been controversial. In our laboratory, we have not observed of 100–500. Transfection with the target ASO or siRNA was
reproducible Ago2 mediated siRNA activity in the nucleus performed 144 h post shRNA adenovirus treatment as de-
(36). Micro-RNAs (miRNA) have also been shown to de- scribed previously.
grade mRNA, but in contrast to siRNAs, miRNA medi-
ated degradation does not involve endonucleolytic cleav-
Western blot
age of the mRNA. Instead, miRNA mediated degrada-
tion of the mRNA occurs in P-bodies requiring the RNA- At 24 h post-transfection, cells were lysed in RIPA buffer
binding protein GW182 and the DCP1:2 decapping com- (Sigma or Santa Cruz Biotechnology) for 15–30 min on
plex (37). DNA-based antisense oligonucleotides (ASOs) ice. The lysate was cleared by top speed centrifugation be-
have been shown to degrade mRNAs and nuclear-retained fore the addition of 4X LDS sample buffer (Life Technolo-
RNAs via RNase H1-mediated endoribonucleolytic cleav- gies) and heated at 95◦ C for 3 min. Samples were separated
age (38). Surprisingly, despite decades of use, the mech- in a 4–12% Bis-Tris polyacrylamide gel (Life Technologies)
anisms by which these mRNA cleavage products are de- according to the manufacturer’s instructions. The proteins
graded remain poorly understood. were transferred to a nitrocellulose membrane via the iBlot
To better understand the mechanisms involved in de- system (Life Technologies). The nitrocellulose membrane
grading the cleavage products generated by RNase H1- was blocked in a solution of PBST (PBS containing 0.1%
activating ASOs and siRNAs, the levels of the key fac- Tween) and 5% non-fat milk for 1 h. The blot was then
tors involved in RNA surveillance were reduced in cells incubated in a solution containing primary antibody di-
using siRNAs or an shRNA. The cells were then treated luted 1:500–1:1000 in PBST overnight (Santa Cruz Biotech-
with ASOs or siRNA targeting a nuclear-retained non- nology for XRN1: sc-98459, XRN2: sc-99237, GAPDH:
coding RNA, pre-mRNA or mRNAs. The stabilities of sc-365062. Abcam for EXOSC5 : ab168804, Histone H3:
the cleavage products were determined by PCR, 5 -RACE ab1791. Sigma for Tubulin: T6199). The blot was washed
or 3 -RACE. The binding interactions between the target four times in PBST followed by incubation with secondary
RNA cleavage products and the 5 -cap and poly(A) pro- antibody conjugated to horse-radish peroxidase (Bio-Rad
tein complex were determined by immunoprecipitation with Laboratories) diluted 1:2500 in PBST for 1 h. Following
PABPC1 or eIF4E. In this study, we identified both the fac- three washes in PBST and a final wash in PBS, ECL reagent
tors involved in the degradation of the upstream and down- (GE Healthcare or Millipore) was applied to the membrane
stream cleavage products as well as the proteins bound to for 5 min followed by exposure to Kodak film from 30 sec
the cleavage products. to 20 min.

MATERIALS AND METHODS RNA isolation and RT-qPCR

Preparation of oligonucleotides At 24 h post-transfection, total RNA was isolated using
Synthesis and purification of modified ASOs were per- TRIzol (Life Technologies) per the manufacturer’s instruc-
formed using an Applied Biosystems 380B automated DNA tions. Briefly, growth medium was removed from cells, and
synthesizer as described previously (38). DNA and RNA cells were washed once with PBS. TRIzol was added directly
oligonucleotide probes used for 5 - or 3 -RACE and quan- to the plate, and cells scraped and harvested. The aqueous
titative real-time PCR (RT-qPCR) are listed in Supple- phase of a phenol-chloroform extraction was mixed with
mentary Figure S1 and Supplementary Materials. The se- isopropanol to precipitate the total RNA. The RNA was
quences or catalog numbers of siRNAs used in this study further treated with DNase I (Life Technologies) to avoid
are listed in Supplementary Materials. false-positives caused by DNA contamination and then re-
precipitated. For RT-qPCR, 1–2ug total RNA was used to
synthesize cDNA using StepOne Real-Time PCR System
Transfection of HeLa cells with siRNA, shRNA and ASOs
at 48◦ C for 30 mins, followed by qPCR using Express One–
HeLa cells were maintained in Dulbecco’s Modified Ea- Step Superscript RT-qPCR Kit (Life Technologies). Primer
gle Media (DMEM, Life Technologies), supplemented with probe sets include 2 primers (a forward and reverse) as well
10% fetal bovine serum and 1% penicillin/streptomycin as an internal probe and were selected to be either upstream
at 37◦ C in an atmosphere of 7.5% CO2 /92.5% air. Cells or downstream of the amplicon site (Supplementary Figure
 Nucleic Acids Research, 2016, Vol. 44, No. 7 3353

S1). For all RT-qPCR reactions, we report means and stan- Poly(A) isolation and immunoprecipitation
dards deviations from 3 independent experiments with trip-
Poly(A) isolations were performed using 20 ␮g of total
licate samples in each experiment and the data are normal-
RNA and the Oligotex mini kit (QIAGEN) following the
ized to Ribogreen (Life Technologies). The primer probe
manufacturer’s instructions. Poly(A) RNA was quantified
sets used in RT-qPCR are listed in Supplementary Figure
by RT-qPCR using StepOne Real-Time PCR System (Life
S1 and Supplementary Materials.
Technologies). The primers and probes used for RT-qPCR
are listed in Supplementary Figure S1.
Nuclear/cytoplasmic fractionation and PCR PABPC1 and EIF4E were immunoprecipitated from
lysates following the Magna RIP Protocol (Millipore).
Untreated or treated cells were washed briefly in PBS to re-
Briefly, magnetic Protein A/G beads (Millipore) contain-
move any residual media and pelleted. The cell pellet was
ing PABPC1 antibody (Millipore 03–101), EIF4E antibody
resuspended in NP-40 lysis buffer (150 mM NaCl, 0.6% NP-
(Abcam76256) or Rabbit IgG (Millipore) were incubated
40, 10 mM Tris HCl, pH 8.0 and 0.1 mM EDTA) and incu-
with equal amounts of the treated lysate for 2 h at 4◦ C.
bated on ice for 10 min. The lysate was spun at 4400 RPM
The beads were washed five times in wash buffer provided in
for 1.5 min, and the supernatant (cytoplasmic fraction) was
the kit followed by a final wash in PBS. Following the final
collected. The pellet (nuclear fraction) was washed in DOC
wash, samples were treated with TRIzol and the RNA was
wash buffer (150 mM NaCl, 0.6% NP-40, 10 mM Tris HCl,
isolated as described previously. RNA in the supernatant
pH 8.0, 0.1 mM EDTA and 0.5% deoxycholate), spun at
was analyzed using RT-qPCR, 5 -RACE or 3 -RACE.
7500 RPM for 1.5 min and lysed in RIPA buffer. Cytoplas-
mic and nuclear RNAs were prepared using TRIzol. Puri-
ties of the fractions were determined by western blot analy- RESULTS
sis or RT-qPCR of known nuclear and cytoplasmic proteins
Downstream cleavage products are degraded by the XRN ex-
and RNAs, respectively. To assess target reduction in each
oribonucleases
fractionated compartment, equal amounts of RNAs (≈50
ng) from each preparation were used in RT-qPCR reaction We designed ASOs to target α-Actinin and PTEN mRNAs
with gene-specific primer probe sets and AgPath-ID On- and Malat 1, a nuclear retained long non-coding RNA, and
estep RT-PCR kit (Life Technologies), based on the manu- an siRNA to target PTEN mRNA (Figure 1A). The ac-
facturer’s procedures. RT-qPCR was performed in Step-one tivities of the ASOs and siRNA were determined by RT-
qPCR system, in following programs: 48◦ C, 30 min, 94◦ C, qPCR using primer/probe sets (PPS) designed to bind ei-
10 min, following by 40 cycles of 94◦ C, 20” and 60◦ C, 1’. ther the upstream or downstream cleavage products (Sup-
The primer probe sets used in RT-qPCR are listed in Sup- plementary Figure S1A). The ASOs and siRNAs used in
plementary Figure S1 and Supplementary Materials. this manuscript are the products of extensive screens to
identify the most potent and selective reagents and typically
several siRNAs and ASOs were studied and these served
5 - and 3 -RACE
as controls (data not shown). The ASOs and siRNA re-
RACE was performed using Life Technologies RACE sys- duced complementary target RNAs by 80–97% compared
tem according to the manufacturer’s instructions with the to the levels in control cells treated with transfection reagent
following modifications: The CIP and TAP procedures were alone (Figure 1B). The upstream and downstream cleavage
not performed due to the fact that RNase H- and Ago2- products appeared to be degraded with similar efficiencies
mediated cleavages generate 5 -phosphorylated RNA re- (Figure 1B). Nuclear and cytoplasmic fractions were pre-
quired for ligation of the 5 -RACE adapter. Total RNA in- pared and the purity of the fractions was determined by RT-
put was increased to 2–5 ␮g and was ligated to the spe- qPCR using PPS designed to bind Malat1 or NEAT1 nu-
cific adapter using RNA ligase (Life Technologies). Reverse clear lncRNAs and the cytoplasmic RN7SL1 RNA (Figure
transcription and amplification was performed as described 1C–F, left panels, and Supplementary Figure S1A; 39,40).
in the materials and methods for PCR with the cycling ex- Note that RN7SL1 RNA was barely detected in the nu-
tended to 37X. Following outer PCR, nested PCR was per- clear fractions, indication that there was little cytoplasmic
formed. For 3 -RACE, the upstream fragment contains a contamination in the nuclear fractions. The targeted RNAs
3 -OH after RNase H and Ago2 cleavage so the 3 -adapter were quantified in nuclear and cytoplasmic fractions using
contains both a 5 - and a 3 -Phosphate (this is to prevent primers designed to bind downstream of the ASO or siRNA
ligation of the adapter to itself to try to maximize ligation binding sites. Malat1 RNA was observed to be enriched in
to the target RNA). the nuclear fraction, and treatment with the Malat1 ASO re-
The sequences of the primers are listed in Supplemen- sulted in the loss of the Malat1 RNA in the nuclear fraction
tary Figure S1. The PCR products were gel purified in a 2% (Figure 1C, right panel). The α-Actinin and PTEN mRNAs
agarose TBE gel and extracted and purified using QIAquick were detected in both the cytoplasmic and nuclear fractions,
gel extraction kit (QIAGEN) following the manufacturer’s though the majority of the mRNAs were observed in the
instructions. The samples were ligated to pCR 4-TOPO vec- cytoplasmic fraction, as expected (Figure 1D,E, right pan-
tor by TA cloning (Life Technologies). After transforma- els). Treatment with their respective ASOs resulted in loss
tion into Mach1 cells, plasmid DNA was isolated from sev- of the mRNA from both fractions (Figure 1D,E, right pan-
eral colonies. The correct inserts were verified by EcoRI di- els). Consistent with the expected Ago2-mediated cleavage
gestion and further verified by sequencing (Retrogen or Ge- upon treatment of cells with siRNA, reduction of PTEN
newiz). mRNA was observed in the cytoplasmic fraction, but not
3354 Nucleic Acids Research, 2016, Vol. 44, No. 7

Figure 1. ASO- or siRNA-mediated reduction of target RNA and subcellular localization of targeted RNAs. (A) In sequences of the ASOs and siRNA,
the orange, blue, red and black letters represent, respectively, 2 -methoxyethylribonucleotides, 2 -constrained ethylribonucleotides, 2 -ribonucleotides and
2 -deoxyribonucleotides. The siRNA was prepared by annealing the guide strand (top sequence) with complementary length matched RNA (bottom
sequence). ASOs had phosphorothioate linkages; linkages in the siRNA were phosphodiester. (B) Target RNA levels were determined 24 h post ASO or
siRNA treatment by RT-qPCR and are shown as a percentage of untreated controls. Dark and light gray bars correspond to, respectively, the upstream
and downstream target RNA cleavage products. (C–F) Cells treated with ASOs targeting Malat1 (C), ␣-Actinin (D), PTEN (E) or with a siRNA targeting
PTEN (F) were collected and cytoplasmic and nuclear fractions were separated. The purity of the nuclear and cytoplasmic fractions (left panels) was
determined by RT-qPCR using primers targeting, respectively, the cytoplasmic RNA RN7SL1 and nuclear NEAT1 or Malat1 RNA. The levels of the
RN7SL1 RNA are shown as a percentage of the level in the cytoplasmic fraction in control cells; whereas the levels of NEAT1 or Malat1 RNA are shown
as a percentage of the level in the nuclear fraction in control cells. RT-qPCR analyses of the target RNA downstream cleavage products were performed
on the cytoplasmic and nuclear fractions from control cells treated with transfection reagent only (UTC) or ASO/siRNA treated cells (right panels, Y-axis
is shown as logarithmic scale). The qPCR primers are located downstream to the cleavage sites on the target RNAs. The PPS used for the RT-qPCR are
listed in Supplementary Figure S1A. The predicted sizes (base pairs) of the PCR products are: Malat1 495; ␣-Actinin 432; PTEN ASO 435; PTEN siRNA
435. The error bars represent standard deviation from three independent experiments. Statistics analysis was performed using unpaired t-test. *, P < 0.05;
**, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

obviously in the nuclear fraction, of the cells treated with stream cleavage products, XRN1 and XRN2 would de-
PTEN siRNA (Figure 1F, right panel). Similar results were grade, respectively, cytoplasmic and nuclear RNA frag-
obtained using primers targeting the regions upstream of ments. To evaluate this, siRNAs targeting the XRN mR-
the ASO and siRNA hybridization sites (data not shown). NAs were used to reduce XRN levels in cells. The activ-
Consistent with previous reports, western blot analysis ities of the siRNAs targeting the XRN mRNAs were de-
of the XRN proteins following cell fractionation showed termined by qRT-PCR (Figure 2B). Greater than 90% re-
that XRN1 was predominantly localized in the cytoplasm ductions in XRN proteins were observed 48-h post siRNA
and XRN2 was predominantly in the nucleus (Figure 2A). treatment (Figure 2C). After treating cells with an XRN-
Thus, if XRNs are involved in the degradation of the down- targeted siRNA, cells were treated with ASO or siRNA tar-
 Nucleic Acids Research, 2016, Vol. 44, No. 7 3355

Figure 2. XRN exonucleases degrade downstream RNA cleavage products. (A) Subcellular localization of XRNs was determined western blots performed
on cytoplasmic (Cyto) and nuclear (Nuc) fractions of HeLa cells. Purity of the fractions was determined using antibodies to histone H3 and ␣-tubulin. (B)
siRNA-mediated reduction of XRN mRNAs were quantified 48 h post treatment. Cells were transfected with 3-fold serial dilutions of the siRNAs ranging
in concentration from 14 pM to 10 nM. The mRNA levels were determined by RT-qPCR and are shown as percentages of control cells (UTC) treated with
transfection reagent only. The mean and standard errors reported are based on three trials. The PPS used for the RT-qPCR are listed in Supplementary
Figure S1A. Note that the XRN1 siRNA had no effect on XRN2 or vice versa. (C) Western blot analyses of XRN proteins from untreated (UTC) and
siRNA-treated cells 48 h post siRNA treatment. A non-specific band (*) serves as a control for loading. (D–H) Cells were either treated or not with siRNA
targeting XRN1 or XRN2 mRNA for 4 h, or were treated with transfection reagent only (UTC). Cells were then treated with Malat1 ASO (panel D), PTEN
siRNA (Panel E), PTEN ASO (Panel F), ␣-Actinin ASO (Panel G) or AR ASO (Panel H), and 5 -RACE was performed 48 h later to detect downstream
cleavage products. The adapter and primers used for the 5 -RACE are listed in Supplementary Figure S1B. RN7SL1 RNA was detected by RT-qPCR from
10% of input RNA for RACE and used as loading controls, as listed below each panel of the 5 RACE results. The error bars represent standard deviation
from three independent experiments. The predicted sizes (base pairs) of the 5 -RACE products are: Malat 1 504; ␣-Actinin 470; PTEN ASO 331; PTEN
siRNA 266.
3356 Nucleic Acids Research, 2016, Vol. 44, No. 7

geting α-Actinin, PTEN, or Malat1, and the downstream 5 -RACE products were observed for targeted RNAs in
cleavage products were detected by 5 -RACE. Ligation of cells treated with Malat1 ASO (Figure 3B), PTEN ASO
the 5 -RACE adaptor requires 5 -phosphorylated RNA and (Figure 3C), ␣-Actinin ASO (Figure 3D) or PTEN siRNA
RNase H and Ago2 generate downstream cleavage prod- (Figure 3E). A faint 5 -RACE band was observed in cells
ucts with a 5 -phosphate; therefore, only the downstream treated with PTEN ASO; however, sequence analysis of
cleavage products and not intact RNA or upstream cleav- the product showed no sequence homology with PTEN
age products were amplified. As a control, RN7SL1 RNA mRNA (data not shown). The failure to detect enrichment
was detected by RT-qPCR from 10% of input RNAs used of 3 fragment in PARN reduced cells appears not to be
in the 5 -RACE, and the relative levels were comparable, as due to insufficient reduction of the protein, since similar
shown below each panel of the 5 -RACE products (Figure treatment with the PARN siRNA led to accumulation of
2D–H). polyadenylated snoRNA ACA45 (Supplementary Figure
5 -RACE products corresponding to the Malat1 RNA S3), a class of snoRNAs known to be targets of PARN (41).
downstream cleavage product following the reduction of 5 -RACE products were only observed with the reduction
XRN2 but not after reduction of XRN1 were detected (Fig- of XRN exoribonucleases suggesting that the deadenylase
ure 2D). The stabilization of the cleavage product in the ab- does not contribute significantly to degradation of RNA
sence of XRN2 suggests that XRN2 degrades the down- downstream cleavage products (Figure 3B–E). As a control,
stream cleavage products of Malat1. The degradation of the relative levels of RN7SL1 RNA detected by RT-qPCR
the Malat1 RNA downstream cleavage products by XRN2 from 10% of input RNAs for 5 -RACE were comparable, as
is consistent with the nuclear localization of both the en- presented below each panel.
zyme and Malat1 RNA (Figure 1D). A second nuclear tar- Given that deadenylases did not appear to be involved
get was then analyzed. An ASO was designed to target an in the degradation of the downstream cleavage products,
intronic region of the androgen receptor (AR) pre-mRNA we evaluated the polyadenylation states of the downstream
(Figure 2H). 5 -RACE products were again observed in ex- cleavage products. Specifically, poly(A) isolations were per-
tracts of cells pretreated with siRNA targeting XRN2 but formed from cells treated with the XRN siRNAs prior to
not those pretreated with XRN1 siRNA (Figure 2H). Se- treatment with Malat1 ASO, ␣-Actinin ASO, or PTEN
quencing of the 5 -RACE products corresponding to the ASO or siRNA (Figure 4A). qRT-PCR amplification was
Malat1 RNA and the androgen receptor pre-mRNA con- performed using PPS that hybridize to regions up or down-
firmed the identity of the RACE products and showed that stream of the ASO/siRNA hybridization sites (Supplemen-
the sites of cleavage were within the regions complementary tary Figure S1A). A previous report showed that Malat1
to the ASOs, consistent with an RNase H1-mediated cleav- RNA does not have a poly(A) tail, and little to no poly(A)
age (Supplementary Figures S2A and S2B). RNA was detected with either Malat1 RNA primer set (Fig-
In contrast, XRN1 appears to be involved in the degra- ure 4A; 42). The levels of poly(A) mRNA isolated from cells
dation of the PTEN mRNA downstream cleavage product treated with the target specific ASOs or siRNA ranged from
that resulted from treatment with the PTEN siRNA. 5 - 7% to 27% of the levels in control cells treated with transfec-
RACE products were observed in cells deficient in XRN1, tion reagent alone when analysis was performed using the
and no 5 -RACE products were observed when XRN2 lev- downstream PPS (Figure 4A). Similar results were obtained
els were significantly reduced (Figure 2E). siRNA-mediated when the upstream PPS was used to amplify mRNA, sug-
Ago2 cleavage activity occurs in the cytoplasm, and the gesting that these co-immunoprecipitated poly(A) mRNAs
results reported here are consistent with the cytoplasmic likely correspond to the full-length mRNAs (Figure 4A).
localization of XRN1 (Figure 1E). Sequencing of the 5 - We then depleted XRN proteins from cells prior to treat-
RACE product confirmed the specificity and showed that ment with ASO or siRNA and analyzed poly(A)-isolated
the sites of cleavage were at mRNA nucleotides expected to RNA using upstream and downstream PPS (Supplemen-
be opposite the tenth and eleventh nucleotides from the 5 - tary Figure S1A). Target mRNA levels detected with the up-
terminus of the siRNA guide strand in the mRNA:siRNA stream PPS were similar when cells were treated with both
duplex (Supplementary Figure S2C). the XRN siRNAs and target-specific ASOs or siRNA or
Consistent with the observation, that ASO-mediated only target-specific ASOs or siRNA (Figure 4A). The up-
degradation of PTEN and α-Actinin RNAs occurred in stream PPS is likely detecting full-length mRNA. In con-
both the nucleus and the cytoplasm (Figure 1D), both trast, when the targeted mRNA was detected using the
XRN1 and XRN2 appear to be involved in the degradation downstream PPS, levels of poly(A) isolated mRNA ranged
of downstream cleavage products resulting from treatment from 60% to 120% for cells treated with both the XRN
of cells with PTEN and ␣-Actinin ASOs (Figure 2F,G). siRNAs and target ASOs or siRNAs compared to the val-
Sequencing of 5 -RACE products were consistent with an ues for the control cells treated with transfection reagent
RNase H1-induced cleavage mechanism (Supplementary alone (Figure 4A). This indicated that a significant frac-
Figures S2D and S2E). tion of the downstream cleavage products was polyadeny-
Next we determined whether degradation of the down- lated supporting our hypothesis that deadenylases do not
stream RNA cleavage products involved deadenylation of contribute significantly to the degradation of downstream
the RNA by reducing the levels of the deadenylases PARN cleavage products.
and CNOT proteins in the cells prior to treatment with tar- Next we determined whether the polyadenylated down-
get ASOs or siRNA. The siRNA treatments reduced the stream cleavage products were bound to PABP proteins by
levels of the targeted RNAs by at least 80% (Figure 3A). immunoprecipitating PABPC1 and associated RNAs from
In cells depleted of PARN or CNOT RNAs, no detectable
 Nucleic Acids Research, 2016, Vol. 44, No. 7 3357

Figure 3. Deadenylases do not contribute significantly to degradation of downstream RNA cleavage products. (A) Cellular levels of XRN1, XRN2, and
indicated deadenylase mRNAs 48 h post siRNA treatment. The mRNA levels were determined by RT-qPCR and are shown as a percentage of untreated
control. The mean and standard errors reported are based on three trials. The PPS used for the RT-qPCR are listed in Supplementary Figure S1A. (B–E)
Cells were treated with individual or combined XRN or deadenylase siRNAs. After 4 h, cells were treated with Malat1 ASO (Panel B), PTEN ASO (Panel
C), ␣-Actinin ASO (Panel D) or PTEN siRNA (Panel E). 5 -RACE of the RNA downstream cleavage products was performed after 48 h. RN7SL1 RNA
was detected by RT-qPCR from 10% of input RNA for RACE and used as loading controls, as shown below each panel of the RACE products. The error
bars represent standard deviation from three independent experiments. The adapter and primers used for the 5 -RACE are listed in Supplementary Figure
S1B.
3358 Nucleic Acids Research, 2016, Vol. 44, No. 7

Upstream cleavage products are degraded by the exosome and

XRNs
The levels of the catalytic components Dis3 and EXOSC10
and the key structural component EXOSC5 of the exo-
some were reduced in cells using siRNAs in order to de-
termine whether the exosome was involved in the degrada-
tion of the target RNA upstream cleavage products. siR-
NAs targeting the 3 to 5 exoribonucleases Dis3L2 and
Dis3L1 were also tested. The siRNAs used reduced levels
of the mRNAs encoding the exosome subunits to less than
30% of levels in untreated cells (Figure 5A). To evaluate
the stabilities of upstream cleavage products in conditions
in which exosome components were depleted, we used 3 -
RACE with 5 -phosphorylated RACE adaptors since the
upstream cleavage products contain a 3 hydoxyl. Under
these conditions only the upstream cleavage products but
not the intact mRNA or the downstream cleavage prod-
ucts were amplified. 3 -RACE products were observed for
the nuclear-retained Malat1 RNA when nuclear Dis3, EX-
OSC10 or EXOSC5 were reduced prior to treating the cells
with the Malat1 ASO (Figure 5B). 3 -RACE products of the
α-Actinin mRNA in cells treated with ␣-Actinin ASO were
observed only when cells were pretreated with nuclear Dis3,
cytoplasmic Dis3L1 or EXOSC5 siRNAs (Figure 5B). Fi-
nally, cells treated with the PTEN siRNA exhibited PTEN
Figure 4. Downstream target RNA cleavage products are polyadenylated 3 -RACE products only after reduction of EXOSC5 (Figure
and bound to PABPC1. (A) Poly(A) RNA was isolated from control cells
treated with transfection reagent alone (gray bars), cells treated with tar- 5B). EXOSC5 is required for the formation of the exosome
get ASOs or siRNA alone (red bars), or from cells treated with both tar- core, and reducing the levels of this key structural subunit
get ASOs/siRNA and XRN1 and XRN2 siRNAs (blue bars). (B) Levels in the cell likely inhibits exosome activity by preventing as-
of the target RNA cleavage products associated with immunoprecipitated sembly of the core structure (18,19). Taken together, these
PABPC1 from untreated cells (gray bars), cells treated with target ASOs
or siRNA alone (red bars) or cells treated with both target ASOs/siRNA
data suggest that the exosome participates in the degrada-
and XRN1 and XRN2 siRNAs (blue bars). Levels of the target RNA cleav- tion of the upstream cleavage products in the nucleus and
age products were determined 48 h post treatment by RT-qPCR and are cytoplasm.
shown as a percentage of untreated controls. The mean and standard er- Given the importance of EXOSC5 protein in the activ-
rors reported are based on three trials. Light and dark hues bars represent, ity of the exosome and to further validate the role of the
respectively, the target RNA upstream and downstream cleavage products.
The PPS used for the RT-qPCR are listed in Supplementary Figure S1A. exosome in the degradation of the target RNA upstream
cleavage products, we treated cells with an shRNA targeting
EXOSC5. The shRNA reduced EXOSC5 mRNA levels by
greater than 95% compared to those in control cells treated
with transfection reagent alone (Figure 5C). In addition,
shRNA-mediated reduction of EXOSC5 protein was ob-
cells depleted of XRN proteins and treated with ASOs
served for up to 144 h (Figure 5D). Cells treated with the tar-
or siRNA. Although eukaryotic cells contain one nuclear
get ASOs or siRNA alone resulted in little to no 3 -RACE
and four cytoplasmic PABP proteins, PABPC1 has been
products (Figure 5E). Conversely, 3 -RACE products were
shown to be the most abundant isoform (43). Approxi-
observed in cells treated with both the EXOSC5 shRNA
mately 10% of the Malat1 RNA, which lacks a poly(A)
and target ASOs or siRNA (Figure 5E), indicating that the
tail, co-immunoprecipitated with PABPC1 (Figure 4B; 42).
exosome is involved in degradation of upstream cleavage
Absent XRN reduction but with ASO or siRNA treat-
fragments. The 3 -RACE products were confirmed by se-
ment, the levels of α-Actinin and PTEN mRNAs that co-
quencing and were consistent with expected cleavage mech-
immunoprecipitated with PABPC1 ranged from 12% to
anisms (Supplementary Figure S2F–I). We also reduced a
22% of the levels in control cells treated with transfec-
number of other nucleases such as ERI1 and CNOT6L and
tion reagent alone (Figure 4B). In contrast, the amounts of
reduction of these did not stabilize either 5 or 3 fragment
α-Actinin and PTEN mRNA co-immunoprecipitated with
(Supplementary Figure S4).
PABPC1 from cells treated with both the XRN siRNAs and
To determine whether upstream cleavage products asso-
the target ASOs or siRNA were 4–5-fold higher than those
ciate with PABP, cells were treated with the shRNA tar-
in cells treated with the target ASOs or siRNA alone (Figure
geting EXOSC5. To ensure that the downstream cleavage
4B). As a negative control, immunoprecipitation using IgG
product/PABP complex remained intact, cells were also
only co-selected less than 2% of input Malat1, PTEN or Ac-
treated with siRNAs targeting XRNs. Finally, cells were
tinin RNAs (data not shown). These data suggest that a sig-
treated with either ASOs or siRNAs targeting α-Actinin
nificant fraction of the polyadenylated downstream cleav-
or PTEN mRNAs, and the levels of upstream cleavage
age products bind PABPC1.
 Nucleic Acids Research, 2016, Vol. 44, No. 7 3359

Figure 5. Exosome complex degrades upstream cleavage products. (A) Cellular levels of indicated mRNAs 48 h post siRNA treatment. The mRNA levels
were determined by RT-qPCR and are shown as a percentage of untreated controls. The mean and standard errors reported are based on three trials.
(B) 3 -RACE of the target RNA upstream cleavage products from ASO or siRNA treated and control cells (UTC) treated with transfection reagent only.
3 -RACE was performed 48 h post treatment with Malat 1, ␣-Actinin or PTEN ASOs or siRNA alone or with both target-specific ASO/siRNA and
exosome/exonuclease siRNAs. The predicted sizes of the 3 -RACE products are: Malat1 378; ␣-Actinin 423; PTEN ASO 439; PTEN siRNA 386. (C)
Cellular level of EXOSC5 mRNA 48 h post shRNA treatment. (D) Western blot analysis of EXOSC5 protein from untreated (UTC) and siRNA treated
cells 48 h to 144 h post siRNA treatment. (E) 3 -RACE of upstream cleavage products was performed on extracts of cells treated with Malat1, ␣-Actinin,
or PTEN ASOs or siRNA alone or first treated 144 h with EXOSC5 shRNA and then with target-specific ASO/siRNA. The PPS used for the RT-qPCR
are listed in Supplementary Figure S1A. The adapter and primers used for the 3 -RACE are listed in Supplementary Figure S1C.

products co-immunoprecipitated with PABPC1 were deter- products were observed in extracts of cells depleted of EX-
mined using the upstream PPS (Supplementary Figure S1). OSC5 (Figure 6C). Importantly, the signal from 3 -RACE
Unlike the target RNA downstream cleavage products, the products appeared to be enhanced when both EXOSC5 and
upstream cleavage products did not co-immunoprecipitate XRN protein levels were reduced compared to EXOSC5
with PABPC1 (Figure 6A). alone suggesting that both the exosome and XRNs partic-
Next, we immunoprecipitated the 5 -cap binding pro- ipate in the degradation of upstream cleavage products re-
tein eIF4E and associated RNA from cells treated with sulting from ASO or siRNA treatment (Figure 6C).
the EXOSC5 shRNA, XRN siRNAs, and/or target ASOs
or siRNA. eIF4E was chosen since it binds cytoplasmic
mRNAs which represent majority of mature mRNAs, even DISCUSSION
though a small portion of mRNAs may associate with The RNA surveillance mechanism relies predominately on
CBC20/80 complex in the nucleus (44). The upstream cleav- exoribonuclease activity to degrade unnecessary or aberrant
age products of α-Actinin and PTEN mRNAs did not co- RNA. Both antisense oligonucleotides and siRNAs have
immunoprecipitate with eIF4E (Figure 6B). The lack of significant clinical potential. These agents induce cleavage
binding between the upstream cleavage products and ei- of a targeted RNA by recruiting RNase H1 and the RNAi
ther PABPC1 or eIF4E suggests that the 5 terminus of machinery, respectively. We sought to determine the fates of
the upstream cleavage products may be susceptible to 5 the products of ASO- and siRNA-mediate cleavage.
to 3 exonuclease degradation. To determine whether the mRNAs are protected from exonucleases with a 5 7-
XRNs participate in the degradation of the upstream cleav- methylguanosine cap and 3 -poly(A) tail, whereas non-
age products, we performed 3 -RACE from cells in which coding RNAs often have stable structures that protect
the levels of the EXOSC5 only or both EXOSC5 and XRNs against exoribonuclease degradation. For example, the
were reduced prior to treatment with the target ASOs or nuclear-retained long non-coding RNA Malat1 has a 7-
siRNA. The number of amplification cycles was reduced in methylguanosine cap and a triple helical structure, instead
order to better differentiate the 3 -RACE signals. 3 -RACE of a poly(A) tail, protects the 3 terminus (42). Given the
3360 Nucleic Acids Research, 2016, Vol. 44, No. 7

Figure 6. Upstream cleavage products are not bound by PABPC1 or eIF4E and are degraded by both XRNs and the exosome complex. (A and B) Levels
of the target RNA upstream cleavage products associated with (A) PABPC1 or (B) eIF4E from untreated cells (gray bars), cells treated with target ASOs or
siRNA alone (red bars), or cells treated first with XRN siRNAs and EXOSC5 shRNA and then with ASO or siRNA (blue bars). Levels of the target RNA
cleavage products were determined 48 h post treatment by RT-qPCR and are shown as a percentage of their respective untreated controls (transfection
reagent only). (C) Cells were treated with EXOSC5 shRNA or XRN siRNAs and EXOSC5 shRNA and then with ASO or siRNA. 3 -RACE of the target
RNA upstream cleavage products was performed 48 h post treatment. The PPS used for the RT-qPCR are listed in Supplementary Figure S1A. The adapter
and primers used for the 3 -RACE are listed in Supplementary Figure S1C.

complex nature of mRNA biogenesis, these RNAs contain orientation for an inline nucleophilic attack of the scissile
additional stabilizing factors. Some mRNAs are further phosphate results in cleavage products with a 3 hydroxyl
protected in the cytoplasm against exoribonuclease degra- and 5 phosphate (46).
dation by a protein complex containing 5 -cap and poly(A) Given their prominent role in the RNA surveillance ma-
binding proteins, which effectively sequester the termini of chinery, it was not surprising that the XRN exoribonu-
the mRNA. As a result, degradation of these mRNAs be- cleases were involved in the degradation of upstream and
gins with dissociation of the cap/poly(A) protein complex downstream products resulting from ASO- and siRNA-
from the mRNA by shortening of the poly(A) tail, decap- mediated cleavage. The XRN enzymes degraded the tar-
ping the mRNA or initiation of translation (4). get RNA upstream cleavage products, presumably follow-
An effective RNA degradation mechanism that circum- ing decapping of the target RNA; our analysis indicated
vents the prerequisite for disruption of the 5 -cap/poly(A) that these products did not contain the 5 -cap binding pro-
complex involves production of unprotected termini tein eIF4E and likely were decapped (Figure 6B). Nuclear
through endoribonucleolytic cleavage mediated by en- XRN2 was responsible for degrading the unprotected 5 ter-
doribonucleases RNase H1 and Ago2. These unprotected minus of the downstream cleavage products generated by
termini are ideal substrates for exoribonucleases since cleav- ASOs targeting the nuclear-retained Malat1 and androgen
age by RNase H1 and Ago2 produces a 5 -phosphorylated receptor pre-mRNA. Consistent with the observed cyto-
terminus, the preferred substrate for XRN exoribonu- plasmic activity of the siRNA targeting the PTEN mRNA,
cleases (15,16). The substrate preferences of the XRN the downstream cleavage of the PTEN mRNA was de-
enzymes are likely the result of the synergistic activities graded by cytoplasmic XRN1 (Figure 2D). Conversely, the
between the XRNs and the decapping enzymes DCP1 and downstream cleavage products of the α-Actinin and PTEN
2, which also produce unprotected 5 -phosphorylated RNA mRNAs generated by ASO treatment were degraded by
(45). Whether the cleavage products generated by RNase both XRN1 and XRN2 (Figure 2D). Consistent with these
H1 and Ago2 evolved to function synergistically with the observations and the fact that RNase H1 is found in both
XRN enzymes is unclear, but the structure of the RNase the nucleus and cytoplasm, we observed ASO-mediated
H1 and Ago2 substrates suggest that it is unlikely. RNase cleavage of mRNAs in both the nucleus and cytoplasm (Fig-
H1 and Ago2 cleave the RNA within a double-stranded ure 1D). As ASOs are capable of targeting regions of pre-
structure and the position of the scissile phosphate within mRNAs, XRN2 may degrade downstream cleavage prod-
the double-stranded structure is such that the only possible ucts resulting from these events.
 Nucleic Acids Research, 2016, Vol. 44, No. 7 3361

Figure 7. Degradation pathway for RNA cleavage products generated by treatment with ASO or siRNA. In the nucleus, XRN2 and either the exosome
containing Dis3 and /or EXOSC10 degrades the nuclear-retained target RNA upstream cleavage products and XRN2 degrade the downstream cleavage
products. The same pathway may also be involved in the degradation of mRNA cleavage products generated by ASOs in the nucleus and/or these cleavage
products may be exported to the cytoplasm for processing by XRN1, the exosome and/or Dis3L1. In the cytoplasm, mRNA cleavage products resulting
from treatment with ASOs or siRNAs are degraded by XRN1, the exosome and/or Dis3L1.

The exosome complex containing the nuclear Dis3 not play a significant role in the degradation of downstream
and/or EXOSC10 catalytic subunits was responsible for cleavage products (Figure 4).
the 3 to 5 degradation of the nuclear-retained RNA up- Taken together, our analysis reveals the major degrada-
stream cleavage products (Figure 5B,E). Both the nuclear tion pathways for cleavage products generated by treatment
Dis3 exosome complex and cytoplasmic Dis3L1 exoribonu- of cells with ASOs and siRNAs (additional minor path-
clease were responsible for the 3 to 5 degradation of the ways may exist, as we have not studied all possible nucle-
mRNA upstream cleavage products suggesting that degra- ases; Figure 7). Following siRNA-mediated Ago2 cleavage
dation likely occurs in both cellular compartments (Figure of mRNA in the cytoplasm, the downstream cleavage prod-
5B,E). Interestingly, cytoplasmic Dis3L1 did not appear to ucts, containing a poly(A) tail and bound by PABPC1, are
play a role in the degradation of the upstream cleavage prod- degraded predominantly in the 5 to 3 direction by XRN1.
ucts generated by the siRNA suggesting that different path- The upstream cleavage products, which were not bound by
ways maybe involved in the degradation of siRNA and ASO the 5 -cap binding protein eIF4E, are presumably decapped
cleavage products (Figure 5B). For example, RISC compo- and then degraded in the 5 to 3 direction by XRN1 and
nents are known to localize in P-bodies where siRNA me- in the 3 to 5 direction by the exosome complex. A similar
diated mRNA cleavage and degradation may occur (47), mechanism was observed for products of the ASO-mediated
whereas RNase H1 was not detected in P-bodies (our un- RNase H1 cleavage of the target mRNAs which mainly lo-
published data). Perhaps due to the redundant nature of calize in the cytoplasm. Consistent with these observations,
activities, reduction of either of the catalytic subunits of the the 3 to 5 cytoplasmic exoribonuclease Dis3L1 also ap-
exosome alone did not significantly enhance the stability of peared to be involved in the degradation of the mRNA up-
the target RNA upstream cleavage products (Figure 5B). stream cleavage products generated by ASOs (Figure 5B).
The most consistent enhancement in the stability of the up- In the case of the nuclear-retained target RNAs, degrada-
stream cleavage products were observed following the re- tion of the downstream cleavage products involved XRN2,
duction of EXOSC5. Reductions in levels of this scaffold- whereas the upstream cleavage products were degraded by
ing protein presumably inhibited assembly of an active ex- the exosome complex containing nuclear Dis3 and/or EX-
osome complex (5B and 5E). Surprisingly, the exosome did OSC10 and exoribonuclease XRN2. Our data do not sup-
port exclusion of the possibility that for some RNAs, RNase
3362 Nucleic Acids Research, 2016, Vol. 44, No. 7

H1 could cleave the target in both the nucleus and cyto- 16. Geisler,S.C. and J. (2012) In: Chanfreau,GF and Tamanoi,F (eds).
plasm and some fragments generated in the nucleus might Eukaryotic RNases and Their Partners in RNA Degradation and
Biogenesis. Academic Press, Vol. 31, pp. 97–114.
escape nuclear degradation and be exported to the cyto- 17. Houseley,J., LaCava,J. and Tollervey,D. (2006) RNA-quality control
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ACKNOWLEDGEMENTS exosome. EMBO J., 29, 2358–2367.
21. Liu,Q., Greimann,J.C. and Lima,C.D. (2006) Reconstitution,
We thank Donna Parrett for excellent administrative sup- activities, and structure of the eukaryotic RNA exosome. Cell, 127,
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22. Tomecki,R., Kristiansen,M.S., Lykke-Andersen,S., Chlebowski,A.,
Reigle for figure preparation. Larsen,K.M., Szczesny,R.J., Drazkowska,K., Pastula,A.,
Andersen,J.S., Stepien,P.P. et al. (2010) The human core exosome
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FUNDING hDIS3L. EMBO J., 29, 2342–2357.
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Ionis Pharmaceuticals has funded the work for this Dompenciel,R.E. and Schoenberg,D.R. (1998) A polysomal
manuscript. Funding for open access charge: Ionis Pharma- ribonuclease involved in the destabilization of albumin mRNA is a
ceuticals, Inc. novel member of the peroxidase gene family. RNA, 4, 1537–1548.
Conflict of interest statement. None declared. 24. Yang,F., Peng,Y. and Schoenberg,D.R. (2004)
Endonuclease-mediated mRNA decay requires tyrosine
phosphorylation of polysomal ribonuclease 1 (PMR1) for the
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