Nucleic Acids Research Advance Access published September 15, 2010
 

Nucleic Acids Research, 2010, 1–12
doi:10.1093/nar/gkq776

Human box C/D snoRNAs with miRNA
like functions: expanding the range of
regulatory RNAs
Markus Brameier1,*, Astrid Herwig1, Richard Reinhardt2, Lutz Walter1 and
Jens Gruber1,*

1Primate Genetics Laboratory, German Primate Center, 37077 Go¨ ttingen and 2Max-Planck-Institute for
Molecular Genetics, Berlin, 14195 Germany

Received February 10, 2010; Revised August 14, 2010; Accepted August 18, 2010

ABSTRACT

INTRODUCTION

Small nucleolar RNAs (snoRNAs) and microRNAs
are two classes of non-protein-coding RNAs with
functions in RNA modification or post-
distinct
transcriptional gene silencing.
In this study, we
insights to RNA-induced gene
introduce novel
activity adjustments in human cells by identifying
numerous snoRNA-derived molecules with miRNA-
like function,
including H/ACA box snoRNAs and
C/D box snoRNAs. In particular, we demonstrate
that several C/D box snoRNAs give rise to gene
regulatory RNAs, named sno-miRNAs here. Our
data are complementing the increasing number of
studies in the field of small RNAs with regulatory
functions. In massively deep sequencing of small
RNA fractions we identified high copy numbers
of sub-sequences from >30 snoRNAs with lengths
of 18 nt. RNA secondary structure prediction
indicated for a majority of candidates a location in
predicted stem regions. Experimental analysis
revealed efficient gene silencing for 11 box C/D
sno-miRNAs,
indicating cytoplasmic processing
and recruitment to the RNA silencing machinery.
Assays in four different human cell lines indicated
variations in both the snoRNA levels and their pro-
cessing to active sno-miRNAs. In addition we show
that box D elements are predominantly flanking
at least one of the sno-miRNA strands, while the
box C element locates within the sequence of the
sno-miRNA guide strand.

A variety of small non-protein coding RNAs (npcRNAs),
including microRNAs (miRNAs), small interfering RNAs
(siRNAs) and Piwi-interacting RNAs (piRNAs) are im-
portant mediators of gene regulation (1–3). piRNAs
control retrotransposition (4,5) and are expressed in
testes only (3). siRNAs and miRNAs are targeting
mRNAs in a sequence-speciﬁc manner and guide transla-
tional
inhibition, degradation or deadenylation of
target-mRNAs (6–9). A role in transcriptional regulation
for an endogenous miRNA was recently discovered
(10,11) and previously shown for exogenously delivered
siRNAs (12,13). Furthermore, a general transcriptional
regulation mechanism was recently postulated for tran-
scription start site (TSS) associated tiny RNAs, called
tiRNAs
the RNA induced
silencing
the Ago
proteins, act in concert with small npcRNAs to regulate
gene expression at the various levels mentioned earlier.

(14,15). Components of

in particular

complex

(RISC),

(rRNAs) or

A new source of regulatory npcRNAs was recently
discovered to originate from small nucleolar RNAs
(snoRNAs) (14,16). SnoRNAs are ubiquitously expressed
small npcRNAs (also named sRNAs here) that function in
maturation and modiﬁcation of other npcRNAs such as
ribosomal RNAs
small nuclear RNAs
(snRNAs) (17). Box H/ACA snoRNAs trigger the site
speciﬁc synthesis of pseudouridines in a broad variety of
ribosomal and spliceosomal RNAs (18). In addition these
snoRNAs play a role in nucleolytic processing of precur-
sor rRNA (pre-rRNA) and also function in synthesis of
telomeric DNA (19). Box C/D snoRNAs, which are the
primary source of sno-miRNAs in this study, contain
conserved Box C (UGAUGA) and Box D (CUGA)

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*To whom correspondence should be addressed. Tel: +49 551 3851 418; Fax: +49 551 3851 372; Email: brameier@dpz.gwdg.eu
Correspondence may also be addressed to Jens Gruber. Tel: +49 551 3851 481; Fax: +49 551 3851 372; Email: jgruber@dpz.eu

ß The Author(s) 2010. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Nucleic Acids Research, 2010

‘work-environment’. Ender et al.

elements located closely to the 50- and 30-ends, respective-
ly. Internal copies of these elements are termed Box C0 and
Box D0
(20,21). An interaction between the 50- and
30-termini allows the formation of a stem bringing the
Boxes C and D elements together to form a hairpin struc-
ture. Box C/D snoRNAs serve as guides for 20-O-ribose
methylation of
ribosomal RNA. Pseudouridylation
and 20O-ribose methylation are important steps during
maturation of ribosomal RNAs, thus playing a fundamen-
tal role in key processes of all cells. With exception of U3
all box C/D snoRNAs presented in this study are
intron-encoded, as it is the general pathway for the bio-
genesis of this class of snoRNAs (22). However, apparent-
ly some snoRNAs have an additional function outside
their normal
(16)
demonstrated processing of the cajal body speciﬁc RNA
ACA45 to cytoplasmic, Ago protein associated 20- to
25-nt RNAs with gene silencing activity. The identiﬁcation
of CDC2L6 as a target mRNA, which is down-regulated
by the miRNA function of ACA45 underlined the physio-
logical relevance of the dual function. Independence of
nuclear micro-processing by Drosha/DGCR8 indicates
that the mechanism is distinct from the classic miRNA
pathway that was formulated in the early years of
miRNA research (23) and processing factors and their
regulations are constantly being reﬁned (24,25). Today
we know more about alternative processing of
the
primary miRNA transcripts (pri-miRNAs) in a Drosha-
independent fashion (26,27) and regulation of the micro-
processor complex itself (25,28,29). Recently, the possibil-
ity of processing box C/D snoRNAs to small RNA
molecules with miRNA-like features and functions was
postulated from intensive analysis of deep sequencing
data and predicted for numerous candidates (30). The
box C/D snoRNA MBII-52 was shown to give rise to
shorter RNAs with a regulatory function in alternative
splice site selection (31). We addressed the crucial task
of providing further experimental evidences for regulatory
snoRNA- mechanisms in human cells. In the ancient eu-
karyote Giardia lamblia a box C/D snoRNA was shown
to be processed by Dicer and performed gene silencing in
reporter gene assays (32) and similar snoRNA effects were
postulated for humans (33).

By second generation sequencing of small RNA frac-
tions (up to 40 nt)
from human T lymphocytes we
identiﬁed 22 fragments of C/D box snoRNAs by bioinfor-
matics analysis. These may serve as substrates for Dicer
processing as described in ref. (27) and may enter the
RNA silencing machinery as it is usually observed for
(pre-)miRNAs (34). For these candidate sequences we
functionally characterized the putative miRNA-like
activity by in vitro experiments. Two small RNAs
snoRNA HBII-99b and
derived from C/D box
SNORD126 are reported as miRNAs
in the latest
release of the miRNA database (miRBase).

Furthermore, we identiﬁed miRNAs in miRBase, which
de facto are box H/ACA snoRNA-derived RNAs
(sdRNAs). These snoRNAs, i.e. ACA34, ACA36b and
HBI-61, share common features in terms of predicted
structures, processing and silencing capacity as was
described for
cajal body speciﬁc RNA

small

the

(scaRNA) ACA45 (16) and additional box H/ACA
sdRNAs (35). Based on the striking similarity shared by
the two RNA species the theory was introduced that some
miRNAs may have evolved from ancient box H/ACA
snoRNAs (35). In their study Scott et al. (35) provide a
list of 14 snoRNAs, which give rise of sdRNAs, including
the three miRBase listed candidates (miR-1291/ACA34,
miR-1248/HBI-61, miR-664/ACA36b). ACA36b was
already predicted to have a miRNA-like activity that
may compare to the miRNA-activity of ACA45 (16) and
we provide experimental data for this function in three
human cell lines. In this study we present an expanded
range of candidate npcRNAs with putative miRNA-
features,
silencing
activity of human box C/D sno-miRNAs. In addition,
we show functional data for three previously discovered
box H/ACA sno-miRNAs. Moreover, we provide experi-
mental evidence for cell
type speciﬁc activities of
sno-miRNAs, suggesting a general and controlled par-
ticipation of snoRNAs in gene regulation in a cell type
dependent manner.

including evidence

the gene

for

In a detailed analysis of snoRNA-derived sequences
from our small RNA libraries we identiﬁed an interesting
feature. In 15 out of 17 box C/D snoRNAs at least one
of the sequenced (and functionally tested) sno-miRNA
candidates was ﬂanked by the box D element (CUGA)
at the 30-end and/or a box D0 element close to the
50-end. The box C element (UGAUGA) was identiﬁed in
12 snoRNA sequences and located within the sequence of
11 sno-miRNAs. This pattern could be conﬁrmed more
frequently for 107 snoRNAs that express sequences from
our sRNA libraries. Our ﬁndings indicate a relation of
the functional snoRNA elements to the processing and
activity of sno-miRNAs.

MATERIALS AND METHODS

Small npcRNA cloning and sequencing

(PAA)

denaturing

15% polyacrylamide

Small RNA cloning was
carried out by Vertis
Biotechnology (Weihenstephan, Germany) and has been
described earlier (36). The small RNA species were
isolated from total RNAs using the mirVana miRNA
isolation kit (Ambion). The small RNAs were separated
on
gels.
Oligonucleotides of 18- to 40-nt size were poly(A)-tailed
using poly(A) polymerase, and an adaptor was ligated to
the 50-phosphate of the miRNAs: 50-end adaptor GCCTC
CCTCGCGCCATCAGCTNNNNGACCTTGGCTGTC
ACTCA. Next, ﬁrst-strand cDNA synthesis was per-
formed using an oligo(dT)-linker primer and M-MLV-
RNaseH reverse transcriptase 30-end adaptor GCCGGG
GCGATGTCTCGTCTGAGCGGGCTGGCAAGGC.
The resulting cDNAs were PCR ampliﬁed in 16 cycles
using the high-ﬁdelity Phusion polymerase (Finnzymes).
The 120–135-bp ampliﬁcation products were conﬁrmed
by polyacrylamide gel electrophoresis (PAGE) analysis.
sRNA library sequencing was performed in a Roche
FLX System (Roche Diagnostics, Mannheim, Germany)
and yielded 550 000 reads in total.

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sRNA extraction from library

sno-miRNA identiﬁcation

Nucleic Acids Research, 2010 3

Each library sequence (read) comes with a unique identi-
ﬁer and is composed of a ﬁxed-length 50-adaptor,
a transcribed sRNA sequence of maximum 40 nt,
a variable-length poly(A)
sequence A+NA+, and a
30-adaptor (see above). The following protocol is used to
extract the sRNA sequences from the library:

(i) The start position is simply found by removing the
ﬁxed-length 50-primer. Only in a few cases the
precise position may be missed in this way due to
single nucleotide deletions (or insertions) inside the
adaptor as a result of sequencing errors.

(ii) The identiﬁcation of

the sequences at

the end position is more
challenging due to the poly(A) string that precedes
the 30-primer. To cope with its variable length and
patterns, we used Perl regular expression matching.
In >99% of
the
subexpressions AA+NA+, A+NAA+, AAA+N,
NAAA+ or AAAAAA+ matches. Here, we have
to accept
sRNAs ending on
A+, cutting at the matching position is imprecise.
together
The non-matching reads are discarded,
with all
that are
empty or
further
processing (<5%).

extracted sRNA sequences
just

least one of

too short

(<15 nt)

for

that at

least

for

for

candidates

To discover both (i) expressed/transcribed fragments
of known snoRNAs in the short RNA library and
(ii) novel
snoRNA-derived RNAs
(sno-miRNAs), we aligned all extracted sRNA sequences
against all 402 known snoRNAs from snoRNABase (37)
using the ofﬂine version of NCBI BLAST. Only perfect
alignments (without gaps or mismatches) are accepted
which cover at least 17 nt. This is the minimum length
found for mature human miRNAs. From the BLAST
analysis a snoRNA-expression proﬁle was
created
counting the frequencies of matching sequences from the
library (9500 in total). From this proﬁle we selected suf-
ﬁciently expressed snoRNAs which include a subsequence
that (i) is minimum 25-nt long; (ii) is covered by a signiﬁ-
cant number of expressed snoRNA fragments; and (iii)
whose most conserved part
is located in a
hairpin stem of the snoRNA folding. Secondary structure
folding was calculated with RNAfold (rna.tbi.univie.ac.at/
cgi-bin/RNAfold.cgi) and sRNA sequences were aligned
with ClustalW2 (38). Table 1 lists all C/D box snoRNAs,
which were identiﬁed in this way for further functional
(miRNA) analysis. To test whether
some known
miRNAs may actually be derived from snoRNAs all 718
mature human miRNAs from miRBase (39) (release 14.0,
www.mirbase.org) are matched against
the ﬁltered

(20 nt)

Table 1. snoRNA derived sRNAs (sno-miRs), positions on original snoRNA sequences and miRNA-like activity

snoRNA

ACA45
snR39b
U3
U3-3
U3-4
U78
HBII-336
HBI-43
HBII-142
U48
U51
U21
U27

U44
HBII-429
U59b
U83a
U15a
U74
U45AC

HGNC symbol
(host gene)

SCARNA15 (Hs.513091)
SNORD2 (EIF4A2)
X14945
SNORD3
SNORD3
SNORD78 (GAS5)
SNORD93 (EST cluster)
SNORD17 (SNX5)
SNORD66 (EIF4G1)
SNORD48 (C6orf48)
SNORD51 (EEF1B2)
SNORD21 (RPL5)
SNORD27 (U22 Host

Gene, UHG)

SNORD44 (AL110141)
SNORD100 (RPS12)
SNORD59b (ATP5B)
SNORD83a (RPL3)
SNORD15a (RPS3)
SNORD74 (GAS5)
SNORD45aSNORD45c

(RABGGTB)

Type

H/ACA
C/D
C/D
C/D
C/D
C/D
C/D
C/D
C/D
C/D
C/D
C/D
C/D

C/D
C/D
C/D
C/D
C/D
C/D
C/D

miRbase listed snoRNAs
ACA34 hsa-miR-1291
HBI-61 hsa-miR-1248
ACA36b hsa-miR-664
HBII-99b hsa-miR-1259
SNORD126

hsa-miR-1201

SNORA34 (FLJ20436)
SNORA81 (EIF4A2)
SNORA36b
SNORD12b (C20orf199)
SNORD126 (CCNB1IP1)

H/ACA
H/ACA
H/ACA
C/D
C/D

Sno-miR
pos. (major)

Sno-miR
*pos. (minor)

miRNA activity
(H/J/R)

References

64–85
2–29
193–214
45–66
75–96
32–61
40–62
207–233
47–70
1–25
41–65
2–27
43–67

2–26
4–36
44–67
4–26
2–27
40–68
57–73

2–25
1–27
106–128
16–36
52–75

102–127
36–63
(93–127)

1–24
2–23
2–29
1–23
40–61
2–39
52–86
2–24

28–57
37–70
6–25
n.d.
114–137
3–30
1–29

(39–63)
n.d.
68–91
n.d.
n.d.

+/+/O
+/+/ 
+/O/+
 /O/n
O/+/+
+/+/O
 /+/+
O/–/n
O/+/n
 /O/n
 /O/n
 / /n
 /+/ 
 /O/n
+/O/n
 /O/n
 /+/+
+/+/n
 /+/n
O/ /n

+/+/+
+/+/+
+/O/ 
n.d.
n.d.

(16)

This study

Identiﬁed
(35)

This study

Host gene, pre-mRNA source of intron-encoded snoRNA; sno-miR, small RNA originating from snoRNAs; major, dominant sRNA, found in high
copy numbers by deep sequencing; minor, sRNA found in signiﬁcantly lower copy numbers; var., various minor sRNAs; n.d., not detected; miRNA
activity: HeLa, Jurkat, RPMI8866 +, <60% remaining Renilla luciferase activity; O, 60–80%;  , no reduction.

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4 Nucleic Acids Research, 2010

snoRNA fragments. The ﬁve matches found are also listed
in Table 1.

RESULTS

Pool of ncRNAs in human T lymphocytes

Cell culture
Human CD4+ T cells (Jurkat) and B cells (RPMI8866)
were maintained in RPMI 1640 Medium supplemented
with 10% FCS and 1% antibiotics solution (Pen/Strep).
Human HeLa and MCF7 cells were maintained in
DMEM (Dulbecco’s modiﬁed Eagle’s Medium) supple-
mented with 10% FCS and antibiotics. RNA preparation
was performed with TRIzol reagent (Invitrogen, Carlsbad,
CA, USA) according to manufacturer’s guidelines.

Reporter gene assays

snoRNA derived sRNA was
Silencing activity of
measured in the dual
luciferase reporter gene system
psiCHECK-2 (Promega, Madison, WI, USA). Fully com-
plementary target sites for sRNAs were synthesized with
ﬂanking overhangs for NotI and XhoI restriction sites,
annealed in isostoichiometric ratio in annealing buffer
(10 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA)
and ligated into the 30-UTR of the Renilla luciferase gene
in the XhoI/NotI-digested psiCHECK-2 vector. For a
complete list of ligated target site oligonucleotides see
the Supplementary data. Transient
transfection of
human cell lines was performed with either nucleofection
(suspension cell lines Jurkat and RPMI8866) according to
manufacturer’s protocols (Amaxa) or with lipofection
(adherent HeLa and MCF7 cells) utilizing FuGENE HD
transfection reagent
(Roche Diagnostics, Mannheim,
Germany). Luciferase activities were determined with
either the Dual-Glo Luciferase Assay or the two individ-
ual assays for Renilla and Fireﬂy luciferase (all Promega).
psiCHECK-2 transfected cells were treated in accordance
to manufacturer’s protocols and luminescence was
assayed on a Victor2 microplate reader (Perkin Elmer).
Luminescence
sno-miRNA-target
associated Renilla luciferase were normalized against the
simultaneously examined ﬁreﬂy luciferase values
to
minimize the risk of transfection and assay artefacts.
Normalized R/F values were standardized with two
negative controls,
i.e. the empty dual luciferase vector
and a scrambled, non-cognate target sequence.

intensities

the

for

Northern blot analysis

onto

transferred

Total RNA was isolated from cell cultures by the TRIzol
method (Invitrogen Life Technologies). An amount of
10 mg of total RNA were size-separated on denaturing
8% acrylamide/bisacrylamide (19:1; 7 M Urea) gels
(Invitrogen),
nylon membranes
(BrightStar-Plus, Ambion), UV- crosslinked (Stratagene
crosslinker) and pre-hybridized for 1 h at 40C in 1M
sodium phosphate
7% SDS.
Hybridizations to 32P-ATP, end-labelled oligonucleotides
complementary to the respective npcRNAs
(Supple-
mentary Data) were performed in 1 M sodium phosphate
(pH 6.2), 7% SDS for 12 h at 38C. Membranes were
washed twice for 30 min at room temperature in 2
SSC, 0.05% SDS buffer and exposed to Kodak MS-1
ﬁlms for 3 h–2 days.

(pH 6.2),

buffer

240 000

sequences

originating

Interestingly,

the previously

Small RNA fractions (up to 40 nt) were generated from
RNA extracts of human CD4+ T lymphocytes and sub-
jected to massive parallel pyrosequencing. We identiﬁed
approximately
from
known human miRNAs, reﬂecting >60% of all small
RNAs in the deeply sequenced library. In addition we
identiﬁed more than 9000 RNAs of 18–28 nt in length
that originate from bona ﬁde snoRNAs. Among these
sequences 20 box C/D snoRNAs were selected using the
procedure described in ‘Materials and Methods’ section
and are enriched in human Jurkat cells (Table 1),
besides
characterized H/ACA box
snoRNA (ACA45).
the majority of
sno-miRNAs with increased frequencies match perfectly
in regions of 18-nt and display only minor differences
in containing additional 1- to 5-nt in the 30- or 50-ﬂanking
region. When compared to secondary structure predic-
tions we found most of
the sno-miRNA sequences
located in regions that are likely to form stem-loops
when thermodynamics is the sole folding determinant.
Three structurally distinct C/D box snoRNAs and their
corresponding sno-miRNAs are shown in Figure 1. For
U48 we include the alignment of sno-miRNA sequences,
the dominant or guide strand is highlighted with a red bar,
the aligned minority or passenger strand is marked with a
blue bar. The location of sno-miRNAs on predicted
snoRNA structures of U15a, U48 and U21 is indicated,
red marks the major, blue the minor sno-miRNA-strand.
The RNAfold-predicted structures of all 20 snoRNAs
with embedded candidate sno-miRNAs are provided in
Supplementary Figure S1. We frequently identiﬁed the
sno-miRNA guide strand to contain the box C element
of the snoRNA, while guides as well as the passenger
strands that were only present in very low copy numbers
in the small RNA libraries were in most cases ﬂanked by
the box D element in their 30-end (compare Table 2).

Functional characterization of candidate sno-miRNA
sequences

Selected candidates were tested for their gene silencing
capabilities in dual luciferase based reporter gene assays.
Fully complementary target sites for putative sno-miRNA
guide strands were cloned into the 30-UTR of the Renilla
luciferase gene of the psiCHECK-2 reporter vector. The
second luciferase gene (Fireﬂy, Photinus pyralis) remained
unaffected and served as an internal control for subse-
quent normalization of the data obtained. Data variations
due to different transfection efﬁciencies can be excluded,
since the reporter gene and the control gene for normal-
ization of the data are encoded on the same plasmid.
Upon RISC incorporation of sno-miRNAs the transcripts
were recognized and subjected to ago2 cleavage and sub-
sequent degradation. A target for the previously described
H/ACA box snoRNA ACA45 served as positive control,
the unmodiﬁed (empty) psiCHECK-2 vector or insertion
of a non-cognate target site as negative control.

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Nucleic Acids Research, 2010 5

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Figure 1. Box C/D snoRNA derived sno-miRnas. Predicted secondary structures of the snoRNAs U48, U15a and U21 were calculated with
RNAfold. Secondary structure sequences are given in dot-bracket notation with base pairings represented by two complementary parentheses ‘(’
and ‘)’ and non-pairing bases by dots ‘.’. A multiple sequence alignment of U48 derived sequences that were identiﬁed by 454 sequencing displays
multiple copies of 23–25 nt of the 50-region of U48, a minority of sequences was identiﬁed as 30-fragments or full length U48. The positions of high
frequency sequences from snoRNAs U15a, U48 and U21 are indicated with red bars, the corresponding low frequency strand positions are marked
with blue bars. Box C and D elements are given in bold in the sequences.

6 Nucleic Acids Research, 2010

Table 2. Sequences of box C/D snoRNAs with indicated sno-miRNAs that were functionally tested

snoRNA

HGNC

Sequence (50!30-direction)

ACA45

SCARNA15

CUGGAGACUAAGAAAAUAGAGUCCUUGAAAUCAAGCUGACUCUGCUUUUAGCCUCCUAAAUGAAAAGGUAGAUAGAACAGGUC

snR39b
U3

SNORD2
X14945

U78
U74
HBII-336
HBI-43

SNORD78
SNORD74
SNORD93
SNORD17

HBII-142
U48
U51
U21

U27
U44
HBII-429
U59b
U83a

SNORD66
SNORD48
SNORD51
SNORD21

SNORD27
SNORD44
SNORD100
SNORD59b
SNORD83a

UUGUUUGCAAAAUAAAUUCAAGACCUACUUAUCUACCAACAGCA

AAGUGAAAUGAUGGCAAUCAUCUUUCGGGACUGACCUGAAAUGAAGAGAAUACUCAUUGCUGAUCACUUG
AAGACUAUACUUUCAGGGAUCAUUUCUAUAGUGUGUUACUAGAGAAGUUUCUCUGAACGUGUAGAGCACCGAAAACCACGAGGA
AGAGAGGUAGCGUUUUCUCCUGAGCGUGAAGCCGGCUUUCUGGCGUUGCUUGGCUGCAACUGCCGUCAGCCAUUGAUGAUCG
UUCUUCUCUCCGUAUUGGGGAGUGAGAGGGAGAGAACGCGGUCUGAGUGGU

GUGUAAUGAUGUUGAUCAAAUGUCUGACCUGAAAUGAGCAUGUAGACAAAGGUAACACUGAAGAA
CUGCCUCUGAUGAAGCCUGUGUUGGUAGGGACAUCUGAGAGUAAUGAUGAAUGCCAACCGCUCUGAUGGUGG
UGGCCAAGGAUGAGAACUCUAAUCUGAUUUUAUGUGCUUCUGCUGUGAUGGAUUAAAGGAUUUACCUGAGGCCA
GUGAAAUGAUGAUUCAGUUUAUCCAUUCGCUGAGUGCGCUGCACUGACCUUCUUCCAAGCCUCAGUUCCUGUUCUAGGAACUUG
AGGCUAUGUAGCCUGAAAAUGCCCUGCAGUCUGCAGUGUUCUACUGUGAACUGCUUGUGUGUUGGCAGGCUACCGGUAAGAA
UGGUUGGUGUCAGCAGGGACGGGGCCCUCUGAGACCCAUCUCACAAAGAUGAGUGGUGAAAAUCUGAUCAC

CUGCCACGUGUCUGGGCCACUGAGACACCAUGAUGGAACUGAGGAUCUGAGGAA
AGUGAUGAUGACCCCAGGUAACUCUUGAGUGUGUCGCUGAUGCCAUCACCGCAGCGCUCUGACC
GUUGCAUGAUGAAUAAAAUCAAAUCACCAUCUUUCGGCUGAGUUCGUGAUGGAUUUGCUUUUUUCUGAUU
GCUGAAUGAUGAUAUCCCACUAACUGAGCAGUCAGUAGUUGGUCCUUUGGUUGCAUAUGAUGCGAUAAUUGUUUCAAGACGGGA

CUGAUGGCAGC

ACUCCAUGAUGAACACAAAAUGACAAGCAUAUGGCUGAACUUUCAAGUGAUGUCAUCUUACUACUGAGAAGU
CCUGGAUGAUGAUAAGCAAAUGCUGACUGAACAUGAAGGUCUUAAUUAGCUCUAACUGACUAA
GCUGUACAUGAUGACAACUGGCUCCCUCUACUGAACUGCCAUGAGGAAACUGCCAUGUCACCCUUCUGAUUACAGC
UAUUCCUCACUGAUGAGUACGUUCUGACUUUCGUUCUUCUGAGUUUGCUGAAGCCAGAUGCAAUUUCUGAGAAGG
GCUGUUCGUUGAUGAGGCUCAGAGUGAGCGCUGGGUACAGCGCCCGAAUCGGACAGUGUAGAACCAUUCUCUACUGCCUUCC

UUCUGAGAACAGC

U15a

SNORD15a

CUUCGAUGAAGAGAUGAUGACGAGUCUGACUUGGGGAUGUUCUCUUUGCCCAGGUGGCCUACUCUGUGCUGCGUUCUGUGGCAC

AGUUUAAAGAGCCCUGGUUGAAGUAAUUUCCUAAAGAUGACUUAGAGGCAUUUGUCUGAGAAGG

U45A

SNORD45a

GGUCAAUGAUGUGUUGGCAUGUAUUAUCUGAAUCUAUUGCUGAUGUGUAAUAACACUUUAGCUCUAGAAUUACUCUGAGACCU

Sequences that were found in small RNA libraries are shown in bold, the putative guide strand (higher frequencies) is underlined. Blue indicates
possible box C and C0 consensus sequences, red indicates consensus box D and D0 elements.

Figure 2 displays the general exemplar for the combined
results of analysing the C/D box snoRNA HBII-336. The
RNAfold-predicted secondary structure of HBII-336 is
shown in Figure 2A. The major sno-miRNA strand, as
was determined by frequencies in deep sequencing of
small RNA libraries from human T cells is highlighted
in red in (Figure 2B), nucleotides of the minority strand
(sno-miRNA*) are shown in blue colour. Sequence
alignments of deep-sequenced small RNAs are shown in
Figure 2B, the 30-ﬂanking box D element is indicated with
an arrow. The high degree of sequence stability taken
together with the putative location in stable stem regions
of the source-snoRNA and their abundance in small RNA
libraries were selection criteria for functional analysis of
the candidates. RISC incorporation and RNA silencing
activity of the candidate sequences were determined in
luciferase assays. The HBII-336 sno-miRNA was efﬁcient
in silencing the reporter gene upon transfection into
Jurkat cells and displayed miRNA activity that was
directly comparable to that of the ACA45 sno-miRNA
(Figure 2C).

Total RNA extracts from human HeLa and Jurkat
cells were analyzed by northern blots with probes
against sno-miRNAs U74, U3, U78, U21 and HBI-43
(Figure 3). The snoRNAs were processed to sno-
miRNAs in a cell type speciﬁc manner, e.g. U74 was pro-
cessed only in Jurkat, but not in HeLa cells. Reporter gene
assays for the sno-miRNA activities indicated a possible
correlation between presence of the sno-miRNA and gene
silencing activity (Figure 3A). The snoRNA U21 was
not detected as a corresponding sno-miRNA and gene
silencing was not observed in Jurkat and HeLa cells.

A correlation of the expression level of the host snoRNA
to the sno-miRNA activity could not be concluded from
the results. U78 was the most abundant snoRNA that was
present as a sno-miRNA but still the silencing efﬁciency
was lower when compared to the U3 sno-miRNA.

miRNA-activity assays for 22 snoRNA derived
small RNAs

types,

We tested 21 putative sno-miRNAs, including 18 box C/D
and three box H/ACA snoRNA-derived, in dual luciferase
assays in three human cell
i.e. HeLa (cervix
carcinoma) line, Jurkat (T cells) and RPMI8866 (B cell).
Eleven of the box C/D sno-miRNA candidates tested
showed miRNA activity, which was directly comparable
to that of the ACA45 sno-miRNA in at least one of the
cell lines. All three box H/ACA sno-miRNAs were efﬁ-
cient in gene silencing. A summary for all sno-miRs,
including positions within the parental snoRNAs and
gene silencing efﬁciencies is given in Table 1. A more
detailed view on the
sequences of box C/D
sno-miRNAs and the relevant box C and D elements is
provided in Table 2.

full

Interestingly we identiﬁed the functional box C element
within the guide sequences of many sno-miRNAs that were
capable of gene silencing. The corresponding passenger
strands were in most cases stopped prior to or after the
ﬁrst two nucleotides of the box D element. Some passenger
strands were in addition ﬂanked by a box D0 element on the
50-end. Guide and passenger strand positions are shown in
Table 2, guides are shown in bold and underlined, passen-
gers only in bold. Box C elements are highlighted in blue,

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Nucleic Acids Research, 2010 7

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Figure 2. Structural and functional analysis of the box C/D snoRNA HBII-336 sno-miRNA. (A) Secondary structure prediction for HBII-336 was
calculated using RNAfold. A dashed red line marks the guide-sno-miRNA strand, the dashed blue line indicates the positions of the passenger
strands. (B) The dominant sno-miRNA sequence found in deep sequencing of Jurkat small RNAs is highlighted in red, the minor sequence is marked
with a blue bar. The sno-miRNA is located in a very stably base paired stem that can possibly be recognized and processed by Dicer. ClustalW
alignments of the guide and passenger HBII-336- sno-miRNAs show homogenous distributions of the sequences found in small RNA libraries. The
guide sno-miRNA (red) shows length variability of 21- to 27-nt, while the length distribution for the minor species (or the passenger-strand, blue) is
from 17- to 27-nt. (C) Functional investigation of the HBII-336 sno-miRNA in human HeLa cells by the dual luciferase assay shows similar silencing
effects when compared to the previously described ACA45 sno-miRNA. Insertion of a scrambled target site sequence into the Renilla luciferase gene
did not lead to signiﬁcant luciferase reduction when compared to the unmanipulated vector. Data represent normalized outcomes of three individual
experiments.

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box C and C0 elements in red colour. To conﬁrm that this
feature is a general property, we scanned all 402 snoRNAs
in snoRNABase version 3 (37) and found 107 candidates
where (i) the box C element (UGUGUA) occurs inside an
expressed subsequence and/or (ii) the box D element
(CUGA) is ﬂanking (and often partly contained in) the
small RNA on the 30-end (Table 3). Appropriate folding
of the intron-borne box C/D snoRNAs seems to be crucial
for processing of sno-miRNAs. Expression of full length
snoRNAs from a H1 polymerase III promotor did not
further
endogenous
sno-miRNA activity. Five box C/D snoRNAs were
tested in overexpression assays combined with the corres-
ponding psiCHECK-2-vectors and resulted in a mild cyto-
toxicity in HeLa cells without increasing silencing of the
Renilla luciferase gene (Supplementary Figure S2).

increase

effects

the

of

the

lines. Apparently some of

Figure 4 displays the relative silencing activity of the
C/D box snoRNA-derived sRNAs in comparison to
controls in four cell
the
sno-miRs were active only in some, hardly ever in all
cell types. Examples are snR39b, which is silencing in
HeLa and Jurkat, but not in RPMI8866, or U83a and
U27, which were silencing well
in Jurkat but did not
show signiﬁcant silencing effects in HeLa and MCF7
cells. However, some of the candidates that appeared
like promising Dicer substrates (e.g. U21) were incapable
of miRNA activity and did not interfere with their com-
plementary targets (Table 1 and Figure 3).

Functional characterization of box H/ACA sno-miRNAs

Three miRNA entries in the Sanger miRBase were dis-
covered to originate
from H/ACA box snoRNAs

8 Nucleic Acids Research, 2010

Figure 3. Sno-miRNA analysis in human HeLa and Jurkat cells. Five
individual snoRNAs were probed for expression and processing via
northern blots with total RNA extracts from HeLa and Jurkat cells
as shown in (B). Processed sno-miRNAs can be identiﬁed in both cell
types. In Hela extracts sno-miRNAs from U3, U78 and HBI-43 can
be seen, in Jurkat cell extracts the sno-miRNAs U74 and U78 were
identiﬁed. The total sizes of
length snoRNAs are indicated.
Reporter gene assays with complementary target sites for the sno-
miRNAs indicate a correlation of snoRNA processing to gene silencing
activity as displayed in (A). A randomized non-cognate target sequence
served as negative control,
the ACA45 sno-miRNA as a positive
control. The empty psiCHECK-2 vector was used for normalization.
Data represent the average outcome of four (HeLa, Jurkat) or three
(RPMI8866, MCF7) individual experiments.

full

with very similar features when compared to ACA45
(Figure 5A). Arrows indicate the location of mature
miRNA guide strands. In contrast to previous ﬁndings
(35) the sno-miRNA guide strands locate either to 30- or
50-terminal
the snoRNAs. We addressed
luciferase targets to the described mature miRNA se-
quences of miRs 664, 1248 and 1291 and found all of
them to be expressed and active in RPMI8866 and HeLa
cells (Figure 5B). Sno-miRNA ACA34 performed well
only in RPMI8866 cells.

regions of

Which known miRNAs are actually sno-miRNA?

We also tested whether some known miRNAs are actually
derived from snoRNAs. Therefore, the mature human
miRNAs in miRBase were matched against the ﬁltered
snoRNA fragments (‘Materials and Methods’ section).
Among these, we found three miRNAs
located in
H/ACA box snoRNAs [previously reported in ref. (35)
and two in C/D box snoRNAs (Table 1). In all cases,
the corresponding precursor sequences in miRBase fully
matched the snoRNA sequences, providing additional
evidence that these miRNA are misclassiﬁed. The pre-
dicted secondary structures of
two C/D box
snoRNAs represent rather atypical miRNA-precursors
or unstable hairpins among the snoRNAs tested here,
when comparing global binding patterns
(compare
Supplementary Figure S1A).

the

Table 3. snoRNAs with box C sequence (completely) inside and/or
box D sequence (right) ﬂanking sequences from the small RNA
libraries

snoRNA

C box
inside
sRNA

D box
Flanking
sRNA

snoRNA

C box
inside
sRNA

D box
ﬂanking
sRNA

HBI-43
HBII-108B
HBII-13
HBII-135
HBII-142
HBII-180B
HBII-180C
HBII-202
HBII-210
HBII-251
HBII-276
HBII-289
HBII-295
HBII-296B
HBII-336
HBII-419
HBII-429
HBII-436
HBII-85-12
HBII-85-16
HBII-85-17
HBII-85-18
HBII-85-19
HBII-85-21
HBII-85-22
HBII-95
HBII-99B
SNORD119
SNORD121B
U101
U102
U104
U105
U105B
U106
U15A
U15B
U16
U18A
U18B
U20
U21
U22
U24
U25
U26
U27
U28
U29
U3
U3-2
U3-2B
U3-3
U3-4
U30

x
x
x
x
x

x
x
x
x
x
x

x
x
x

x
x
x

x
x
x
x
x
x
x
x

x
x
x
x

x
x

x

x
x
x
x
x
x
x

x

x
x
x
x
x

x
x

x
x
x
x
x

x
x
x
x
x
x

x
x
x
x

x
x
x
x
x

x

x
x
x

x
x
x
x

x
x
x
x
x
x

x

x
x

x
x

x
x

x
x

x
x
x
x
x

x
x

x
x
x
x
x
x
x
x

x
x
x

x
x
x

x

U31
U35A
U36B
U36C
U37
U38A
U38B
U42B
U43
U44
U45B
U45C
U48
U49A
U50
U50B
U51
U52
U55
U58A
U58C
U59B
U60
U62A
U62B
U63
U65
U74
U76
U77
U78
U79
U8
U80
U81
U82
U83A
U83B
U84
U86
U87
U88
U96a
Z17B
mgU2-19
mgU6-47
mgU6-53B
mgh18S-121
snR38A
snR38B
snR38C
snR39B

x
x
x
x
x
x
x
x

x
x
x
x
x
x
x
x
x

x

x
x
x
x
x
x
x

x
x
x
x
x
x
x
x

x
x
x

x
x

x
x
x
x
x

The expressed subsequences may act as guide or passenger strands of
putative sno-miRNAs.

DISCUSSION

We identiﬁed numerous so far undiscovered box C/D
sno-miRNAs that exhibit processing, and in particular,
mRNA silencing features similar to those of miRNA

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Nucleic Acids Research, 2010 9

Figure 4. Functional analysis of snoRNA generated sdRNAs in different human cell lines. Reporter gene assays were performed on human HeLa
(light grey), Jurkat T (black), RPMI8866 B (white) and mammary carcinoma cell line MCF7 (dark grey). The snoRNA ACA45 served as positive
control, non-cognate target and empty dual luciferase as negative control and for normalization. General gene silencing can be seen for ACA45,
U3 and U3-4. HBII-336 and U83a did not induce any reduction of the Rluc gene in HeLa cells, but were very efﬁciently silencing the same target in
both Jurkat and RPMI8866 cells. Note the differences of some sno-miRNA target sensors on the different cell types (U27, HBII-336, U83a, snR39b).
Data for HeLa, Jurkat and RPMI8866 cells are from three individual experiments.

its

functions

in pre-rRNA processing,

molecules. These sno-miRNAs originate from relatively
short snoRNAs, such as U27 or HBII-336 as well as
from structurally more complex snoRNAs with >150 nt
in length, e.g. U3. The latter is particularly interesting
since U3 is a very well characterized snoRNA in terms
of
including
the shuttling of U3–snRNP
pre-rRNA capping (40),
complex between cytoplasm and nucleoli (41) and also
the post-transcriptional regulation of the snoRNA U3
itself
and experimental
determination of U3 snoRNP (43,44) characterize the
U3 snoRNA as complex and highly structured RNA
molecule (compare also Supplementary Figure S1B),
assembling into the U3 snoRNP complex. However, we
ﬁnd sequences of the U3 30-hairpin structure that are, at
least in HeLa and RPMI8866 cells, capable of targeting
complementary sequences for efﬁcient RNA silencing.

(42). Structure predictions

sequences

In functional analysis we demonstrate that in total
11 candidate
from C/D box snoRNAs
snR39b, U3 (2 sno-miRNAs), U78, HBII-336, HBII-429,
HBII-142, U27, U83a, U74 and U15a, are capable of
entering the silencing machinery and perform efﬁcient
gene silencing on reporter-gene mRNAs (Table 1). Our
results are indicating that snoRNA-derived small RNAs
can function like miRNAs and may therefore be termed
sno-miRNAs. We tested a broad variety of human cell
lines, including T- and B-lymphocytes, HeLa cells and
the mammary carcinoma cell line MCF7 and observed
general gene-silencing activity for 14 sno-miRNAs
including box H/ACA sno-miRNAs. In some cases we
observed a positive regulation of the target gene. This
effect may be due to an increased stability of
the
transcribed mRNA after insertion of the target site. In
absence of a loaded RISC attacking this mRNA R/F
values >1 may occur (compare Figure 4). However, a

number of the candidate sno-miRNAs tested were only
active in some cell types such as sno-miRNA U3-1 only
in HeLa and RPMI8866, sno-miRNA U83a in Jurkat
and RPMI8866 cells or sno-miR U27 only in Jurkat
(Figure 3).

The silencing activity differs among cell types, indicat-
ing discrete processing of snoRNAs that resembles the
post-transcriptional regulation of pre-miRNAs described
for rodent miRNAs (45). Differential snoRNA expression
among different cell types and tissues was shown for 5S
snoRNA genes (46) and regulation can be linked to host
genes in which the snoRNA are located. One example for
this effect is the increased level of U14 snoRNA that can
be observed upon co-expression of its host gene HSC70
during stress response in Chinese hamster ovary cells (47).
In general snoRNAs appear to have an extremely high
degree of expression ﬂexibility considering the modes of
transcription (individual and intronic), genomic organiza-
tion and processivity (48). A proﬁling of the expression
levels of box C/D snoRNAs in 11 different human tissues
was recently published and displayed strong variations of
copy numbers for some snoRNAs/sno-miRNAs from our
study (including snR39b and U3) (49). By testing total
RNA samples from human cells on northern blots with
radioactively labelled probes for different sno-miRNA
guide strands we see indications of different expression
levels of the snoRNAs between human HeLa and Jurkat
cells. Also, we succeeded in correlating existence of the
sno-miRNA to its function in RNA silencing. Due to
the low abundance of snoRNAs when compared to e.g.
miRNAs the signals are rather faint and only slightly
above the detection threshold. However, determinants
for generating sno-miRNAs
remain
elusive and will be subjected to future studies. Minimum
free energy based secondary structure predictions for all

from snoRNAs

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10 Nucleic Acids Research, 2010

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Figure 5. Box H/ACA originating miRNAs in functional analysis. (A) The predicted secondary structures of three miRbase listed snoRNAs
(miR-664/ACA36b, miR-1291/ACA34 and miR-1248/HBI-61, top right) share similar features with ACA45, shown top left. Two rather stable
hairpins are connected via a ﬂexible hinge loop, one of the hairpins is giving rise to a sno-miRNA (arrows). Sno-miRNA guide strand positions
are marked with dashed red lines and locate to the 30-stem of ACA45 and ACA36b, but to the 50-stems of ACA34 and HBI-64. (B) Target sites for
the sno-miRNA guide strands were checked in RPMI8866 (grey), Jurkat (black) and Hela (white) cells and compared to silencing activity of
ACA45-sdRNA. The most efﬁcient silencing on all cell types was observed for miR-664/ACA36b-sdRNA, miR-1291/ACA34 and miR-1248/
HBI-61 displayed only mild silencing, in particular in Jurkat cells. Values represent normalized data from two individual experiments.

snoRNAs that were tested for miRNA activity are
provided in the Supplementary data. These structures
are indicating possible alternatives to the functional
snoRNAs, which may be structurally different in their
native nucleolar environment. Increasing the expression
levels of the ‘naked’ snoRNAs by plasmid driven polymer-
ase III transcription did not lead to additional processing
of sno-miRNAs and an increase of the gene silencing
activity. This ﬁnding indicates that native processing of
the naturally intron-borne box C/D snoRNAs is a
crucial pre-requisite for sno-miRNA production.

early

evolved

from the

It is still very likely, that both snoRNAs and miRNAs
share a common ancestor, or even more plausible that
miRNAs
‘housekeeping’-
snoRNAs, as was claimed by Scott et al.
(35). An
overview on
ancestral RNA-silencing mechanisms
is provided in (50) and is indicating common origins
for various classes of regulatory non-protein coding
small RNAs. The miRNA function of a sno-miRNA
(miRNA2) originating from the box C/D snoRNA
GlsR17 in the ancient Giardia lamblia is supporting the
idea that biogenesis and processing of both snoRNAs
and miRNAs is performed by the same machinery (32).

export of

subsequent

However, mammalian miRNA biogenesis is rather
restricted when considering secondary structural features
of pri-miRNAs and pre-miRNAs. MiRNA function
through accurate RISC loading is dependent on successful
processing in the nucleus by Drosha/Pasha as already
described in 2004 (23),
the
pre-miRNA to the cytoplasm followed by Dicer process-
ing prior to RISC loading (51). The processing of the
H/ACA box sno-miRNA ACA45 is Drosha-independent
(16), indicating alternative routes for the biogenesis of
small RNAs with miRNA-like functions. Box C/D
sno-miRNAs display a variety of predicted structures,
including
(Supplementary
Figure S1). A proper Dicer processing remains question-
able since the preferred structure for the ribonuclease III is
a stable RNA stem (52,53) and structural requirements for
accurate miRNA processing are well deﬁned in mammals
(54). Recently a novel processing scheme for miRNA
biogenesis was introduced and could probably explain
processing and RISC loading of snoRNAs. Processing
of
the human miR-451 resulted in overlapping and
loop-spanning mature miRNAs in a presumably ago2
mediated and Dicer independent mechanism (55,56).

rather unstable

hairpins

MiRNA biogenesis in plants harbours some principle
differences and the secondary structure of appropriately
processed miRNA precursors is much more diverse
when compared to animal miRNAs (57,58). A link
from snoRNA structure to a miRNA-like function in
gene silencing cannot be concluded from our data.
Nevertheless, we provide an alternative, which is based
on the location of box D elements (CUGA) right
ﬂanking one mature sno-miRNA strand in most of the
snoRNAs tested here (Table 2). Box C elements with the
consensus sequence UGAUGA are often located within
the other mature sno-miRNA sequence. The same
pattern could be identiﬁed in 107 from 402 snoRNA se-
quences which are currently contained in snoRNABase
(Table 3). This may indicate a controlled RNA processing
based on recognition of the functional elements of box C/
D snoRNAs rather than on structural elements as it is the
case for miRNA biogenesis. We ﬁnd some sno-miRs
originating from RNA hairpin regions; some do not at
all display this feature. However, for both types of second-
ary structures in the parental snoRNAs we identiﬁed can-
didates with and without miRNA like functions.

Carefully observing the recent literature the reader will
ﬁnd numerous publications on the rapidly growing
number of novel non-coding RNA species with regulatory
or so far unknown function (59). New members of this
family are tiRNAs, which apparently are involved in
promoter transcriptional gene regulation via TSS inter-
action (14) or, very recently, tRNA transcript-derived
RNA fragments, or tRFs, which are not yet functionally
characterized, but aberrantly induce proliferative defects
when lost (60). Our study is now adding an additional
class of dual-action non-coding RNAs by introducing
the gene regulatory effects of human box C/D snoRNAs.
The spectra of novel ncRNA, the bandwidth of their
functions,
their abundance through the kingdoms of
life and the ever increasing range of new discoveries
is implying a Kuhnian revolution in understanding the
molecular processes in life sciences.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

ACKNOWLEDGEMENTS

The authors thank Dr Timofey Rhozdestvensky and
Prof. Ju¨ rgen Brosius for fruitful discussion. Christina
Albrecht and Meike Hermes
contributed valuable
comments on the article and hands-on help. Ellen
Eckermann-Felkl contributed expert technical assistance,
Martina Reitz, Alexandra Juckert and Michaela Micke
contributed to experimental work.

FUNDING

Funding for open access charges: German Federal
Ministry of Education and Research joint
research
project ‘RNomics in Infectious Diseases’ in the frame of
the National Genome Research Net (NGFN-Plus).

Nucleic Acids Research, 2010 11

Conﬂict of interest statement. None declared.

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