A Role for Small RNAs in DNA Double-Strand Break Repair

Please cite this article in press as: Wei et al., A Role for Small RNAs in DNA Double-Strand Break Repair, Cell (2012), doi:10.1016/j.cell.2012.03.002

A Role for Small RNAs in DNADouble-Strand Break RepairWei Wei,1,2,9 Zhaoqing Ba,2,3,9 Min Gao,4 Yang Wu,2 Yanting Ma,2 Simon Amiard,5 Charles I. White,5

Jannie Michaela Rendtlew Danielsen,4,6 Yun-Gui Yang,4 and Yijun Qi2,7,8,*1Graduate Program, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China2National Institute of Biological Sciences, Number 7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China3College of Life Sciences, Beijing Normal University, Beijing 100875, China4Genome Structure and Stability Group, Disease Genomics and Individualized Medicine Laboratory, Beijing Institute of Genomics,

Chinese Academy of Sciences, Number 7 Beitucheng West Road, Chaoyang District, Beijing 100029, China5Genetique, Reproduction et Developpement, Unite Mixte de Recherche Centre National de la Recherche Scientifique 6247,

Clermont Universite, Institut National de la Sante et de la Recherche Medicale U931, Aubiere 63177, France6The Novo Nordisk Foundation Center for Protein Research, Ubiquitin Signalling Group, Faculty of Health Sciences, Blegdamsvej 3b,

2200 Copenhagen, Denmark7Tsinghua-Peking Center for Life Sciences, Beijing 100084, China8School of Life Sciences, Tsinghua University, Beijing 100084, China9These authors contributed equally to this work

*Correspondence: [email protected]

DOI 10.1016/j.cell.2012.03.002

SUMMARY

Eukaryotes have evolved complex mechanisms torepair DNA double-strand breaks (DSBs) throughcoordinated actions of protein sensors, transducers,and effectors. Here we show that �21-nucleotidesmall RNAs are produced from the sequences inthe vicinity of DSB sites in Arabidopsis and in humancells. We refer to these as diRNAs for DSB-inducedsmall RNAs. In Arabidopsis, the biogenesis of diR-NAs requires the PI3 kinase ATR, RNA polymeraseIV (Pol IV), and Dicer-like proteins. Mutations in theseproteins as well as in Pol V cause significant reduc-tion in DSB repair efficiency. In Arabidopsis, diRNAsare recruited by Argonaute 2 (AGO2) to mediate DSBrepair. Knock down of Dicer or Ago2 in human cellsreduces DSB repair. Our findings reveal a conservedfunction for small RNAs in the DSB repair pathway.We propose that diRNAs may function as guidemolecules directing chromatin modifications or therecruitment of protein complexes to DSB sites tofacilitate repair.

INTRODUCTION

DNA double-strand breaks (DSBs) are deleterious forms of DNA

damage that can cause mutations, genome instability, and cell

death. Efficient repair of DSBs is thus critical for themaintenance

of genome integrity and cell survival. DSBs are mainly repaired

by nonhomologous end joining (NHEJ) or homologous recombi-

nation (HR) (Ciccia and Elledge, 2010; Lieber, 2010; Moynahan

and Jasin, 2010; Puchta, 2005; San Filippo et al., 2008; Sasaki

et al., 2010). NHEJ repairs DSBs in an efficient but error-prone

fashion. It can cause deletions or insertions at the break site

because of the modification of DNA ends before joining (Lieber,

2010). In contrast, HR is considered error free, but it requires

resection of the DSB and a sister chromatid as template for

repair (Moynahan and Jasin, 2010; San Filippo et al., 2008; Sa-

saki et al., 2010). Single-strand annealing (SSA) is a particular

type of HR that takes place when DSB resection occurs at repet-

itive sequences, providing complementary single strands that

can then anneal (Ciccia and Elledge, 2010; Hartlerode and

Scully, 2009). These repair pathways all require well-regulated

and coordinated enzymatic actions of protein sensors, trans-

ducers, and effectors in the DSB signaling cascade (Ciccia and

Elledge, 2010; Huen and Chen, 2008; Polo and Jackson, 2011).

Small RNAs have emerged as key players in various aspects of

biology. Three major classes of small RNAs have been discov-

ered in eukaryotes: microRNAs (miRNAs), small interfering

RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs). miRNAs

and siRNAs are processed by RNase III domain-containing Dicer

or Dicer-like (DCL) proteins from their double-stranded RNA

(dsRNA) precursors. Via association with members of the Argo-

naute (AGO) family, small RNAs mediate transcriptional or post-

transcriptional regulation of gene expression (Baulcombe, 2004;

Carthew and Sontheimer, 2009). AGO proteins contain several

characteristic domains: a variable N-terminal domain and con-

served PAZ, MID, and PIWI domains (Tolia and Joshua-Tor,

2007). The PAZ and PIWI domains bind to the 30 and 50 ends of

small RNAs, respectively (Ma et al., 2004; Ma et al., 2005), and

the PIWI domain has endonuclease activity when an Asp-Asp-

His catalytic triad is present (Rivas et al., 2005; Song et al., 2004).

In plants, the most abundant small RNAs are heterochromatic

siRNAs (hc-siRNAs) derived from transposons and other repeti-

tive sequences. Single-stranded RNA transcripts are presum-

ably generated from DNA repeats by DNA-dependent RNA

polymerase IV (Pol IV) (Herr et al., 2005; Onodera et al., 2005)

and converted by RNA-dependent RNA polymerase 2 (RDR2)

Cell 149, 1–12, March 30, 2012 ª2012 Elsevier Inc. 1

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http://dx.doi.org/10.1016/j.cell.2012.03.002


into dsRNAs (Xie et al., 2004), which are in turn processed

by DCL3 into 24 nucleotide (nt) hc-siRNAs (Xie et al., 2004).

Hc-siRNAs are associated with AGO4 subfamily members and

direct DNA methylation by the de novo DNA methyltransferase

DRM2 through a pathway known as RNA-directed DNA methyl-

ation (RdDM) (Law and Jacobsen, 2010; Matzke et al., 2009).

Functionally analogous to plant hc-siRNAs, piRNAs are specific

to animals and most often specifically generated in the germ line

to inactivate transposons (Malone and Hannon, 2009). In addi-

tion to these three classes of small RNAs, other types of small

RNAs have also been found in various organisms. Tetrahymera

thermophila contains scan RNAs (scnRNAs) that are involved

in developmentally regulated DNA elimination (Mochizuki and

Gorovsky, 2004; Yao and Chao, 2005). More recently, high-

throughput approaches have identified promoter-associated

short RNAs (PASRs) (Kapranov et al., 2007), termini-associated

short RNAs (TASRs) (Kapranov et al., 2007; Kapranov et al.,

2010), and transcription-initiation RNAs (tiRNAs) (Taft et al.,

2009) in animals.

In light of the expanding universe of small RNAs and their

increasingly diverse biological roles, we explored whether small

RNAs could play a role in DSB repair. Using well-established

reporter assays for DSB repair in Arabidopsis thaliana and

human cells, we found that DSBs trigger the production of small

RNAs from the sequences in the vicinity of DSB sites and these

small RNAs are required for efficient DSB repair. Our results

reveal an unsuspected and conserved role for small RNAs in

the DSB repair pathway.

RESULTS

DCLs Are Required for Efficient DSB Repairin Arabidopsis

We used an Arabidopsis transgenic line carrying the DGU.US

reporter in which expression of the endonuclease I-SceI,

induced by crossing, introduces a single DSB in the genome

(Mannuss et al., 2010; Orel et al., 2003). Repair of the DSB

through SSA mechanism restores b-glucuronidase (GUS)

expression, which provides a visible and quantitative readout

of DSB repair events (Figure 1A). Confirming published results

(Mannuss et al., 2010; Orel et al., 2003), crossing the DGU.US

reporter (R) line with a DSB-triggering (T) line that expresses

I-SceI induced DSB repair with a repair rate about two orders

of magnitude higher than that of uncrossed R line (Figures 1B,

1D, and 1E). We then introduced, via crossing, this reporter

system into an Arabidopsis atr mutant background to assay

the DSB repair rate in this mutant (Figure S1, available online).

ATR encodes a phosphatidyl inositol 3-kinase-like protein kinase

(PI3 kinase) that primarily responds to stalled replication forks

and also acts with ATM (Ataxia Telangiectasia Mutated, another

PI3 kinase) in DSB response (Amiard et al., 2010; Culligan et al.,

2004; Culligan and Britt, 2008; Culligan et al., 2006; Jazayeri

et al., 2006). We found that the repair efficiency was greatly

reduced in the atr mutant as determined by GUS staining or

PCR detection of repaired DNA (Figures 1B, 1D, and 1E).

DCLs are required for small RNA biogenesis in Arabidopsis

(Xie et al., 2004). We next examined whether DCLs are involved

in DSB repair. We introduced the GUS reporter system into dcl2,

2 Cell 149, 1–12, March 30, 2012 ª2012 Elsevier Inc.

dcl3, and dcl4mutant backgrounds. Intriguingly, compared with

those in the wild-type controls, the repair rates were reduced by

42%, 90%, and 44% in the dcl2, dcl3, and dcl4 mutant back-

grounds, respectively (Figures 1C, 1D, and 1E), suggesting that

DCL2, DCL3, and DCL4 play partially redundant roles in DSB

repair and that DCL3 is the major player.

DSBs Trigger Production of Small RNAs fromSequencesFlanking the DSB SitesThe requirement of DCLs for efficient DSB repair suggested

the involvement of small RNAs in this process. Thus, we sought

to examine whether small RNAs were produced from the

sequences around the DSB site by northern blot analysis. When

a probe that recognizes the �450 nt sequence flanking the

I-SceI site (Figure 1A) was used, small RNAs were barely detect-

able in theR or T line but were readily detected in the progenies of

RxT cross (Figure 2A). These small RNAswere nameddiRNAs for

DSB-induced small RNAs. Deep sequencing analyses revealed

that diRNAs were specifically derived from both sense and anti-

sense strands of the sequence around the I-SceI site (Figure 2B).

Confirming thenorthernblot results,�40 timesmorediRNA reads

were obtained from the RxT sample compared to those from the

uncrossed R line (Figure 2B), whereas the expression profiles of

endogenous small RNAs (miRNAs and siRNAs) were comparable

in these two samples (Table S1). Moreover, we found that the

production of diRNAs was dependent on ATR. In the atr mutant

background, the diRNAs were barely detectable either by

northern blot (Figure 2C) or deep sequencing (Table S2).

We extended the diRNA analysis to another DSB repair

reporter (DU.GUS) system (Orel et al., 2003). In this system,

the DSB site generated by I-SceI digestion is repaired by the

HR pathway that involves gene conversion through synthesis-

dependent strand annealing (SDSA). We detected the produc-

tion of diRNAs in the DU.GUSxT plants by deep sequencing

(Figure 2D). The amount of diRNAs produced in the DU.GUSxT

plants was approximately one-fifth of that detected in the RxT

plants (Figures 2B and 2D). This is in correlation with the lower

DSB repair frequency in the DU.GUS line, which has been

reported to be about five times lower than that in the DGU.US

line (Orel et al., 2003).Taken together, these observations

demonstrate that DSBs induce the production of diRNAs specif-

ically around the DSB sites.

diRNAs Play a Role in DSB RepairTo investigate the role of diRNAs in DSB repair, we first examined

the production of diRNAs in the dcl mutants that had reduced

repair rates (Figures 1C, 1D, and 1E). Compared to the wild-

type controls, dcl2, dcl3, and dcl4 accumulated many fewer

diRNAs (Figure 3A and Table S2). In particular, the production

of diRNAs was reduced by 98% in the dcl3 mutant, consistent

with its predominant effect on the repair efficiency (Figures 1C,

1D, and 1E). DCL2, DCL3, and DCL4 are known to process

dsRNAs into 22 nt, 24 nt, and 21 nt siRNAs, respectively (Qi

et al., 2005; Xie et al., 2004). It is intriguing that the production

of both 21 nt and 24 nt siRNAs was greatly reduced in each of

the dcl mutants (Figure 3A). This suggests that diRNAs might

be processed from their dsRNA precursors through coordinated

actions of DCL2, DCL3, and DCL4. Coordinated actions of DCLs

Figure 1. DCLs Are Required for Efficient DSB Repair in Arabidopsis

(A) Schematic representation of DSB repair in the DGU.US reporter system. The DGU.US reporter (R) line harbors an I-SceI site located within the direct repeats

(U) of a nonfunctional GUS. Crossing R with the DSB-triggering (T) line that expresses the I-SceI endonuclease introduces a single DSB in the genome of F1

progenies (RxT). Repair of the DSB restores the functional GUS.

(B and C) Representative GUS staining images for DSB repair analysis in the T, R, and RxT plants and RxT plants in the indicated mutant backgrounds (�/�) or

their corresponding wild-type (+/+) backgrounds. See Figure S1 for the crossing scheme.

(D) The relative repair rate in the indicated plants determined by GUS staining. For each genetic background, at least 30 plants from three independent

experiments were stained and blue sectors were counted. The DSB repair rates in the mutant plants (�/�) are presented in relation to those of the corresponding

wild-type (+/+) controls (arbitrarily set to 1.0). Error bars indicate standard error of the mean (SEM), and the asterisks indicate a significant difference between the

indicated groups (t test, p value < 0.001).

(E) Detection of repaired DNA in the indicated plants by PCR using primers p1 and p2 depicted in (A), Histone H4 was also amplified and used as the internal

control. See also Figure S1.


have been previously shown for the biogenesis of longmiRNAs in

rice (Wu et al., 2010).

The generation of diRNAs from both sense and antisense

strands at approximately equal frequency (Figure 2B) suggested

that they were processed from dsRNA precursors, the produc-

tion of which usually requires RDRs (Xie et al., 2004). RDR2 is

required for the production of hc-siRNAs (Xie et al., 2004),

whereas RDR6 plays a role in the biogenesis of trans-acting

siRNAs (Peragine et al., 2004; Vazquez et al., 2004). We found

that mutations in RDR2 and RDR6 caused 87% and 82%

reductions in diRNA production, respectively (Figure 3A and

Table S2). However, these mutations had no significant effects

on the repair efficiency (Figures 3B and 3C and Figure S3),

which could indicate a redundancy between RDR2 and RDR6

or that other RDRs are involved. Alternatively, these findings

could also imply that there might be a threshold of diRNA

abundance required for its function in DSB repair and that the

levels of diRNAs present in the rdr2 and rdr6 mutants are

sufficient.

We next examined whether Pol IV and Pol V are required for

diRNA biogenesis and DSB repair. Pol IV and Pol V are both

involved in the Arabidopsis RdDM pathway. Pol IV is required

for hc-siRNA biogenesis (Herr et al., 2005; Kanno et al., 2005;

Onodera et al., 2005), whereas Pol V generates nascent scaffold


Figure 2. DSB Induces the Production of diRNAs Specifically Around the DSB Site in Arabidopsis

(A) Detection of small RNAs in the F1 progenies (RxT), as well as the parental plants (R and T) by northern blot analysis. The probe used for detection of

GUS-derived diRNAs is depicted in Figure 1A. miR173, SIMPLEHAT2 hc-siRNAs, and U6 were probed as controls. nt is an abbreviation for nucleotide.

(B) Deep sequencing analysis of diRNAs generated from DGU.US reporter construct. The y axis represents the number of normalized small RNA reads per

10 million sequences, numbers in (+) and (�) values represent the reads of small RNAs derived from sense and antisense strands, respectively. The nucleotide

positions (bp) of the components in the DGU.US construct are shown.

(C) Northern blot detection of diRNAs in RxT in Col-0 background (WT), atr mutant (�/�) and its corresponding wild-type (+/+) backgrounds.

(D) Deep sequencing analysis of diRNAs generated from DU.GUS reporter construct. The y axis represents the number of normalized small RNA reads per 10

million sequences, numbers in (+) and (�) values represent the reads of small RNAs derived from sense and antisense strands, respectively. The nucleotide

positions (bp) of the components in the DU.GUS construct are shown. See Figure S2 for the diagram of DU.GUS reporter construct.



Figure 3. A Role for diRNAs in DSBRepair in

Arabidopsis

(A) Northern blot analysis of diRNA accumulation

in the indicated mutant (�/�) and the corre-

sponding wild-type (+/+) backgrounds. miR173,

ta-siR255, and SIMPLEHAT2 hc-siRNAswere also

probed and used for verification of respective

mutant backgrounds. U6 was detected and used

as a loading control. nt is an abbreviation for

nucleotide.

(B) The relative DSB repair rates in the indicated

plants determined by GUS staining. For each

genetic background, at least 30 plants from three

independent experiments were stained and blue

sectors were counted. The repair rates in the

mutant plants (�/�) are presented in relation to

those of the corresponding wild-type (+/+)

controls (arbitrarily set to 1.0). Error bars indicate

SEM, and asterisks indicate a significant differ-

ence between the indicated groups (t test,

p value < 0.001).

(C) Detection of repaired DNA in the indicated

plants by PCR.

(D) Detection of diRNAs in the ago4 mutant (�/�)

and the wild-type control (+/+) plants.

(E) The repair rates in the ago4mutant (�/�) and its

corresponding wild-type control (+/+) determined

by GUS staining (upper panel) and PCR (lower

panel).

(F) The repair rates in the drm1/drm2 double

mutant (�/�) and its corresponding wild-type

control (+/+) determined by GUS staining (upper

panel) and PCR (lower panel). See also Figures S3

and S4.


transcripts upon which RdDM effector complexes are assem-

bled but has no direct role in hc-siRNA biogenesis (Wierzbicki

et al., 2008). Intriguingly, diRNA production was greatly compro-

mised in nrpd1 (NRPD1 encodes the largest subunit of Pol IV) but

increased in nrpe1 (NRPE1 encodes the largest subunit of Pol V)

(Figure 3A and Table S2). Repair rates were reduced by 80% and

50% in the nrpd1 and nrpe1 mutant backgrounds, respectively

(Figures 3B and 3C and Figure S3). These data indicate that

Pol IV and Pol V are involved in DSB repair through regulating

diRNA biogenesis and functioning, respectively.

diRNA-Mediated DSB Repair Does Not Involve the RdDMPathwayDCL3, Pol IV, and Pol V are all components in the RdDMpathway

(Law and Jacobsen, 2010). The dependence of DSB repair on

Cell 149, 1

these genes raised a possibility that

diRNAs function through RdDM to

mediate DSB repair. To test this possi-

bility, we investigated whether AGO4

(the major RdDM effector protein that

binds hc-siRNAs) (Qi et al., 2006; Zilber-

man et al., 2003) and DRM2 (the de

novo DNA methyltransferase that cata-

lyzes RdDM) (Cao and Jacobsen, 2002;

Matzke et al., 2009) are involved in DSB

repair. In the ago4 mutant, the accumulation of diRNAs

was not reduced but instead mildly increased (Figure 3D and

Table S2), and DSB repair efficiency was also not affected (Fig-

ure 3E). Similarly, mutation in DRM1/2 did not have an obvious

effect on DSB repair (Figure 3F).

The reduced DSB repair efficiency observed in dcl3, nrpd1,

and nrpe1 could be caused by dysregulation of genes involved

in DNA damage response in these mutants. To test this possi-

bility, we used quantitative RT-PCR (qRT-PCR) to measure the

expression levels of several genes (MRE11, RAD50, NBS1,

ATM, ATR, RAD51, RPA1, BRCA1, BRCA2, RAD54, RECQ4A,

RAD5A, and RPA2b) that play key roles in DNA damage

response. We found that the expression levels of all the exam-

ined genes were comparable in wild-type and mutant plants

(Figure S4).

–12, March 30, 2012 ª2012 Elsevier Inc. 5

Figure 4. AGO2 Is an Effector Protein of diRNAs

(A and B) AGO2 expression was induced by g-irradiation

as measured at both mRNA and protein levels by qRT-

PCR (A) and western blot (B) analyses, respectively. Error

bar indicates SEM and the asterisk (*) indicates a signifi-

cant difference between the indicated samples (t test, p <

0.001). In (B) tubulin was detected in parallel and used as

a loading control.

(C) Detection of diRNAs in the immunopurified AGO2

complex from the RxT plants by northern blot analysis.

The silver-stained gel shows comparable amounts of

AGO2 complexes were used for RNA extraction.

(D and E) Northern blot (D) and deep sequencing (E)

analyses of diRNAs in the ago2 mutant (�/�) and the

corresponding wild-type (+/+) plants. In (E), reads per

10 million sequences are shown after being normalized

with reads of endogenous miRNAs.

(F) The effect of mutation in AGO2 on the DSB repair rate

was determined by GUS staining. Representative images

are shown in the left. Relative repair rate was calculated

and shown in the right. For each genetic background, at

least 30 plants from three independent experiments were

assayed. Error bars indicate SEM, and the asterisk indi-

cates a significant difference between the indicated

groups (t test, p value < 0.001).

(G) Detection of repaired DNA by PCR in the indicated

plants. See also Figure S5.


These results argue against the possibility that diRNAs

mediate DSB repair through the RdDM pathway or through the

regulation of genes involved in DSB response.

AGO2 Is an Effector Protein of diRNAsTo dissect the mechanism through which diRNAs act in DSB

repair, we sought to identify the effector protein that recruits

diRNAs. It has been previously reported that the expression of

AGO2 can be induced by g-irradiation, a potent DSB inducer

(Culligan et al., 2006). Consistent with the published results,

we found that expression of AGO2, but not the expression of

other AGOs, was highly induced in plants upon g-irradiation at

both mRNA and protein levels (Figures 4A and 4B and Fig-

ure S5A). This suggested AGO2 as a potential candidate effector

that recruits diRNAs. To test this, we immunopurified AGO2

complexes from the RxT plants as well as the uncrossed R line

(Figure 4C) and examined whether they contained diRNAs.

Northern blot analysis detected diRNAs in the AGO2 complexes

isolated from the RxT plants but not in those from the uncrossed

R line (Figure 4C). Confirming the northern blot results, deep

sequencing analysis revealed that GUS-matching diRNAs

accounted for �3% of the AGO2-bound small RNAs, and there

was a 6.2-fold enrichment of diRNAs in AGO2 relative to those

in the total extracts (Figures S5B and C). In agreement with the

role of AGO2 in recruiting diRNAs, the ago2mutant had dramat-

ically reduced accumulation of diRNAs (Figures 4D and 4E) and

reduced repair rate (Figures 4F and 4G). These results indicate

that AGO2 is an effector protein of diRNAs and plays a role in

DSB repair.


We reasoned that some diRNAs might be produced from

endogenous loci upon g-irradiation. We immunoprecipitated

AGO2 complexes from plants with or without the treatment of

g-irradiation and analyzed the coimmunoprecipitated small

RNAs by deep sequencing. We identified 150 loci that produced

two times more small RNAs in the g-irradiated plants than in the

unirradiated plants (Table S3). As g-irradiation triggers DSB

randomly in the chromosomes, we were unable to determine

the DSB sites in the g-irradiated plants. It remains to be

confirmed whether these induced small RNAs were DSB

associated.

diRNAs Are Not Involved in the Phosphorylation of H2AXPhosphorylation of histone H2AX, referred to as g-H2AX, is one

of the earliest events in the response to DSBs. g-H2AX plays a

key role in recruiting repair and chromatin remodeling factors

at the sites of DNA damage (Fillingham et al., 2006; Paull et al.,

2000; Podhorecka et al., 2010) and has emerged as a highly

specific and sensitive molecular marker for monitoring DSBs

and their repair (Amiard et al., 2010; Kinner et al., 2008). In

Arabidopsis, phosphorylation of H2AX is dependent on both

ATM and ATR in response to DSBs caused by ionizing radiation,

and ATM has a dominant role (Friesner et al., 2005).

We examined whether diRNAs are required for the phosphor-

ylation of H2AX at DSB sites induced by g-irradiation. We

performed g-H2AX immunofluorescence staining with nuclei

isolated from Arabidopsis leaves. As expected, g-H2AX foci

were not detectable in the nuclei of unirradiated plants. When

plants were irradiated with 25 Gy, 100% of the nuclei from

Figure 5. diRNAs Are Not Involved in the

Phosphorylation of H2AX

(A) Detection of g-H2AX by fluorescent immuno-

staining in the nuclei of wild-type (Col-0) and the

indicated mutant plants without (0 Gy, upper

panels) or with (25 Gy, lower panels) the treatment

of 25 Gy g-irradiation. DNA was stained with DAPI

(blue), and g-H2AX foci were colored in red.

Representative pictures are shown. The scale

bar = 5 mm.

(B) Graphic representation of the number of

g-H2AX foci detected in the unirradiated or irra-

diated wild-type (Col-0) and mutant plants. The

numbers of foci in at least 30 nuclei were counted

for each sample and used to generate the

diagrams. In the right panel, error bars indicate

SEM, and the asterisks (*) indicate a significant

difference between the indicated groups (t test,

p value < 0.001).

See also Figure S6.


wild-type plants showed g-H2AX foci with a mean of 31 foci per

nucleus (Figures 5A and 5B). The proportion of nuclei showing

g-H2AX foci and the number of foci per nucleus were dramati-

cally reduced in the irradiated atm mutant plants and slightly

but significantly decreased in the irradiated atr mutant plants

(Figures 5A and 5B). However, the amounts of g-H2AX foci in

irradiated nrpd1, dcl3, and ago2mutant plants were comparable

to those in irradiated wild-type plants (Figures 5A and 5B). Immu-

nofluorescence staining with mitotic root tip nuclei showed that

mutations in AGO2 and DCL3 did not decrease the numbers of

g-H2AX foci in M-phase or interphase nuclei (Figure S6). These

data indicate that diRNAs are not involved for the generation of

g-H2AX and diRNAs most likely function downstream of H2AX

phosphorylation.

Cell 149, 1

Detection of diRNAs and Their Rolein DSB Repair in Human CellsOur findings point to a key role for diRNAs

in the DSB repair pathway in plants.

Becausemultiple aspects of this pathway

are highly conserved, we asked whether

diRNAs could also be involved in DSB

repair in mammalian cells. We employed

human U2OS cells carrying a DR-GFP

substrate (DR-GFP/U2OS), which con-

tains two nonfunctional GFP open

reading frames, including one GFP-

coding sequence that is interrupted by

a recognition site for the I-SceI endonu-

clease. Expression of I-SceI leads to

formation of a DSB in the I-SceI GFP

allele, which is repaired by HR using a

nearby GFP lacking N- and C-terminal

GFP-coding sequences, thereby pro-

ducing functional green fluorescent pro-

tein (GFP) that can be readily detected

by flow cytometry (Pierce et al., 1999).

DSBs were induced by the expression

of I-SceI in DR-GFP/U2OS cells (Figure S7). Deep sequencing

analyses demonstrated that, as in Arabidopsis, DSBs in human

cells induced diRNA production from sense and antisense

strands of the sequence close to the DSB site (Figure 6A).

diRNAs appeared to be produced from the vicinity of the DSB

but not directly around it as in Arabidopsis. To examine the

impact of diRNA production on DSB repair in human cells, we

investigated the effect of Dicer or Ago2 depletion on DSB repair

efficiency. Whereas DR-GFP/U2OS cells treated with control

siRNAs displayed efficient repair resulting in robust production

of GFP-positive cells after I-SceI expression, a significant reduc-

tion in HR of DNA DSB repair was observed after Dicer or Ago2

depletion (Figures 6B and 6C). We tested the protein expression

levels of several DSB repair proteins and found that the levels

–12, March 30, 2012 ª2012 Elsevier Inc. 7

Figure 6. diRNA Production and Regulation of DSB Repair in Human Cells(A) Deep sequencing analysis of diRNAs generated from the DR-GFP reporter construct. The y axis represents the number of normalized small RNA reads per 10

million sequences, numbers in (+) and (�) values represent the reads of small RNAs derived from sense and antisense strands, respectively. Small RNA reads

from RNA isolated from DR-GFP/U2OS cells at 12, 16, 20, or 24 hr following I-SceI transfection or at 20 hr following transfection with control vector (Control) are

shown. The nucleotide positions (bp) of the components in the DR-GFP construct are shown.

(B and C) Relative repair rate in DR-GFP/U2OS cells after treatment with the indicated siRNAs. Forty-eight hours after siRNA treatment, DR-GFP/U2OS

cells were transfected with I-SceI plasmid for 48 hr and processed for flow cytometric analysis of GFP. The repair efficiency was scored as the percentage of

GFP-positive cells in control, Dicer, or Ago2 siRNA-treated cells. Graph represents the mean of three independent experiments. Error bars indicate SEM.

(D) DR-GFP/U2OS cells were treated with Control, Dicer, or Ago2 siRNAs for 48 hr and analyzed by immunoblotting with the indicated antibodies.

See also Figure S7.


were comparable in control and Ago2 or Dicer siRNA-treated

cells (Figure 6D). Taken together, the identification of diRNAs

in both plants and human cells points to a conserved role for

the small RNA pathway in DSB repair.


DISCUSSION

Damaged DNA is repaired through coordinated activation of

cell cycle checkpoints and DNA repair machineries, which

Figure 7. A Model for diRNA-Mediated DSB Repair in Arabidopsis

This model is proposed on the basis of genetically identified components

required for diRNA-mediated DSB repair and their roles extrapolated from their

known functions in the RdDM pathway in Arabidopsis. Single-stranded RNA

transcripts (ssRNAs) are presumably generated by RNA polymerase IV

(NRPD1 and NRPD2) from the sequences in the vicinity of a DSB. Redundant

activities of RNA-dependent RNA polymerases (RDRs) convert the ssRNAs

into double-stranded RNAs (dsRNAs), which are processed into diRNAs by

coordinated actions of Dicer-like proteins (DCL2, DCL3, and DCL4). diRNAs

are then incorporated into Argonaute 2 (AGO2). AGO2/diRNA complexes are

localized to the DSB site through interaction with scaffold transcripts that are

made by Pol V (NRPE1 and NRPE2). AGO2/diRNA complexes may recruit

chromatinmodifying complexes tomodify local chromatin (A) or directly recruit

DSB repair proteins to the DSB site (B) to facilitate DSB repair. Further

experiments are required to test thismodel. The gray dots represent chromatin

modifications.


involve protein sensors, transducers, and effectors (Ciccia and

Elledge, 2010; Huen and Chen, 2008; Polo and Jackson, 2011).

In this study, we established an important role for small RNAs

in DSB repair, adding an unsuspected RNA component to the

DSB repair signaling pathway. Importantly, we demonstrated

that this layer of DSB repair regulation is conserved: diRNAs

are produced in both plant and human cells and interfering

with their production has severe effects on DSB repair. It is

also noteworthy that in the filamentous fungus Neurospora

crassa, QDE-2-interacting small RNAs (qiRNAs) derived from

rDNA repeats have been detected in cells treated with DNA

damaging agents (Lee et al., 2009). It was proposed that these

small RNAs contribute to DNA damage response by inhibiting

rRNA biogenesis and protein translation (Lee et al., 2009). In

light of our current findings, an alternative interpretation may

be considered. rDNAs are arrayed in tandem in the Neurospora

genome (Galagan et al., 2003), which makes them perfect

targets for HR. Upon exposure to DNA damaging agents,

DSBs may be introduced within the rDNA repeats. This could

then trigger the production of rDNA-specific small RNAs

that mediate DSB repair on damaged repetitive rDNAs. Inter-

estingly, in the fly female germline, mutations in the repeat-

associated siRNA (rasiRNA, an analog of piRNA in mammals)

pathway resulted in elevated g-H2AX levels (Klattenhoff et al.,

2007), raising the possibility that rasiRNAs are involved in

DSB repair.

RDRs andPol IVwere found to be involved in diRNA biogenesis

in Arabidopsis (Figure 3). Based on their roles in making dsRNA

precursors of hc-siRNAs in the RdDM pathway (Law and Jacob-

sen, 2010;Matzke et al., 2009), we speculate that single-stranded

DNA (ssDNA) generated by resection of the DSB might serve as

the template for Pol IV/RDRs-mediated generation of double-

stranded diRNA precursors (Figure 7). In accordance with this

hypothesis, it has previously been shown that the Neurospora

RDR (QDE-1) can produce dsRNA from ssDNA (Lee et al.,

2010). We found that no diRNAs could be detected in atr mutant

plants (Figure 2C). This could suggest that ATR-dependent phos-

phorylation of components of the DSB repair machinery is

required for the recruitment of the diRNA biogenesis machinery

to the DSB sites. Alternatively, ATR-dependent phosphorylation

of components of the diRNA biogenesis machinery itself could

be required for their activityor recruitment tositesofDNAdamage.

diRNAs were specifically generated from the regions close to

the DSB sites (Figures 2 and 6), implying that diRNAs mediate

DSB repair in cis. We showed that DSB repair is not compro-

mised in the ago4 and drm2 mutants (Figures 3E and 3F), sug-

gesting that diRNAs do not function through changing the DNA

methylation at the DSB sites to mediate DSB repair. Accumu-

lating evidence indicates that DSBs trigger a number of histone

modifications around the DSB sites and these modifications

may facilitate DSB repair (Lukas et al., 2011; Polo and Jackson,

2011).We propose that diRNAsmay function as guidemolecules

for these histone modifications at the DSB site, analogous

to hc-siRNAs in the RdDM pathway (Figure 7A). Alternatively,

diRNAs may play a more direct role in recruiting DSB repair

complexes to DSB sites through their effector protein AGO2 (Fig-

ure 7B). The phosphorylation of H2AX around the DSB sites is

one of the earliest events in response to DSBs and facilitates

local recruitment and retention of DSB repair and chromatin

remodeling factors (Fillingham et al., 2006; Paull et al., 2000;

Podhorecka et al., 2010).We found no evidence of compromised

phosphorylation of H2AX in the diRNA-deficient mutants (Fig-

ure 5 and Figure S6), demonstrating that the very early step in

DSB recognition is intact and that the diRNAs most likely affect

events downstream of H2AX phosphorylation.

In summary, we have demonstrated that small RNAs gener-

ated from the sequences flanking a DSB are important for

efficient DSB repair. It will be very exciting to have future studies

dissecting the molecular and biochemical underpinnings of

diRNAs in DSB repair.

EXPERIMENTAL PROCEDURES

Plant Materials, Human Cells, and Growth Conditions

Arabidopsis mutants and lines used in this study have been previously

described. See Table S4 for references and detailed information about these

mutants. All plants were grown in soil or Murashige and Skoog (MS) medium

at 16 hr light/8 hr dark photoperiod.

Human DR-GFP/U2OS cells (U2OS-derivative cell line harboring an inte-

grated HR reporter construct [DR-GFP]) (Pierce et al., 1999) were grown in

Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum.

DSB Repair Reporter Assay

DGU.US-1 line (DSB reporter line) and 2X35S:I-SceI-8 line (DSB-triggering

line) were used for assaying DSB repair efficiency in Arabidopsis (Mannuss

et al., 2010; Orel et al., 2003). To compare the DSB repair efficiency in a mutant



background with that in the corresponding wild-type background, we first

crossed both the DGU.US reporter (R) line and DSB-triggering (T) line express-

ing I-SceI with the mutant of interest (m/m) independently. After F2 segrega-

tion, the following progenies were identified by genotyping: those homozygous

for either the recombination substrate locus from the R line or the I-SceI-

expressing locus from the T line in the respective homozygous mutant

background (named R/R; m/m and T/T; m/m, respectively) and those in the

WT background (named R/R; M/M and T/T; M/M, respectively). Then, plants

R/R;m/mwere crossedwith T/T;m/m, whereas the correspondingWT siblings

R/R; M/M were crossed with T/T; M/M. The seeds of these crosses were

harvested, sterilized, and sown on MS medium. Thirteen-day-old seedlings

were collected for GUS staining or PCR analysis. For GUS staining, seedlings

were infiltrated with 50 mM sodium phosphate (pH 7.0), 10 mM EDTA, and

0.5 mg/ml X-gluc (Apollo Scientific), followed by incubation at 37�C in the

dark overnight. Then plantlets were cleared in ethanol and the blue sectors

in each plantlet were counted under a stereomicroscope (Nikon) and represen-

tative pictures were taken. To determine the DSB repair rate at the DNA level,

200 ng of genomic DNA were digested with recombinant I-SceI (NEB) at 37�Covernight to remove DNAs that were not repaired. Repaired DNAs were

amplified using p1 and p2 primers as depicted in Figure 1A. The sequences

of the primers are listed in Table S5.

DR-GFP/U2OS cells (Pierce et al., 1999) were used for assaying DSB repair

efficiency in humans. DR-GFP/U2OS cells were transfected with pCBASce

plasmid expressing I-SceI for 48 hr and processed for flow cytometric analysis

of GFP. The repair efficiency was scored as the percentage of GFP-positive

cells. To examine the role of Dicer and Ago2 in DSB repair, DR-GFP/U2OS

cells were treated with siRNAs targeting Dicer and Ago2 prior to the transfec-

tion with pCBASce. The sequences of the siRNAs are listed in Table S5.

All siRNAs duplexes were used at a final concentration of 30 nM and trans-

fections were performed using Lipofectamine RNAiMAX (Invitrogen) according

to the manufacturer’s instructions.

RNA Analyses

Arabidopsis total RNA was extracted from 13-day-old seedlings with Trizol

reagent (Invitrogen). Northern blot analysis with enriched small RNAs or

RNAs extracted from the purified AGO2 complex was carried out as described

(Qi et al., 2005). To detect small RNAs generated from the DSB region, we

amplified a 444 bp PCR fragment from the DGU.US construct, randomly

labeled with 32P-a-dCTP, and used as probe. miR173, SIMPLEHAT2, and

ta-siR255 were probed with end-labeled oligonucleotides. The sequences of

the primers and probes are listed in Table S5. For qRT-PCR, total RNA was

treated with RNase-free DNase I (Promega) and reverse-transcribed by

M-MLV Reverse Transcriptase (Promega) with oligo (dT). qRT-PCR was

performed with SYBR Premix EX Taq (TAKARA) on Applied Biosystems

7500 Fast. The GAPDH gene was detected in parallel and used as the internal

control. The sequences of the primers are listed in Table S5.

Total RNA from human DR-GFP/U2OS cells that were transfected with

pCBASce plasmid was extracted using Tri-reagent (Sigma) at 12, 16, 20,

and 24 hr posttransfection. Cells transfected with a plasmid having the same

vector backbone but missing I-SceI were collected at 20 hr posttransfection

and used as a negative control.

Immunopurification of the AGO2 Complex and Associated Small

RNAs

Immunopurification of the AGO2 complex was performed as previously

described (Mi et al., 2008). A small fraction of the immunoprecipitates was

separated on an 8%SDS-PAGE gel and examined by silver staining for quality

control. The associated small RNAs were extracted from the purified AGO2

complex by Trizol reagent (Invitrogen), resolved on a 15% denaturing PAGE

gel, and visualized by SYBR-Gold (Invitrogen) staining. Gel slices within the

range of 18–28 nt were excised, and the RNAs were eluted and purified for

small RNA library construction.

Small RNA Cloning and Deep Sequencing

Cloning of small RNAs from Arabidopsis and human cells was carried out

essentially as described (Mi et al., 2008). The Illumina GA IIx was used for

sequencing. A detailed protocol is available upon request.


Bioinformatic Analysis of Small RNAs

All reads obtained from Illumina GA IIx were sorted into respective libraries

by parsing their barcodes. Then the adaptor sequences were removed

using ‘‘vectorstrip’’ in the EMBOSS package. Small RNA reads with length

of 18–28nt were mapped to the Arabidopsis or human nuclear genome.

Unaligned reads were mapped to the DGU.US, DU.GUS, or DR-GFP

sequence. Small RNA densities along the DGU.US, DU.GUS, or DR-GFP

sequence were calculated and plotted within the 100 bp sliding windows

with a step size of 1 bp.

Protein Immunoblots

Thirteen-day-old seedlings of pAGO2::3HA-AGO2 Arabidopsis transgenic line

expressing HA-tagged AGO2 under its native promoter (Montgomery et al.,

2008) were treated with 100 Gy g-irradiation and placed in a growth chamber

at 22�C for 1.5 hr. Seedlings without g-irradiation treatment were used as

controls. Total proteins were extracted with 2X SDS-PAGE loading buffer

(100 mM Tris-HCl [pH 6.8], 100 mM b-mercaptoethanol, 4% [w/v] SDS,

0.2% [w/v] bromophenol blue, and 20% [v/v] glycerol). Proteins in human cells

were extracted in RIPA buffer (20 mM Tris-HCl [pH7.4], 20% glycerol, 0.5%

NP40, 1 mM MgCl2, 0.5 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and

1 mM PMSF). Protein samples were separated by SDS-PAGE, transferred to

PVDF membrane, incubated with antibodies in TBST containing 5% nonfat

dried milk, and, after incubation with HRP-conjugated secondary antibodies,

detected by ECL Western Blotting Detection Kit (BD Bioscience). Antibodies

used in this study included: rabbit anti-Ku80 antibody (CTS 2753), mouse

anti-RPA32 antibody (Abcam, ab2175), mouse anti-Rad50 antibody (Upstate,

05-525), mouse anti-Ago2 antibody (Abcam, ab57113), mouse anti-Dicer anti-

body (Abcam, ab14601), mouse anti-b-tubulin antibody (Sigma T5293 and

T5168), and mouse anti-HA antibody (Roche, 11666606001).

g-H2AX Immunofluorescence Staining and Fluorescence

Microscopy

Arabidopsis leaf nuclei were isolated essentially as previously described

(Onodera et al., 2005). g-H2AX immunofluorescence staining with isolated

leaf nuclei or mitotic root tip nuclei was performed as previously described

(Amiard et al., 2010; Friesner et al., 2005).

Images of the nuclei were acquired with a Nikon Eclipse 90i epifluorescence

microscope equipped with a Nikon DS-Qi1Mc monochrome quantitative

digital camera and filters for Alexa 568, exciter, 545/30 nm/nm; emitter,

610/75 nm/nm and for DAPI, exciter, 360/40 nm/nm; emitter, 450/60 nm/nm.

ACCESSION NUMBERS

Data sets of small RNAs generated in this study are deposited in the National

Center for Biotechnology Infromation Gene Expression Omnibus (http://www.

ncbi.nlm.nih.gov/geo/) under accession number GSE 36338.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

seven figures, and five tables and can be found with this article online at

doi:10.1016/j.cell.2012.03.002.

ACKNOWLEDGMENTS

We thank Drs. A. Britt, L. Du, M. Carmell, and B. Ding for critical reading of the

manuscript and stimulating discussions. We are grateful to Dr. H. Puchta for

DGU.US, DU.GUS and I-SceI trigger lines and Dr. M. Jasin for DR-GFP/

U2OS cell line and PCBASce plasmid. This work was supported by National

Basic Research Program of China (973 program 2012CB910900,

2011CB100700, and 2011CB812600 to Y.Q. and 2011CB510103 to Y.G.Y.).

Received: November 3, 2011

Revised: January 15, 2012

Accepted: March 7, 2012

Published online: March 22, 2012

http://www.ncbi.nlm.nih.gov/geo/

http://www.ncbi.nlm.nih.gov/geo/

http://dx.doi.org/doi:10.1016/j.cell.2012.03.002


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A Role for Small RNAs in DNA Double-Strand Break Repair