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RNA Biology 10:10, 1602–1608; October 2013; © 2013 Landes Bioscience
POINTOFVIEW
1602 RNA Biology Volume 10 Issue 10
Non-coding Y RNAs as tethers and gates
Insights from bacteria
Sandra L Wolin,1,2,* Cedric Belair,1 Marco Boccitto,1 Xinguo Chen,1 Soyeong Sim,1 David W Taylor,2 and Hong-Wei Wang2,3
1Department of Cell Biology; Yale School of Medicine; New Haven, CT USA
2Department of Molecular Biophysics and Biochemistry; Yale School of Medicine; New Haven, CT USA
3Tsinghua-Peking Center for Life Sciences; School of Life Sciences ; Tsinghua University; Beijing, P.R. China
Non-coding RNAs (ncRNAs) called
Y RNAs are abundant components
of both animal cells and a variety of bacte-
ria. In all species examined, these ~100 nt
RNAs are bound to the Ro 60 kDa (Ro60)
autoantigen, a ring-shaped protein that
also binds misfolded ncRNAs in some
vertebrate nuclei. Although the function
of Ro60 RNPs has been mysterious, we
recently reported that a bacterial Y RNA
tethers Ro60 to the 3′ to 5′ exoribonuclease
polynucleotide phosphorylase (PNPase)
to form RYPER (Ro60/Y RNA/PNPase
Exoribonuclease RNP), a new RNA degra-
dation machine. PNPase is a homotrimeric
ring that degrades single-stranded RNA,
and Y RNA-mediated tethering of Ro60
increases the effectiveness of PNPase in
degrading structured RNAs. Single par-
ticle electron microscopy of RYPER sug-
gests that RNA threads through the Ro60
ring into the PNPase cavity. Further stud-
ies indicate that Y RNAs may also act as
gates to regulate entry of RNA substrates
into the Ro60 channel. These findings
reveal novel functions for Y RNAs and
raise questions about how the bacterial
findings relate to the roles of these ncRNAs
in animal cells. Here we review the litera-
ture on Y RNAs, highlighting their close
relationship with Ro60 proteins and the
hypothesis that these ncRNAs function
generally to tether Ro60 rings to diverse
RNA-binding proteins.
What Are Y RNAs?
Y RNAs were discovered because these
ncRNAs are complexed with the Ro60
protein, a frequent target of the immune
system in patients suffering from two
common rheumatic diseases, systemic
lupus erythematosus, and Sjogren’s syn-
drome.1–4 Characterization of the four dis-
tinct Y RNAs in human cells revealed that
these ncRNAs (called hY1, hY3, hY4, and
hY5; h stands for human) are 83–112 nt
long and transcribed by RNA polymerase
III.3,5–10 The number of distinct Y RNAs
varies between species. For example,
mouse cells contain only two Y RNAs,
mY1 and mY3, which are orthologs of hY1
and hY3.5 A defining feature of animal
cell Y RNAs is that these RNAs fold into
structures consisting of a large internal
loop and a long stem formed by basepair-
ing the 5′ and 3′ ends of the RNA.6 –8, 11
Near the base of the stem is a conserved
sequence that is the high affinity binding
site for Ro60 (refs. 12–15 and Fig. 1A).
Most Y RNAs in cells are bound to
Ro60, a doughnut-shaped RNA-binding
protein that also binds misfolded ncRNA
precursors in some animal cell nuclei.15 –18
Ro60 orthologs are present in most ani-
mal cells and also in ~5% of sequenced
bacterial genomes.19 Immunoprecipitation
experiments from mouse and human cells
have demonstrated that most Y RNAs are
present as Ro60 RNPs (refs. 7 and 20 and
Fig. 2A and C). As worms, mouse cells,
and bacteria lacking Ro60 all have drasti-
cally reduced Y RNAs, Ro60 is required
for stable accumulation of these RNAs
(refs. 18 and 21–23 and Fig. 2B). Ro60
also stabilizes human Y RNAs, as experi-
ments in which we used siRNAs to deplete
Ro60 by 83% from human keratinocytes
Keywords: non-coding RNAs,
Y RNAs, exoribonucleases,
RYPER, RNA degradation
*Correspondence to: Sandra L Wolin;
Email: sandra.wolin@yale.edu
Submitted: 07/19/2013; Accepted: 08/15/2013
http://dx.doi.org/10.4161/rna.26166
Citation: Wolin SL, Belair C, Boccit to M, Chen
X, Sim S, Taylor DW, Wang H. Non-coding
Y RNAs as tethers and gates: Insights from
bacteria. RNA Biology 2013; 10:In press
©2012 Landes Bioscience. Do not distribute.
www.landesbioscience.com RNA Biology 1603
POINTOFVIEW POINTOFVIEW
revealed that these RNAs were reduced
between 30 and 90%, depending on the
RNA (Fig. 2D and E).
In addition to Ro60, some Y RNAs in
animal cells are bound by the La autoan-
tigen, a nuclear phosphoprotein that binds
all newly synthesized RNA polymerase
III transcripts.5,24 Like other La-bound
RNAs, Y RNAs initially end in uridines,
since RNA polymerase III terminates in a
run of Ts and La recognizes the sequence
UUUOH.24 Because trimming of the
terminal uridines by exoribonuclease(s)
removes the La binding site, the Y RNAs
bound by La are slightly longer at the
3′ end than the bulk of the population
(Fig. 2A). Ro60 and La can bind simul-
taneously to the same RNA.5 However,
Figure1. Potential seconda ry structures for Y RNA s. (A) The four human Y RNAs. A cons erved helix present in all an imal cell Y RNAs is boxed. Nucleot ides
within this helix are important for Ro60 binding.13 –15 The proposed structures are consistent with phylogenetic analyses52 and enzymatic probing
experiments.11,53 (B) X. laevis Y3 RNA. Structural and enzymatic probing studies show that nucleotides in the conserved helix can form two alternate
conformers.14,15 ,53 Sequences present in the X. laevis Ro60/Y3 RNA crystal structure are in bold type. (C) D. radiodu rans Y RNA. Regions involved in Rsr
and PNPase binding are shown.30
©2012 Landes Bioscience. Do not distribute.
1604 RNA Biology Volume 10 Issue 10
since the La binding site can be eliminated
by end trimming, the fraction of Y RNAs
bound by La varies between 0–100%,
depending on the cell type.7,8,20
Assigning Functions to Mammalian
Y RNAs: A Work in Progress
One role of mammalian Y RNAs is to
influence the subcellular location of Ro60.
Ro60 is both nuclear and cytoplasmic, and
its distribution between these compart-
ments is at least partly Y RNA-mediated.
Ro60 exits mouse cell nuclei as a Ro60/
mY3 complex, and binding of the zip-
code binding protein ZBP1 (also known
as IMP1 and IGF2BP1) to mY3 RNA is
important for export of this R NP.25 Addi-
tionally, binding of mY3 to Ro60 masks a
nuclear accumulation signal on the Ro60
surface, thus retaining the RNP in the
cytoplasm.26 Moreover, since both Ro60
and mY3 RNA become strongly nuclear
following UV irradiation,18 the position of
this RNA on Ro60 may be altered during
environmental stress to allow the nuclear
accumulation signal to become accessible.
Since Y RNAs are intimately asso-
ciated with Ro60, our laboratory has
used Ro60 as an entry point to identify
additional roles of these RNAs. Because
Ro60 binds misfolded 5S rRNA precur-
sors and variant U2 small nuclear RNAs
(snRNAs) in some animal cell nuclei, we
proposed that Ro60 functions in ncRNA
surveillance.16 –18 Biochemical and crystal-
lographic studies demonstrated that Ro60
binds misfolded ncRNAs that contain
both single-strand 3′ ends and adjacent
protein-free helices. Structural analyses
revealed that the single-stranded 3′ end
of a misfolded RNA fragment inserts
through the Ro60 cavity, while a helix
contacts the ring outer surface (ref. 27 and
Fig. 3B). Because binding of Ro60 to mis-
folded ncRNAs is not strongly sequence-
specific, Ro60 may scavenge RNAs that
fail to assemble with their correct RNA-
binding proteins. Further, the relative lack
of sequence specificity suggests that Ro60
could potentially bind a wide range of
RNAs.27
Structural and biochemical studies
suggest that Y RNA binding could regu-
late access of misfolded RNAs to Ro60.
A crystal structure of Ro60 complexed
with a Y RNA fragment encompassing
the Ro60 binding site revealed that this
part of Y RNA binds on the outer edge
of the ring (ref. 15 and Fig. 3C). How-
ever, both Y RNAs and misfolded RNAs
are larger than the fragments present in
the crystal structures, and biochemical
experiments indicate that the two RNAs
bind overlapping portions of Ro.15, 27 Since
Y RNAs bind Ro60 with higher affinity
than misfolded RNAs, a bound Y RNA
could sterically prevent misfolded RNA
binding.14,15 ,27 It has also been proposed
that Y RNAs could potentially contribute
to recognition of misfolded ncR NAs and/
or to recruiting helicases or nucleases that
refold or degrade these RNAs.28 Excit-
ingly, studies in bacteria29,30 demonstrate
that Y RNAs both regulate access of Ro60
to some RNA substrates and recruit exori-
bonucleases involved in their degradation
(described below).
Figure2. Ro60 is a stable component of Y RNPs and is important for Y RNA integrity. (A) Mouse
embryonic stem cell lysates were subjected to immunoprecipitation with antibodies against the
trimethylguanosine (TMG) cap that is the 5′ end of many snRNAs (lanes 2–3), anti-Sm antibodies,
which recognize the Sm proteins of the spliceosomal U snRNPs (lanes 4–5), anti-La (lanes 6–7) and
anti-Ro60 antibodies (lanes 8–9). RNAs in immunoprecipitates (lanes 2,4,6,8), supernatants (lanes
3,5,7,9), and an equivalent amount of lysate (lane 1) were subjected to northern blotting to detect
mY1 and mY3. As a control, th e blot was reprobed to detec t the spliceosomal U2 snRNA. Note th at Y
RNAs in the anti- La immunoprecipitate are slightly larger (lane 6) than the Y RNAs remaining in the
supernatant (lane 7). (B) RNA extrac ted from wild-typ e and Ro60−/− embryo nic stem cells (lanes 1–2),
brain (lanes 3–4), testis (lanes 5–6), and ovary (lanes 7–8) were subjected to northern blotting to
detect mY1 and mY3. As a loading control, th e blot was reprobed to detect 5S rRNA. (C) HEK 293 cell
lysates were subjected to immunoprecipitation with anti-Ro60 antibodies or nonimmune isotype
control IgG. RNAs extracted from immunoprecipitates (lanes 4 and 5), supernatants (lane 2 and 3),
and the starting lysate (lane 1) were subjected to northern blotting to detect hY RNAs. As a nega-
tive control, the blot was probed to detect the spliceosomal U6 snRNA. (D and E) To determine if
Ro60 stabilizes hY RNAs, siRNAs against Ro60, or control non-target (NT) siRNAs were transfected
into human keratinocytes. After 72 h, lysates were prepared and subjected to western blotting (D)
to detect Ro60. Actin was used as a loading control. RNA extracted from the lysates was subjected
to northern blotting (E) to detect hY RNAs. U6 snRNA was used as a loading control. Quantitation
revealed that Ro60 was reduced by 83%, while hY1, hY3, hY4, and hY5 were reduced by 90%, 30%,
59%, and 60%, respectively.
©2012 Landes Bioscience. Do not distribute.
www.landesbioscience.com RNA Biology 1605
Y RNAs may also function indepen-
dently of Ro60. Specifically, it has been
reported that vertebrate Y1 and Y3 RNAs
are required for initiation of DNA repli-
cation.31,32 In these experiments, Y RNAs
were found to stimulate DNA replication
when added to isolated G1 phase nuclei.31
Moreover, when added to cell extracts, all
four Y RNAs bound proteins involved in
initiation of DNA replication.33 Although
most of these experiments were performed
in cell extracts, siRNA-mediated deple-
tion of either hY1 or hY3 reduced the
number of replicating HeLa cells31,3 4 and
injection of antisense oligonucleotides into
X. laevis and zebrafish embryos was found
to block DNA replication.35 However, a
role for Y RNAs in DNA replication must
be reconciled with findings that Y RNA
levels are reduced ~30-fold in mouse cells
lacking Ro60, yet these cells grow indis-
tinguishably from wild-type cells,18,36 and
that mice lacking Ro60 are viable.23 One
possibility is that the remaining Y RNAs,
or fragments thereof, are sufficient to sup-
port DNA replication.37
A Bacterial Y RNA Tethers
Ro60 to a Nuclease to Form an
RNA Degradation Machine
Although Ro60 has not been detected
in budding or fission yeast, likely ortho-
logs are encoded in ~5% of sequenced bac-
terial genomes.19 To study Ro60 RNPs in
a genetically tractable single-celled organ-
ism, we chose the first bacterium with a
recognizable ortholog, Deinococcus radio-
durans. D. radiodurans is best known for
its remarkable resistance to severe oxida-
tive stress and its ability to repair massive
DNA damage.38 Our studies revealed that
D. radiodurans lacking the Ro60 ortho-
log Rsr (Ro60-related) exhibit decreased
survival after ultraviolet (UV), but not
γ-irradiation, and are at a competitive
disadvantage during growth in stationary
phase.22,39 Remarkably, as in animal cells,
Rsr associates with a ncRNA resembling Y
RNA (Fig. 1C). Both Rsr and the Y RNA
are upregulated after UV or γ-irradiation,
and also during dessication, heat stress,
and stationary phase.22 ,29,39,4 0 The role of
Ro60 and Y RNA in cell stress responses
is conserved, as mouse cells lacking Ro60
are sensitive to UV irradiation and both
Ro60 and mY3 RNA accumulate in nuclei
after UV irradiation.18 Addition ally, Cae-
norhabditis elegans lacking Ro60 have
abnormalities in dauer formation, a devel-
opmental stage adopted by larvae during
unfavorable growth conditions.41
Molecular analyses revealed that Rsr
and Y RNA function with 3′ to 5′ exori-
bonucleases to modulate RNA metabo-
lism in response to environmental stress.
During heat stress, Rsr, Y RNA, and the
exoribonucleases RNase II and RNase
PH function in 23S rRNA maturation.29
In stationary phase, both Rsr and the
exoribonuclease polynucleotide phos-
phorylase (PNPase) contribute to rRNA
degradation. Although Y RNA was not
investigated in these initial studies, Rsr
and PNPase co-purify, and the associa-
tion of PNPase with ribosomal subunits
requires Rsr.39 Moreover, PNPase exhibits
genetic interactions with both Y RNA and
Rsr during normal growth, growth at low
temperature, and during oxidative stress.29
Characterization of the Rsr/PNPase
complex revealed that Y RNA tethers Rsr
to PNPase to form RYPER, an RNA deg-
radation machine specialized for degrad-
ing structured RNA.30 PNPase forms a
trimeric ring with a degradation cavity
that is capped by single-stranded RNA-
binding S1 and KH domains. These S1/
KH domains bind RNA substrates and
also channel single-stranded RNA into
the PNPase cavity.42,43 In RYPER, the
portion of Y RNA containing the high
affinity Ro binding site interacts with Rsr,
while the other end of Y RNA interacts
with the PNPase S1/KH domains (ref. 30
and Fig. 1C). The Y RNA-mediated teth-
ering of Rsr to PNPase results in a dou-
ble-ringed complex that based on single
particle electron microscopy, is oriented
such that single-stranded RNA could pass
from the Rsr ring into the PNPase cen-
tral channel for degradation (ref. 30 and
Fig. 4). Biochemical analyses revealed that
RYPER degrades structured RNAs such
as rRNAs more efficiently than PNPase,
most likely because threading of RNA
through the Rsr ring contributes to ATP-
independent unwinding.30
Notably, although RYPER is more
effective than PNPase in degrading struc-
tured RNAs, it is less effective on single-
stranded substrates.30 One explanation for
the decreased activity of RYPER on sin-
gle-stranded RNA is that Y RNA-medi-
ated tethering of Rsr to the PNPase KH/
S1 motifs sterically blocks these RNA-
binding domains, replacing the PNPase
RNA-binding surface with that of Rsr.30
Although the RNA-binding specificity of
Rsr has not been characterized, X. laevis
Ro binds RNAs that contain both heli-
ces and single-stranded 3′ ends.27 If Rsr
Figure 3. Structures of Ro60. (A) Molecular
surface representation of X. laevis Ro60. The
hole is 10–15 Å in diameter and binds single-
stranded RNA.15 (B) X. laevis Ro60 bound to a
misfolded pre-5S rRNA fragment consisting
of a short duplex and a single-stranded 3′
extension. The duplex binds on the Ro outer
surface, while the single-stranded end inserts
through the cavity.27 (C) X. laevis Ro60 bound
to a Y RNA fragment.15 The sequence used for
crystallization is shown in bold in Figure 1B.
Studies of Y RNA binding to mutant Ro60 pro-
teins15, 27 suggest that the remainder of the
RNA interacts with portions of Ro60 that over-
lap the misfolded RNA binding site (arrows).
©2012 Landes Bioscience. Do not distribute.
1606 RNA Biology Volume 10 Issue 10
has similar RNA-binding requirements,
RYPER would preferentially bind struc-
tured RNAs.
A key question raised by the discovery
of RYPER is whether similar RNA deg-
radation machines form in other bacteria
with Ro60 orthologs. Preliminary studies
in the human pathogen Salmonella enterica
serovar Typhimurium revealed that the
Ro60 ortholog and a ncRNA also co-
purify with PNPase.30 Notably, both
S. Typhimurium Ro60 and two associ-
ated ncRNAs are encoded within a σ54-
regulated “RNA repair operon” that is
transcribed in response to an unknown
signal.30 Thus, as in D. radiodurans, the
expression of S. Typhimurium Ro60 and
its associated ncRNAs may be regulated
in response to environmental stress.30
A Bacterial Y RNA
May Also Function as a Gate
In addition to their role as tethers, Y
RNAs can potentially regulate access of
other RNAs to the Ro central cavity. As
described above, Y RNA and misfolded
RNAs bind overlapping sites on Ro60 and
a bound Y RNA could sterically inhibit
access of misfolded RNAs to the Ro cav-
it y.15, 27 Consistent with this hypothesis, Y
RNAs and misfolded RNAs compete for
binding to Ro60.14 However, since both
these RNA binding experiments and the
crystal structures employed only purified
Ro60, the possibility that interactions
with other proteins affect Y RNA posi-
tioning was not addressed. Importantly,
the fact that RYPER degrades RNA sub-
strates in the presence of Y RNA implies
that substrates can enter the Ro60 cavity
when PNPase is also complexed.
Single particle electron microscopy of
RYPER suggested a model for how Y RNA
binding can be modulated to allow RNA
substrates to access the Ro60 surface. Spe-
cifically, in the three-dimensional recon-
struction, the Rsr and PNPase rings are
bridged by a rod shaped density (ref. 30
and Fig. 4). If as predicted from the bio-
chemical experiments, this density corre-
sponds to the Y RNA, binding of PNPase
to the distal loops may remove this por-
tion of the Y RNA from the Rsr surface,
rendering the cavity accessible to RNA
substrates (Fig. 5). In this model, a bound
Y RNA would prevent RNA
substrates from entering the
Rsr cavity unless PNPase
was also present.
Studies of the roles of
Rsr and Y RNA during heat
stress support the hypoth-
esis that Y RNAs also func-
tion as gates.29 During
normal growth, maturation
of D. radiodurans 23S rRNA
is very inefficient, as ~40%
of these rRNAs contain
5′ and 3′ extensions. Dur-
ing heat stress, maturation
becomes highly efficient and
requires Rsr, RNase II, and
RNase PH.29 As expected if
Y RNAs block entry of pre-
rRNAs into the Rsr cavity during normal
growth, 23S rRNA is fully matured at all
growth temperatures when either the Y
RNA is deleted or a mutant Rsr that can-
not bind Y RNA is overexpressed.29 These
results also imply that Rsr is capable of
assisting exoribonucleolytic maturation
of at least some RNAs without a Y RNA
tether.
Evidence that Y RNAs Function
as Tethers in Mammalian Cells
In mammalian cells, Y RNA-mediated
tethering may be important for correct
subcellular localization of Ro60. Specifi-
cally, binding of the zipcode-binding pro-
tein ZBP1 to mY3 RNA is important for
nuclear export of the Ro60/mY3 RNP.25
ZBP1, which has well-characterized func-
tions in mRNA post-transcriptional regu-
lation,44 uses two of its four KH domains
to bind mRNAs containing a short “zip-
code” sequence.45 Since mY3 competes
with a zipcode-containing RNA fragment
for ZBP1 binding,46 formation of the
Ro60/Y RNA/ZBP1 RNP may involve
binding of one or both of these KH
domains to the mY3 RNA large internal
loop.
Because Ro60 RNPs are largely cyto-
solic and mammalian PNPase localizes to
the mitochondrial intermembrane space,47
RYPER may not form in mammalian cells.
However, Y RNAs could potentially tether
Ro60 to other RNA remodeling proteins,
such as exoribonucleases, helicases, or
RNA chaperones to assist unwinding of
structured RNAs. In this scenario, Ro60
and their associated Y RNAs would func-
tion as modules that attach in trans to
diverse proteins involved in RNA metabo-
lism. Moreover, the multiple distinct Y
RNAs found in mammalian cells could
allow Ro60 to be tethered to a greater
range of RNA-binding proteins. Consis-
tent with this proposal, several proteins
have been shown to associate with Ro60
Figure4. Model for RYPER based on single particle electron
microscopy reconstruction.30 Portions of the reconstruction
corresponding to Rsr, Y RNA, and PNPase are depicted in
magenta, yellow, and blue, respectively. A possible path for a
duplex-containing RNA substrate is drawn in red.
Figure5. Model for RYPER formation. In the
absence of interacting proteins, the Y RNA
acts as a gate to prevent other RNAs from
accessing the Ro60 cavity. In the presence
of PNPase, the Y RNA loops interact with the
KH and S1 domains, removing this part of the
Y RNA from Ro60, and allowing the single-
stranded ends of RNA substrates to enter the
cavity.
©2012 Landes Bioscience. Do not distribute.
www.landesbioscience.com RNA Biology 1607
RNPs through binding distinct subsets
of Y RNAs. These include two splicing
factors, PUF60 and the polypyrimidine-
tract binding protein PTB1, the multi-
functional protein nucleolin, the putative
helicase MOV10, and the cytidine deami-
nase APOBEC3G.25,2 8,48–50 Major goals of
future studies will be to define the protein
composition and RNA substrates of these
complexes and to elucidate how Ro60 and
Y RNAs contribute to their functions.
Materials and Methods
RNA isolation and northern blotting
Total brain, testis, and ovary tis-
sue was removed from wild-type and
Ro60-/- mice,23 lysed in TRIzol (Invi-
trogen), and RNA isolated as described
by the manufacturer. Wild-type and
Ro60−/− embryonic stem (ES) cells were
cultured as described18 and RNA isolated
using TRIzol as above. For northern blot-
ting, RNAs were fractionated in 5% poly-
acrylamide/8 M urea gels, transferred to
Hybond-N membranes (GE Healthcare),
and hybridized with [γ32-P]ATP-labeled
oligonucleotides as described.51 Oligonu-
cleotide probes were
my1: 5′-AAGGGGGGAA AGT-
GTAG AAC AGGA -3′,
my3: 5′-GAGCGGAGAA GGAA-
CAAAGA AATCTG-3′,
mouse 5s: 5′-TC A G AC -
GAGA TCGGGCGCGT TCAG-3′,
mouse u2: 5′-CAGATAC-
TAC AC TTG ATCT T AGC C-3′,
hy1: 5′-ATCTGTAACT GACTGT-
GA AC A ATC A ATTG A GATA A-3 ′,
hy3: 5′-GGAGA-
AGGAA CAAAGAAATC TGTA-
ACTGGT TGTG AT-3′,
hy4: 5′-GGGTTGTATA CCAACTT-
TAG TG ACAC -3′,
hy5: 5′-GG GAG ACA AT GT TA-
AATCAA CTTAACAATA A-3′,
human u6: 5′-CAC-
GA ATTTG CGTGTCATCC T T-3′.
Immunoprecipitations
Mouse ES cells were maintained as
described above. HEK293 cells were
maintained in Dulbecco’s modified Eagle
medium (Invitrogen) supplemented
with 10% fetal bovine serum (FBS) and
2 mM L-glutamine. Cells were sonicated
in NET-2 (40 mM Tris-HCl, pH 7.5,
150 mM NaCl, 0.1% NP-40) contain-
ing 1 mM phenylmethylsulfonyl fluoride
and 1x protease inhibitor cocktail (Roche
Applied Science). After clearing by cen-
trifugation at 100 000 × g in a Beckman
TLA100.3 rotor for 20 min at 4 °C, ES
cell lysates were incubated as described12
with antibodies bound to Protein A Sep-
harose (GE Healthcare). Antibodies used
were rabbit anti-Ro60,18 human anti-La
(gift of J. Harley, Cincinnati Children’s
Hospital), anti-TMG (Oncogene Sci-
ence), and anti-Sm (Y12; gift of Mei-Di
Shu and Joan Steitz, Yale University).
HEK293 lysates were incubated with
mouse anti-human Ro60 (1F2, Novus
Biologicals) and isotype control IgG
(M2AK), bound to Dynabeads Protein
G (Invitrogen).
siRNA transfections
Adult human epidermal keratinocytes
(Invitrogen) were maintained in EpiLife
medium with 60 μM calcium and Huma n
Keratinocyte Growth Supplement (Invit-
rogen). Cells were transfected with Lipo-
fectamine RNAiMAX (Invitrogen) using
a modified version of the manufacturer’s
protocol for reverse transfection. Anti-
Ro60 siRNA (siGENOME SMARTpool,
Dharmacon) or non-targeting control
siRNA (Ambion) was diluted in 125 μl of
Opti-MEM (Invitrogen) in 10 cm dishes
for a final concentration of 40 nM after
addition of cells. Five μl of Lipofectamine
RNAiMAX was mixed with 125 μl Opti-
MEM, added to the dish and incubated
for 15 min at room temperature. Two
hundred and fifty thousand trypsinized
keratinocytes were then added to each
well in 1.75 ml of growth media. Fresh
media was added the following day, and
the cells incubated for 72 h before har-
vesting. Cells were sonicated in NET-2
containing 1 mM phenylmethylsul-
fonyl fluoride, 200 u/ml RNaseOUT
(Invitrogen), and 1x protease inhibi-
tor cocktail and cleared by sedimenting
for 10 min at 4 °C in a microcentrifuge.
Ten percent of the lysate was removed
for western blotting using a monoclonal
anti-mouse Ro60 antibody as described.23
RNA was extracted from the remaining
lysate using phenol:chloroform:isoamyl
alchohol, precipitated with ethanol,
and subjected to northern blotting as
described above.
Disclosure of Potential Conflicts of Interest
No potential conicts of interest were disclosed.
Acknowledgments
We thank Hong Shi for technical
assistance. This work was supported by
NIH grant R01 GM073863 (to SLW)
and National Basic Research Program of
China grant 2010CB912401 (to H-WW).
DWT is an NSF Graduate Research
Fellow.
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