Roles of DCL4 and DCL3b in rice phased small RNA biogenesis

Xianwei Song1,2,†, Pingchuan Li1,3,4,†, Jixian Zhai1,3,4,†, Ming Zhou1,2,†, Lijia Ma1, Bin Liu5, Dong-Hoon Jeong3,4,

Mayumi Nakano3,4, Shouyun Cao1, Chunyan Liu1, Chengcai Chu1, Xiu-Jie Wang1, Pamela J. Green3,4, Blake C. Meyers3,4

and Xiaofeng Cao1,*

1State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental

Biology, Chinese Academy of Sciences, Beijing 100101, China,2Graduate University of the Chinese Academy of Sciences, Yuquan Road, Beijing 100039, China,3Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19711, USA,4Delaware Biotechnology Institute, Newark, DE 19711, USA, and5Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Received 17 August 2011; revised 27 September 2011; accepted 29 September 2011.*For correspondence (fax +86 10 64873428; e-mail xfcao@genetics.ac.cn).†These authors contributed equally to this work.

SUMMARY

Higher plants have evolved multiple proteins in the RNase III family to produce and regulate different classes of

small RNAs with specialized molecular functions. In rice (Oryza sativa), numerous genomic clusters are

targeted by one of two microRNAs (miRNAs), miR2118 and miR2275, to produce secondary small interfering

RNAs (siRNAs) of either 21 or 24 nucleotides in a phased manner. The biogenesis requirements or the functions

of the phased small RNAs are completely unknown. Here we examine the rice Dicer-Like (DCL) family, including

OsDCL1, -3a, -3b and -4. By deep sequencing of small RNAs from different tissues of the wild type and osdcl4-1,

we revealed that the processing of 21-nucleotide siRNAs, including trans-acting siRNAs (tasiRNA) and over

1000 phased small RNA loci, was largely dependent on OsDCL4. Surprisingly, the processing of 24-nucleotide

phased small RNA requires the DCL3 homolog OsDCL3b rather than OsDCL3a, suggesting functional

divergence within DCL3 family. RNA ligase-mediated 5¢ rapid amplification of cDNA ends and parallel analysis

of RNA ends (PARE)/degradome analysis confirmed that most of the 21- and 24-nucleotide phased small RNA

clusters were initiated from the target sites of miR2118 and miR2275, respectively. Furthermore, the

accumulation of the two triggering miRNAs requires OsDCL1 activity. Finally, we show that phased small

RNAs are preferentially produced in the male reproductive organs and are likely to be conserved in monocots.

Our results revealed significant roles of OsDCL4, OsDCL3b and OsDCL1 in the 21- and 24-nucleotide phased

small RNA biogenesis pathway in rice.

Keywords: phased small RNA, tasiRNA, miRNA, OsDCL4, OsDCL3b, OsDCL1.

INTRODUCTION

Higher plants are rich in two types of endogenous small

RNAs, namely small interfering RNAs (siRNAs) and

microRNAs (miRNAs), which are important in regulating

genome stability, plant development and various biotic

and abiotic stress responses (Baulcombe, 2004;

Ramachandran and Chen, 2008). Most miRNAs are 20–22

nucleotides in length, whereas siRNAs are mostly 21 and

24 nucleotides in length. The biogenesis of small RNAs,

which typically have 5¢-monophosphate and 3¢-OH groups,

requires RNase III-like enzymatic activities. The small RNAs

repress target genes in a sequence-specific manner at

either transcriptional or post-transcriptional levels (Carthew

and Sontheimer, 2009). The miRNAs and siRNAs originate

from different types of precursors: miRNA precursors are

endogenous single-stranded RNA molecules, which form

hairpin-like secondary structures that are imperfectly mat-

ched and predominately generate one species of small

RNAs (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee

and Ambros, 2001; Reinhart et al., 2002; Meyers et al.,

2008); siRNAs, on the other hand, are processed from long

double-stranded RNAs (dsRNAs) with perfect or nearly

perfect complementarity (Zamore et al., 2000; Elbashir

et al., 2001).

The Dicer and Dicer-like (DCL) proteins are core compo-

nents involved in the biogenesis of small RNAs and are

evolutionarily conserved in a diverse set of eukaryotes.

ª 2011 The Authors 1The Plant Journal ª 2011 Blackwell Publishing Ltd

The Plant Journal (2011) doi: 10.1111/j.1365-313X.2011.04805.x

Genetic and biochemical analyses have shown both speci-

ficity and redundancy of Dicer proteins in small RNA

metabolism (Henderson et al., 2006). There are four DCLs

encoded in the Arabidopsis thaliana genome (DCL1–4) and

six DCLs in rice (Oryza sativa), with rice containing one extra

copy of both DCL2 and DCL3 (Liu et al., 2005; Margis et al.,

2006).

OsDCL1 and DCL1 are major enzymes accounting for

miRNA maturation in rice and Arabidopsis, respectively

(Park et al., 2002; Liu et al., 2005). In Arabidopsis, DCL1

processes primary miRNAs (pri-miRNAs) into pre-miRNAs

and subsequently to a miRNA/miRNA* duplex (Park et al.,

2002; Kurihara et al., 2006). In rice, strong loss-of-function

OsDCL1 transgenic lines lead to lethality and weak lines

show pleiotropic phenotypes, similar to the phenotypes of

strong and weak DCL1 loss-of-function alleles in Arabid-

opsis (Schauer et al., 2002; Liu et al., 2005). DCL2 is

mainly responsible for 22-nucleotide siRNA related viral

defenses in Arabidopsis (Xie et al., 2004; Garcia-Ruiz et al.,

2010). DCL3 produces 24-nucleotide siRNAs as triggers for

transcriptional silencing of repeat sequences, mobile

elements and transgenes (Xie et al., 2004). However, dcl2

and dcl3 mutants in Arabidopsis have no obvious devel-

opmental defects under normal growth conditions. In rice,

knock-down of OsDCL2 produced a dwarf plant with

significantly lower spikelet fertility and negatively affected

maintenance of an endogenous dsRNA virus by increas-

ing the accumulation of virus siRNAs (Urayama et al.,

2010). Rice contains two genes that are homologous to

DCL3 of Arabidopsis, OsDCL3a and OsDCL3b. OsDCL3a is

responsible for the biogenesis of 24-nucleotide long

miRNAs (lmiRNA), which can direct cytosine DNA meth-

ylation both in cis and in trans (Wu et al., 2010). However,

the function of OsDCL3b, which exhibited panicle- and

early seed-specific expression (Kapoor et al., 2008),

remains unknown.

Besides processing a few evolutionarily ‘young’ miRNAs

(Rajagopalan et al., 2006), DCL4 is mainly responsible for

processing endogenous 21-nucleotide siRNAs including

trans-acting siRNAs (tasiRNAs) (Dunoyer et al., 2005; Gas-

ciolli et al., 2005; Xie et al., 2005; Yoshikawa et al., 2005) as

well as virus-derived siRNAs (Garcia-Ruiz et al., 2010).

Trans-acting siRNAs are a special type of siRNAs whose

biogenesis is initiated by miRNAs that target the non-coding

TAS transcripts for cleavage. After cleavage, RNA DEPEN-

DENT RNA POLYMERASE 6 (RDR6) converts the sliced

single-stranded RNA into double-stranded with the assis-

tance of SUPPRESSOR OF GENE SILENCING 3 (SGS3).

Subsequently, DCL4 performs a ‘phased’ cleavage at

21-nucleotide intervals starting from the miRNA-directed

cleavage site using the dsRNA as substrate (Vazquez et al.,

2004; Allen et al., 2005; Gasciolli et al., 2005; Xie et al., 2005).

In this context, ‘phased’ indicates that the small RNAs are

generated in a fixed pattern, separated by 21-nucleotides on

both strands, with each duplex of 21-mers having a two-

nucleotide 3¢ overhang.

In Arabidopsis, three DCL1-dependent miRNAs trigger the

initial cleavage event in four TAS families (TAS1–TAS4), with

miR173 acting on TAS1 and TAS2, miR390 on TAS3 and

miR828 on TAS4, respectively (Allen et al., 2005; Rajagopa-

lan et al., 2006). The dual miR390-binding sites present on

TAS3 (only the 3¢ site is cleavable) are conserved in higher

plants and were proposed to explain TAS3 tasiRNA forma-

tion (Axtell et al., 2006). Phylogenetic analysis revealed that

the TAS3 loci are conserved in higher plants (Axtell et al.,

2006) and both OsDCL1 and OsDCL4 are required for

OsTAS3 tasiRNA biogenesis, indicating that the tasiRNA

biogenesis pathway is conserved between rice and Arabi-

dopsis (Liu et al., 2005, 2007). Recently, two independent

lines of evidence showed that both 21- and 22-nucleotide

forms of the same miRNAs can guide cleavage; however,

only the 22-nucleotide miRNAs were capable of triggering

secondary siRNA production, reflecting the unique role of

22-nucleotide RNA in small RNA metabolism (Chen et al.,

2010; Cuperus et al., 2010). We and Nagasaki et al. (2007)

showed that OsDCL4 mutations cause the loss of tasiRNA-

AUXIN RESPONSE FACTORs (tasiR-ARF) and result in

up-regulation of ARFs and miR165/166, which are thought

to establish the adaxial–abaxial axis in lateral organs (Liu

et al., 2007; Nagasaki et al., 2007). Moreover, we also showed

that endogenous 21-nucleotide siRNAs derived from long

hairpin structures are OsDCL4-dependent (Liu et al., 2007).

In rice, many 21- and 24-nucleotide phased small RNA

clusters and superclusters were identified from inflores-

cence small RNA libraries, and miR2118 and miR2275/

miR2775 (known as miR2275 in http://www.mirbase.org/,

and we refer to it as miR2275 hereafter) were predicted to

target the 21- and 24-nucleotide phased small RNA loci,

respectively (Johnson et al., 2009). The biogenesis of

phased small RNAs, especially the 24-nucleotide ones that

were not found in Arabidopsis, is still unknown.

Here, six small RNA libraries were constructed from

seedlings and panicles of 93-11 (a wild-type indica rice

variety) and osdcl4-1, as well as from Nipponbare (a wild-

type japonica rice variety). Sequencing data derived from

93-11 and osdcl4-1 indicated that OsDCL4 has a stronger

impact on 21-nucleotide siRNA accumulation in panicles

than in seedlings. Consistent with previous results (Liu

et al., 2007), tasiRNAs were OsDCL4-dependent in both

tissues tested, and osdcl4 loss-of-function mutants

showed great and moderate reduction of 21- and

24-nucleotide phased small RNAs, respectively. In addi-

tion, we found that the processing of 24-nucleotide

phased small RNAs requires OsDCL3b whose function

was previously unknown, rather than OsDCL3a, which

processes long miRNAs (Wu et al., 2010). The target sites

for cleavage of 21- and 24-nucleotide phased small RNA

loci, which match two families of RNase III-dependent

2 Xianwei Song et al.

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04805.x

22-nucleotide small RNAs (miR2118 and miR2275), respec-

tively, were confirmed by the RNA ligase mediated-5¢rapid amplification of cDNA ends (RLM 5¢-RACE) and a

parallel analysis of RNA ends (PARE)/degradome library.

Both miR2118 and miR2275 were dependent on OsDCL1

and preferentially expressed in stamen and they are likely

to be conserved in monocots. Our results showed

genome-wide effects of osdcl4 on small RNA biogenesis

in rice and revealed the roles of OsDCL4, OsDCL3b and

OsDCL1 in the phased small RNA pathway.

RESULTS

Deep sequencing of small RNA populations

Our previous work showed that OsDCL4 is the major Dicer-

like protein generating 21-nucleotide siRNAs from exoge-

nous transgenic inverted repeats, endogenous inverted

repeats and TAS3 (Liu et al., 2007), indicating that OsDCL4

processes multiple substrates in rice. To further determine

the extent of OsDCL4 action on a genome-wide scale, six

small RNA libraries were constructed from panicles and

seedlings of the wild-type 93-11, the osdcl4-1 null allele in

the 93-11 background (Liu et al., 2007) and another wild-

type, Nipponbare. These libraries were used for deep

sequencing on the Illumina 1G system.

In total, over 20 million raw reads (18–26 nucleotides)

were generated from these six libraries, with each library

represented by 2.3–4.5 million reads. Approximately 73–82%

of the reads, varying by library, could be perfectly mapped to

the reference genomes (93-11 or Nipponbare), and the

numbers of genome-matched non-redundant small RNA

sequences ranged from 692 568 to 2 135 639 (Table S1 in

Supporting Information).

OsDCL4 preferentially affects 21-nucleotide siRNA

biogenesis in panicles

In 93-11 and Nipponbare, 21- and 24-nucleotide small RNAs

were the most predominant sizes (Figure 1a,b), consistent

with the small RNA size distribution typically observed in

Arabidopsis and rice (Henderson et al., 2006; He et al., 2010).

To determine the effect of OsDCL4 loss-of-function on small

RNA metabolism, we compared the small RNA populations

in panicles and seedlings of the osdcl4-1 null mutant with

those of the wild-type 93-11 plants. A marked reduction the

proportion of 21-nucleotide small RNAs was observed in

osdcl4-1 panicles (Figure 1a), but this reduction was smaller

in seedlings (Figure 1b). In the osdcl4-1 panicle sample,

21-nucleotide small RNAs accounted for 6.86% of the total

population, corresponding to an approximately two-fold

reduction compared to the 93-11 panicle library (18.9% of

total). In seedlings, however, the proportion of 21-nucleotide

small RNAs in wild-type 93-11 and osdcl4-1 was 18.6 and

14.3%, respectively, which showed much less of a change

compared with that in the panicles (Figure 1a,b). This is

consistent with two-fold higher expression level of OsDCL4

in panicles compared with seedlings (Figure 1c) and sug-

gests that more OsDCL4-dependent small RNAs exist in

panicles. Further analysis showed that these 21-nucleotide

small RNAs, which exhibited the most significant reduction

in the panicle of osdcl4-1, were preferentially derived from

unannotated regions in the rice genome (Figure 1d);

whereas the 24-nucleotide small RNAs were preferentially

from both repeat-associated and unannotated regions (Fig-

ure 1e), consistent with the previous analysis of rice small

RNA massively parallel signature sequencing (MPSS) data

(Nobuta et al., 2007).

Figure 1. Size distribution and annotation of

small RNAs from various rice genotypes.

(a, b) Small RNA size distribution of 93-11,

osdcl4-1 and Nipponbare in panicles (a) and

seedlings (b). The abundance of small RNA at

each size class is represented by their percentage

relative to the total abundance.

(c) Real-time PCR analysis of the expression level

of OsDCL4 in seedlings and panicles. OsACTIN1

was used for normalization.

(d, e) Annotation of 21- (d) and 24-nucleotide (e)

small RNAs.

Phased small RNA clusters biogenesis in rice 3

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04805.x

OsDCL4-dependent conserved and non-conserved

TAS3 loci in rice

Previous studies showed that OsDCL4 can produce

21-nucleotide tasiRNAs from TAS3 in a phased manner (Liu

et al., 2007). To further investigate whether other siRNAs

were processed in a phased pattern by OsDCL4, we per-

formed a computational analysis to identify phased siRNAs,

using an algorithm modified from a previous study (Howell

et al., 2007) to deal with the higher background caused by

deeper sequencing capacity (Figure S1). Small RNA data

from a published Arabidopsis Col-0 inflorescence study

(Lister et al., 2008) were used to test this modified algorithm.

From the published data, seven phased loci were found with

a phasing score (P-score) higher than 22.5, and all of them

had been previously reported as tasiRNA loci expressed in

flowers (Chen et al., 2007; Howell et al., 2007) (Table S2).

This result indicated that the modified algorithm efficiently

identified phased small RNA patterns within a deep

sequencing dataset. So we adopted a P-score of 25, a more

stringent cut-off, for the identification of phased small RNA.

Applying this algorithm to our six rice libraries, all of the

known rice TAS genes (TAS3a, TASb3b, TAS3c) were

identified with P-scores higher than 25 (Figure S2a) in the

wild types. Notably, TAS3b has two copies (TAS3b1 and

TAS3b2) within an 18 kb region in 93-11 (Figure S2) but one

copy in Nipponbare (data not shown). In addition, two

additional tasiRNA loci, TAS3d and TAS3e, with dual

miR390-binding sites but lower scores (�20) were identified

from both 93-11 and Nipponbare (Figure S2 and data not

shown). The abundances of most tasiRNA loci were dra-

matically reduced in osdcl4-1, and the phasing patterns of

some tasiRNA loci were also impacted (Figure S2a), con-

firming that OsDCL4 is key for tasiRNA biogenesis in rice (Liu

et al., 2007).

Roles of OsDCL4 in the biogenesis of 21- and 24-nucleotide

phased small RNAs

Consistent with a previous report (Johnson et al., 2009),

many 21- and 24-nucleotide phased small RNAs were iden-

tified in wild type panicles, whereas only a few phased small

RNA loci with a score of 25 or higher (mainly known tasiR-

NAs) were identified in seedlings and grains (Figure 2a,c,

Table S3). No other intervals were detected with a signifi-

cant phased pattern (Table S2). The 21-nucleotide phased

small RNAs, represented by 1136 clusters in 93-11 and 1540

clusters in Nipponbare with P-scores over 25 (Figure 2a),

were mainly derived from unannotated regions (Figure 2b).

In osdcl4-1, both the abundance and cluster number of

21-nucleotide phased small RNAs were significantly reduced

compared to the wild type (Table S3, Figure 2a,b). For

example, at 21-nucleotide phased locus No. 1000 in 93-11,

there is a dominant phasing pattern of each cycle to be

coincident with the cleavage site, which was identified by

5¢-RACE (Figure 3a) and further confirmed by PARE/degra-

dome library analysis (Figure 3b) of a degradome library

constructed from young panicles of 93-11 (Zhou et al., 2010).

The phased level within the cluster is reduced in osdcl4-1

due to lower small RNA abundance from each phased po-

sition (Figure 3a). These results suggested that the accu-

mulation of 21-nucleotide phased small RNAs is largely

dependent on the activity of OsDCL4.

The 24-nucleotide phased small RNAs, most of which also

originated from unannotated regions, were identified in

panicles of 93-11 (74 clusters) and Nipponbare (30 clusters)

with P-scores over 25 (Figure 2c,d). Unlike the 21-nucleotide

phased small RNAs, whose abundances and cluster num-

bers were both dramatically impacted in osdcl4-1 (Fig-

ure 2a,b), the abundances of some 24-nucleotide phased

small RNAs were moderately decreased in the osdcl4-1

Figure 2. Cluster numbers and annotation of 21-

and 24-nucleotide (nt) phased small RNAs.

(a, c) Cluster numbers of 21- (a) and 24-nucleo-

tide (c) phased small RNAs in different tissues of

93-11, osdcl4-1 and Nipponbare with a phasing

score (P-score) over 25. Grain data were derived

from (Zhu et al., 2008).

(b, d) Annotation for 21- (b) and 24-nucleotide (d)

phased small RNAs.

4 Xianwei Song et al.

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04805.x

mutant and the cluster numbers were unaffected

(Figure 2c,d). To determine the accumulation pattern of

24-nucleotide phased small RNAs in osdcl4-1, two phased

clusters of No. 57 and No. 56 were selected for further

analysis. Each phased small RNA cluster was validated by

5¢-RACE (Figure 3c) and the PARE/degradome library anal-

ysis (Figure 3d), showing that the predominant cleavage

sites coordinate well with the phasing pattern (Figure 3c,d).

Consistent with the genome-wide data (Table S3, Figure 2c),

the overall phasing patterns of cluster No. 57 and No. 56

were mostly unchanged but with less accumulation of

phased small RNAs in each locus in osdcl4-1 (Figure 3c). In

addition, a moderate reduction of 24-nucleotide phased

small RNAs in osdcl4-1 was also detected by locked nucleic

acid (LNA) labeled probes, LNA53 and LNA42 derived from

cluster No. 57 and No. 56, respectively (Figure 3c,e). The

influence by osdcl4-1 on 24-nuclotide phased small RNAs

might be specific, because 24-nucleotide unphased small

RNAs including one derived from a single copy locus

(detected by LNA12) and another one derived from the

centromeric repeats (CentO siRNAs) (Zhang et al., 2005)

showed no obvious changes in osdcl4-1(Figure 3e). In

addition, the distribution of 21-nucleotide clusters showed

little overlap with that of 24-nucleotide phased small RNA

clusters in the 93-11 genome (Figure S3).

The 24-nucleotide phased small RNAs are processed

by OsDCL3b

Deep sequencing data combined with RNA gel blot results

showed the accumulation of 24-nucleotide phased small

RNAs was partially dependent on OsDCL4, suggesting other

DCL proteins might also be involved. In Arabidopsis, DCL3 is

the major Dicer that processes 24-nucleotide siRNAs (Xie

et al., 2004). Compared with Arabidopsis, two copies of

DCL3, namely OsDCL3a and OsDCL3b, are found in the rice

genome (Figure S4a) (Kapoor et al., 2008). OsDCL3a is

involved in non-canonical long miRNA biogenesis (Wu et al.,

2010). To determine whether OsDCL3a and OsDCL3b play a

role in the production of 24-nucleotide phased small RNAs,

we used RNA interference (RNAi) to knock down either

OsDCL3a or OsDCL3b (Figure S4b). Two OsDCL3a and

OsDCL3b knock-down lines, in which the transcript levels of

OsDCL3a and OsDCL3b were reduced to �20 and �50% of

wild type (WT; Nipponbare) (Figure S4c), respectively, were

used for northern blots to detect phased small RNAs. In the

OsDCL3a RNAi lines (OsDCL3aIRs), the 24-nucleotide

phased small RNAs detected by LNA53 and LNA42 showed

no significant changes (ranging from 0.72 to 1.15-fold)

compared with WT (Figure 4), whereas the signals for 24-

nucleotide unphased small RNAs including the one detected

by LNA12 and CentO siRNAs (Zhang et al., 2005), sharply

decreased (Figure 4). In contrast, the OsDCL3b RNAi lines

specifically affected 24-nucleotide phased but not unphased

small RNAs (Figure 4), suggesting that OsDCL3b is involved

in the processing of 24-nucleotide phased small RNAs and

acts distinctly from OsDCL3a. In addition, we also found that

the knock-down of OsDCL1 did not result in obvious defects

in the accumulation of both 24-nucleotide phased and

unphased small RNAs (Figure 4).

Cleavage sites of 21- and 24-nucleotide phased small RNA

loci share conserved motifs as target sites of miRNAs

Johnson et al. have predicted that miR2118 and miR2275 act

as triggers to initiate formation (Johnson et al., 2009) of 21-

and 24-nucleotide phased small RNAs, respectively. 5¢-RACE

analysis of a 21-(Figure 3a) and two 24-nucleotide phased

small RNAs (Figure 3c), suggested that these phased small

RNAs were initiated from a fixed cleavage site like tasiRNAs.

To explore whether this is common for all phased small

RNAs, we performed 5¢-RACE to locate the cleavage sites;

TAS3c, for which the transcripts are cleaved by miR390, was

used as a positive control (Table S4). Because the tran-

scribed strand of the phased small RNA loci is unknown

(RDR can produce the secondary strand and thus form a

dsRNA template), PCR primers were chosen to amplify only

one of the two strands. Among 30 21-nucleotide phased

small RNA loci chosen based on their abundances, 15 were

successfully mapped with a fixed cleavage site (Figure 5a,

top 15 lines). The cleavage sites exhibit a high degree of

sequence similarity, which defined a 22-nucleotide motif

targeted by members of miR2118 family (Figure 5b). Using

the sequence motif, 9 more 21-nucleotide phased small RNA

loci with this motif were identified and examined by RLM 5¢-RACE; the cleavage sites on these nine transcripts mapped

exactly to the motif region (Figure 5a, bottom 9 lines). MEME

was used to examine whether this motif was common for all

21-nucleotide phased small RNA originating sites (Bailey

and Elkan, 1994). Out of the 1136 clusters, 1116 (98.2%) were

found to contain this 22-nucleotide motif. From the PARE/

degradome library constructed from young panicles of the

93-11 library (Zhou et al., 2010), the cleavage sites of more

than 97% (1084 out of 1116) of the 21-nucleotide phased

small RNA clusters could be confirmed at the 22-nucleotide

motif (Figure 3b, Table S5).

The 24-nucleotide phased small RNA clusters were also

tested by RLM 5¢-RACE to identify cleavage sites (Figure 5c,

Table S6). A different 22-nucleotide motif was found to be

shared by all the tested cleavage sites and can be targeted by

members from the miR2275 family (Figure 5d). We used the

motif-identification program MEME to reveal that almost all

(73 of 74 in 93-11 and 30 of 30 in Nipponbare) 24-nucleotide

phased small RNA clusters contained such a motif

sequence. Moreover, cleavage sites in 67 of 73 (91.8%)

motif-containing clusters in 93-11 could be identified by the

PARE/degradome library (Figure 3d, Table S7). Therefore,

our results confirmed that miR2118 and miR2275 mediate

the biogenesis of 21- and 24-nucleotide phased small RNAs,

respectively, by initiating cleavage of their transcripts.

Phased small RNA clusters biogenesis in rice 5

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04805.x

OsDCL1-dependent small RNAs may trigger 21- and

24-nucleotide phased small RNA initiation

In our panicle small RNA libraries, 21 and 11 miR2118 and

miR2275 miRNAs, respectively, were detected with quite low

abundances (no more than five transcripts per million, TPM)

(data not shown). Among them, three new members of

miR2118 family (miR2118s, miR2118t and miR2118u) and

nine additional members of miR2275 (miR2275c to

miR2275k,) were identified in 93-11 besides previously

6 Xianwei Song et al.

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04805.x

described ones (Johnson et al., 2009) (Figure 6a,b, Tables

S8 and S9). The miR2118 members were mapped to two

small regions on chromosome 4 and 11 and found to be

tandem aligned in 93-11 and Nipponbare (Figure 6a). On

chromosome 4, 17 miR2118 members form nine clusters

within a 20 kb region. On chromosome 11, four members of

the miR2118 family were present in two clusters. In 93-11,

supported by degradome data and bioinformatics predic-

tions, each miR2118 and miR2275 family member could

potentially cleave transcripts and thus trigger the formation

of many 21- or 24-nucleotide phased small RNA clusters

(Figure 6a, b).

In addition, to exclude the possibility that low abundance

of miR2118 and miR2275 family members is a cloning

artifact in our deep sequencing, we treated small RNAs with

5¢-phosphate-dependent exonuclease (Terminator), which

specifically digests RNA containing a 5¢-monophosphate but

does not digest RNAs with a 5¢-triphosphate, 5¢-cap or

5¢-hydroxyl (see Experimental Procedures). The signals of

miR2118 and miR2275 were eliminated after exonuclease

treatment compared with untreated small RNAs from pan-

icles of three rice varieties (93-11, Nipponbare and Zhong-

hua11) (Figure 6c). As a negative control, the signal of U6,

which has a 5¢-triphosphate, was not affected (Figure 6c).

Therefore, we confirmed these motif-targeting small RNAs

were RNase III products with a 5¢-monophosphate.

To determine which DCL protein generates the triggers of

phased small RNA biogenesis, small RNA gel blot analysis

was performed using the LNA probes to detect the levels of

miR2118 (LNA58) and miR2275 (LNA64). As expected,

miR168, the well-recognized and conserved miRNA, was

greatly reduced in OsDCL1 knock-down transgenic plants

(OsDCL1IRs) (Figure 6d). Consistently, both miR2118 and

miR2275 were less accumulated in OsDCL1IRs. We also

noted that the accumulations of both miRNAs were affected

in osdcl4-1 (Figure 6d), which might account for the lower

accumulation of 24-nucleotide phased small RNAs in osdcl4-

1 (Figures 2d, 3c,e) owning to its epistatic influence on

miR2275. Therefore, it seems that the biogenesis of both

triggering miRNAs for 21- and 24-nucleotide siRNAs is

dependent on OsDCL1 and OsDCL4. However, it is worth

noting that loss-of-function osdcl4-1 shows severe spikelet

defects including thread-like lemma and male sterility. It is

also hypothesized that the loss of some organs or cell types

harboring abundant miR2118 and miR2275 in osdcl4-1

might results in losing of both miRNAs.

Figure 4. Effects of OsDCLs on the biogenesis of 24-nucletiode (nt) phased

small RNAs.

Northern blot analysis of the accumulation of the 24-nucleotide phased small

RNAs detected with probes LNA53, LNA42 in Nipponbare, OsDCL1IR1-1,

OsDCL3aIRs and OsDCL3bIRs. LNA12 labels a 24-nucleotide unphased small

RNA. CentO siRNA labels siRNAs derived from 165-bp CentO satellite. U6 was

used as the loading control. The intensity ratio of each signal relative to wild-

type Nipponbare is indicated below each image. The bottom panels represent

expression analysis of OsDCL3a and OsDCL3b in Nipponbare and RNA

interference (RNAi) lines by RT-PCR. The OsACTIN gene was used as an

internal control.

Figure 3. OsDCL4 has different effects on accumulation of 21- and 24-nucleotide (nt) phased small RNAs.

(a, c) The abundance and the phased patterns of selected 21- (a) and 24-nucleotide (c) phased small RNA clusters in the panicles of 93-11 and osdcl4-1. The top panel

represents the abundance of each position. The bottom panel represents the phasing score, plotted in a 10-cycle window. The schematic below the phase plot

represents the genomic position of the phased small RNA clusters. The black arrow indicates a site verified by RNA ligase mediated-5¢ rapid amplification of cDNA

ends (RLM 5¢-RACE) with the frequency of cloned RACE products. The black boxes in (c) indicate the locked nucleic acid (‘LNA’) probe, LNA53 and LNA42, detecting

the most abundant 24-nucleotide phased small RNA in both clusters, respectively.

(b, d) Validation of the cleavage by analysis of a parallel analysis of RNA ends (PARE)/degradome library of a 21- (b) and 24-nucleotide (d) phased small RNA clusters

as shown in (a) and (c), respectively. The red dot represents the cleavage site mapped by highly abundant degradome tag.

(e) Northern blot analysis of the accumulation of the 24-nucleotide phased small RNAs detected with probes LNA53, LNA42 in Nipponbare, OsDCL4IR-1, 93-11,

osdcl4-1. LNA12 labels a 24-nucleotide unphased small RNA. CentO siRNA labels siRNAs derived from 165-bp CentO satellite. U6 was used as the loading control.

The intensity ratio of each signal relative to wild-type Nipponbare is indicated below each image. The genomic locations of LNA53, LNA42 and LNA12 are shown on

right panel. Protein-coding genes and phased small RNA clusters are indicated by green and golden bars, respectively.

Phased small RNA clusters biogenesis in rice 7

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04805.x

The triggers and phased small RNAs are preferentially

expressed in stamen and are conserved in cereal

To determine the hypothesis, we investigated the expres-

sion pattern of both triggering miRNAs (miR2118 and

miR2275). The deep sequencing data from us and a previous

report showed that both 21- and 24-nucleotide phased small

RNAs are a class of panicle-specific siRNAs (Figure 2a,c,

Table S3) (Johnson et al., 2009). Northern blotting signals

for miR2118 and miR2275 as well as 24-nucleotide phased

small RNAs were consistently present in rice panicles but

absent in seedlings (Figure 7a). Furthermore, we found that

in contrast to tasiR-ARFs that accumulated in all detected

tissues and organs, the trigger miR2275 and 24-nucleotide

phased small RNAs were exclusively expressed in the sta-

men (Figure 7a). Compared to miR2275, miR2118 displayed

a distinct pattern and was present predominantly in stamens

but also in the palea and the pistils at low levels (Figure 7a).

Northern blot analysis in maize also revealed that both

miR2118 and miR2275 accumulated in stamens from various

developmental stages (Figure 7b), which was consistent

with the previous identification of both miR2118 and

miR2275 in deep sequencing data of small RNAs from early

developmental stages of maize anthers (Johnson et al.,

2009). Taken together, these findings indicate that 21- and

24-nucleotide phased small RNAs might be stamen-specific

small RNAs conserved in cereal species. So, the develop-

mental defects of osdcl4-1, in which the stamen identity is

disrupted, may account for the lower accumulation of

miR2118 and miR2275 in it.

DISCUSSION

Here we sequenced six small RNA libraries, including

93-11, osdcl4-1 (in 93-11 background) and Nipponbare,

from both panicles and seedling tissues, using Illumina’s

SBS technology. The deep sequencing data showed that

OsDCL4 is responsible for the biogenesis of 21-nucleotide

siRNAs including tasiRNAs, and its role is more promi-

nent in panicles than in seedlings. A genome-wide

phasing analysis revealed that 21-nucleotide phased small

RNAs are completely OsDCL4-dependent, whereas

24-nucleotide phased small RNAs only partially depend

on OsDCL4. 5¢-RACE and PARE/degradome library con-

firmed two 22-nucleotide conserved sequence motifs as

target sites of miR2118 and miR2275, which act to trigger

the biogenesis of 21- and 24-nucleotide phased small

RNAs, respectively. Further experiments demonstrated the

OsDCL1-dependent miR2118 and miR2275 are mainly or

were exclusively expressed in the stamen. In addition, we

revealed that OsDCL3b but not OsDCL3a is the Dicer that

directly processes 24-nucleotide phased small RNAs. To

sum up, our work revealed the roles of OsDCL4, OsDCL3b

and OsDCL1 in the biogenesis of 21- and 24-nucleotide

phased small RNAs.

Figure 5. Cleavage sites of 21- and 24-nucleotide phased small RNAs loci share conserved motifs as target sites of microRNAs (miRNAs).

(a, c) Alignments of sequences flanking the cleavage sites revealed by RNA ligase mediated-5¢ rapid amplification of cDNA ends (RLM 5¢-RACE) for 21- (a) and 24-

nucleotide phased small RNA clusters (c). The black arrows indicate cleavage sites verified by RLM 5¢-RACE. The frequencies of cloned RACE products are listed on

the right. The cleavage sites are marked in yellow. The top 15 sequences (above the black line) in (a) are from the randomly selected phased small RNA loci; the

bottom nine (below the black line) were selected based on the motif sequences for further validation.

(b, d) Conserved 22-nucleotide motifs of the 21- (b) and 24-nucleotide phased small RNA clusters (d) analyzed by WebLogo (Crooks et al., 2004). The diagrams for

conserved motifs were generated from 1084 21-nucleotide and 67 24-nucleotide sequences. The sequence complementarity between miR2118a and the motif of 21-

nucleotide phased small RNAs (b), miR2275a and the motif of 24-nucleotide phased small RNAs (d) are displayed at the bottom. The black arrows indicate cleavage

sites.

8 Xianwei Song et al.

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04805.x

The roles of Dicers in the biogenesis of phased small RNAs

In rice, we and others previously showed that OsDCL1,

OsDCL3a and OsDCL4 are responsible for the production of

canonical miRNAs, non-canonical long miRNAs and

21-nucleotide siRNAs, respectively (Liu et al., 2005, 2007; Wu

et al., 2010). Moreover, both OsDCL1 and OsDCL4 are

required for tasiRNA generation (Liu et al., 2007). In rice

Figure 7. Expression patterns of trigger microRNAs

(miRNAs) and 24-nucleotide phased small RNA in

different tissues.

(a, b) Northern blot analyses of trigger miRNAs in

different tissues of rice (a) and maize (b). Stamen (S),

(M) and (L) represent the anther lengths of �3.0–

3.5 mm, �3.6–4.1 mm and �4.2–5 mm, respectively.

LNA53 detects 24-nucleotide phased small RNAs.

Figure 6. The triggers of 21- and 24-nucleotide phased small RNAs.

(a, b) Genomic locations of miR2118 (a) and miR2275 (b) in the 93-11 genome. The blue box indicates the exon of a coding gene, the light blue box indicates the

intron, the gray line shows the intergenic region and the arrow shows the transcriptional direction. Illustrations below show the hairpin structures of microRNA

(miRNA) precursors. The mature sequences of miRNA are marked in red. The number in each semicircle represents the predicted target clusters by each miRNA.

(c) A northern blot of low molecular weight RNA treated with (+) or without ()) Terminator Exonuclease (TE) was hybridized to detect miR2118 and miR2275. Trans-

acting small interfering RNA-AUXIN RESPONSE FACTORs (tasiR-ARFs) and miR390 were used as the mono-phosphate control, with this 5¢ end conferred via

cleavage by Dicer-like enzymes. U6 was used as the loading control. ZH11 and Nipp represent Zhonghua 11 and Nipponbare accessions.

(d) RNA gel blot analysis of miR2118 and miR2275 in panicles of OsDCL1IRs and osdcl4-1. U6 and 5S/tRNA were used as the loading controls; miR168 was used as

the control for OsDCL1IRs.

Phased small RNA clusters biogenesis in rice 9

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04805.x

phased small RNA biogenesis, our results demonstrated

that the OsDCL1-dependent 22-nucleotide miRNAs trigger

the cleavage for a vast number of phased small RNA pre-

cursors (Figures 5, 6 and 8). After the initial cleavage direc-

ted by the triggers, it would be reasonable to expect that

RDR might be required for second-strand RNA synthesis,

providing suitable substrates for DCLs. Comparing the

moderate and sharp decrease of triggering miR2118 and

mature 21-nucletiode phased small RNAs in osdcl4-1,

respectively (Figures 2b, 3a and 6d), we proposed that

OsDCL4 is the direct DCL to process the maturation of

21-nucleotide phased small RNAs, while the moderate

reduction of triggering miR2118 might be indirect effect

which could be due to lack of certain types of cells in osdcl4-

1 mutant (Figure 8). For 24-nucleotide phased small RNA

biogenesis, the processing was carried out by OsDCL3b but

not OsDCL3a (Figure 4, 8). This is also consistent with Os-

DCL3b being expressed specifically in panicle and early seed

development stages (Kapoor et al., 2008). In addition, that

the 24-nucleotide phased small RNAs show no obvious

change in OsDCL1IRs (Figure 4) may be attributed the weak

RNAi lines that we used, in which the triggering miR2275 is

only moderately decreased (Figure 6d).

Convergent evolution of DCL3b and 24-nucleotide phased

small RNAs in monocots

In rice, two DCL3 family members have distinct roles in the

generation of 24-nucleotide small RNAs. OsDCL3a plays

conserved roles in producing 24-nucleotide unphased

small RNAs, including long miRNAs and heterochromatin

associated siRNAs (hc-siRNAs) such as CentO siRNAs

(Figure 4) (Wu et al., 2010), whereas OsDCL3b specifically

acts in 24-nucleotide phased small RNA biogenesis (Fig-

ure 4). Arabidopsis has a single DCL3, which is more

similar to OsDCL3a (Figure S4a). In addition, both maize

and Brachypodium genomes encode two DCL3 homologs,

which are similar to OsDCL3a and OsDCL3b (Figure S4a).

Clusters of 21- and 24-nucleotide phased small RNAs have

only been reported to exist in the developing young pan-

icles of rice and Brachypodium distachyon (Figure S4a)

(Johnson et al., 2009; Vogel, 2010). Considering that maize

has miR2118 and miR2275 as well as DCL102, the homolog

of OsDCL3b, we speculate that 21- and 24-nucleotide

phased small RNAs also exist in maize (Johnson et al.,

2009) (Figure S4a). We propose that the ancestral DCL3

gene, which has the same functions as that of Arabidopsis,

was subjected to a gene duplication event in monocots

after the divergence of dicots and monocots. During

monocot genome evolution, one paralog, DCL3a, with

constitutive expression patterns may have retained the

original functions of DCL3 in the production of 24-nucleo-

tide hc-siRNAs; whereas DCL3b, which is preferentially

expressed in panicles and early seeds, has acquired the

function of producing stamen-specific 24-nucleotide

phased small RNAs. Therefore, the divergent evolution of

DCL3b might be a prerequisite for the generation of 24-

nucleotide phased small RNAs in monocots, suggesting

the coevolution of DCL3b and 24-nucleotide phased small

RNAs.

Phased small RNAs in rice reproductive development

To date, several types of small RNAs have been reported

to be expressed and function specifically in the reproduc-

tive organs of animals and plants. In animals, piwi-inter-

acting RNAs (piRNAs) and 21U small RNAs are

preferentially expressed in the germline and play impor-

tant roles in gametogenesis (Girard et al., 2006; Batista

et al., 2008). In Arabidopsis, a class of retrotranspo-

son-derived 21-nucleotide siRNAs in pollen regulates

transposon activity in gametes (Slotkin et al., 2009), AGO9-

dependent small RNAs play a crucial role in specifying cell

fates in the Arabidopsis ovule (Olmedo-Monfil et al., 2010),

and a sperm-specific natural cis-antisense siRNA (cis-nat-

siRNA) is essential for double fertilization (Ron et al.,

2010). In addition, a type of 24-nucleotide RNA polymerase

IV (Pol IV)-dependent siRNA is expressed uniparentally in

the developing endosperm, providing a link between

genomic imprinting and RNA silencing (Mosher et al.,

2009). In rice, 21- and 24-nucleotide phased small RNAs

are preferentially expressed in panicles (Figure 7a) (John-

son et al., 2009). Furthermore, we show that the trigger

miRNAs for panicle-specific 21- and 24-nucleotide phased

Figure 8. A model of 21- and 24-nucleotide phased small RNA biogenesis in

rice.

The trigger precursors are processed by OsDCL1 into 22-nucleotide mature

miR2118 and miR2275. The 22-nucleotide triggers can initiate the cleavage of

21- and 24-nucleotide phased small RNA primary transcripts generated by

RNA polymerase II (Pol II). Unknown RDR protein(s) may act on the cleavage

transcripts to synthesize the second strand to form double-stranded RNAs as

a substrates for DCLs. OsDCL4 produces 21-nucleotide phased small RNAs

and OsDCL3b is responsible for 24-nucleotide phased small maturation.

10 Xianwei Song et al.

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04805.x

small RNAs are mainly or exclusively expressed in sta-

mens (Figure 7a), suggesting that both sizes of phased

small RNAs are preferentially present and play roles in

stamens. Here, we tested more than 50 of the potential

21-nucleotide phased small RNA targets by 5¢-RACE and

also did a genome-wide scan using PARE/degradome data;

this analysis found that none of the predicted cleavages on

mRNAs were confirmed, which indicated that 21-nucleo-

tide phased small RNAs might function in a non-cleavage-

based manner. Although the direct targets of phased small

RNAs in rice remain to be elucidated, their tissue-speci-

ficity suggests that they may act at a specific stage during

male reproductive development. What the phased small

RNAs that match to thousands of genomic loci might

contribute to plant reproduction remains a major challenge

for the future.

EXPERIMENTAL PROCEDURES

Plant materials

Rice plants including O. sativa ssp. indica cv. 93-11, osdcl4-1(backcrossed to 93-11 three times from L16S), O. sativa ssp.japonica cv. Nipponbare, OsDCL1IRs, OsDCL3aIRs, OsDCL3bIRs andOsDCL4IRs were grown in the field as previously described (Liuet al., 2005, 2007). For the small RNA libraries, seedling materialsincluded the roots and aerial parts at 6 days after germination(DAG); panicle tissue was �4 cm in length, mainly comprising thefloral organs at differentiation stage In7 (Itoh et al., 2005). All thecollected materials were instantaneously frozen in liquid nitrogenand stored at )80�C until total RNA was extracted. Floral organs ofrice were separated from Zhonghua11 (the same subspecies withNipponbare) in stage An8 (Itoh et al., 2005), and those of maize isfrom B73.

Construction of RNAi vector and plant transformation

The binary OsDCL3a and OsDCL3b RNAi vectors were constructedas previously described (Liu et al., 2005).The fragments to targeteach homologous region of rice OsDCL3a and OsDCL3b wereobtained using primers as follows. OsDCL3a, cx0022: 5¢-CGCGGATCCAGTTCCAGAAATTGTA-3¢. cx0023: 5¢-CCGCTCGAGCATTAGGTACTTTTGATCT-3¢; OsDCL3b, cx0024: 5¢-CGCGGATCCTACTACATGTGTTCTTGCTT-3¢; cx0025: 5¢-CGCGGATCCTACTACATGTGTTCTTGCTT-3¢. Rice transformation and regeneration were performed aspreviously published (Hiei et al., 1994). The generated transgenicplants were confirmed by polymerase chain reaction (PCR) as pre-viously described (Liu et al., 2005).

Small RNA library construction, sequencing and

data analysis

Total RNA was extracted from seedlings and panicles using TRIZOL

reagent (Invitrogen, http://www.invitrogen.com/). Small RNA prep-aration was as previously described (Liu et al., 2005). The purifiedproduct was submitted for sequencing on an Illumina 1G analyzerafter a quality control (QC) assay; sequencing was performed at theBeijing Genomics Institution at Shenzhen.

The data for this article have been deposited at the National Centerfor Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE22763.The Illumina sequencing raw data were trimmed by removingadaptor sequences to generate a set of ‘clean’ reads. All the reads

were mapped to either the 93-11 (http://rice.genomics.org.cn/) orNipponbare (TIGR release version 5; http://www.tigr.org/) genomeusing SOAPaligner (http://soap.genomics.org.cn). Reads in the sizerange of 18–26 nucleotides and perfectly matched to the ricegenome, excluding those matching tRNAs, rRNAs, snRNA andsnoRNA (t/r/sn/snoRNA), were retained for further analysis. Annota-tion was done with Rfam6 (http://www.sanger.ac.uk/Software/Rfam), miRBase 13.0 (http://microrna.sanger.ac.uk/), RepeatMasker(http://www.repeatmasker.org) and the TIGR rice genome annota-tion (TIGR release version 5; http://www.tigr.org/).

After removing t/rRNA matching reads and those with 20 or morematches to the 93-11 or Nipponbare genomes, respectively, theremaining ‘clean’ small RNA reads were used for phasing analysisas described previously but with modifications (Howell et al., 2007;De Paoli et al., 2009).

Phylogenetic analysis

Full-length protein sequences were used for phylogenetic analysis.The sequences were downloaded from ChromDB (http://www.chromdb.org). Alignments of protein sequences were per-formed using ClustalX version 1.81 with default parameters (gapopening, 10.00; gap extension, 0.20; delay divergent sequences,30%; DNA transition weight, 0.50). A bootstrapped phylogenetictree was constructed by the MEGA2 program with the unweightedpair group method and the arithmetic mean method. The number ofbootstrap replicates was 1000.

RNA ligase mediated 5¢-RACE

RNA ligase-mediated 5¢-RACE was performed as previouslydescribed (Yoshikawa et al., 2005). Primers used are listed inTables S4 and S6.

RT-PCR and real-time PCR

Total RNA extraction and reverse transcription were performed aspreviously described (Liu et al., 2005). Transcripts levels weremeasured by RT-PCR and real-time PCR. Real-time PCR analysis wasperformed using the CFX96� Real-Time PCR System (Bio-Rad,http://www.bio-rad.com/) and SYBR Green I (Invitrogen, S-7567).The PCR was performed using hot-start Taq DNA polymerase(DR007B, TaKaRa, http://www.takara-bio.com/). For each sample,quantifications were made in triplicate. Melt curves were read at theend of each amplification by steps of 0.3�C from 65 to 95�C to ensurethat the quantifications were derived from real PCR products andnot primer dimmers. Primers used were as follows: for OsACTIN,cx909 (5¢-CCAATCGTGAGAAGATGACCCA-3¢) and cx910 (5¢-CCATCAGGAAGCTCGTAGCTCT-3¢), for OsDCL4, cx0758 (5¢-GGACTAGTTACACGAACGTCCTCTTCTTTTGGTAGGT-3¢) and cx0857 (5¢-CGATGAGAGAACTTCGAGAGCT-3¢), for OsDCL3a, cx0022(5¢-CGCGGATCCAGTTCCAGAAATTGTA-3¢) and cx0274(5¢-CACTCGCAGTGTTCCACACAAA-3¢), for OsDCL3b, cx0024(5¢-CGCGGATCCTACTACATGTGTTCTTGCTT-3¢) and cx0275(5¢-GCAAGTACATGCTTTCCGTGT-3¢).

Small RNA recognizing motif analysis, folding and

Weblogo generation

The most conserved nucleotides (5¢-TGGGAGGCATCAGGA-3¢, fromthe 7th to 21st position of the motif sequence) of the 21-nucleotidephased small RNA motif were used to scan against all the smallRNAs in our libraries having a size between 20 and 24 nucleotidesand cloned in the set of four 93-11 and Nipponbare panicle libraries.Patscan (Dsouza et al., 1997) was used to identify a conserved motifwith the maximum score at 4.5 (in which a G:U mismatch counts as

Phased small RNA clusters biogenesis in rice 11

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04805.x

0.5 points, and other mismatches count for 1 point). A similarstrategy was applied to search for small RNAs matching the24-nucleotide phased small RNA motif; in this case, ‘GAGAT-ATTGGAG’ was used as the conserved sequence. All the smallRNAs were mapped to both 93-11 and TIGR5 genomes, andpotential precursor sequences were derived using the small RNAsequence plus 150 nucleotides upstream and 150 nucleotidesdownstream; this potential precursor was passed to Mfold (Zuker,2003) for hairpin structure folding. Cleavage sites experimentallyvalidated or predicted by MEME were extracted with 42 nucleotidesupstream and 40 nucleotides downstream to illustrate the con-served motif using Weblogo online tool (http://weblogo.berke-ley.edu/).

Identification of signature derived from motif-containing

phased small RNA clusters in PARE/Degradome library

Small RNA and PARE/degradome signatures (Zhou et al., 2010) weremapped to the 93-11 genome and the most abundant of degradomesignatures in the phasing RNA clusters were kept. From the center ofthe degradome signatures, 82-nucleotide sequence strings weregenerated, using the 22-nucleotide signature plus 30 nucleotidesupstream and 30 nucleotides downstream. The most conserved 17-nucleotide seed sequence, derived from the Weblogo analysis, wasused to scan the tags for candidates with a Perl script performing thepattern search using a maximum penalty score of six (based on themismatches described above). Subsequently, the candidates forgood miRNA/target matches were further filtered by restricting thescore as previously described (Jones-Rhoades and Bartel, 2004).

Small RNA gel blot analysis

All of the small RNA hybridization experiments were carried out aspreviously described (Liu et al., 2005). The LNAs used in this paperwere as follows: LNA53 (gatcaggCccaaCaAggaCa), LNA42(5¢-gtgcttgtcctTgtTgggcCtGata-3¢), LNA12 (5¢-gcttctCtcCtCcccgaag-3¢), LNA58 (5¢-tagGaatggGaggcatcagGaa-3¢), LNA64 (5¢-tgaGatattGgagaaaaaCaag-3¢), LNA3 (tasiR-ARFs) (5¢-gtctcgcCgcttCtcagtCt-ggc-3¢). The uppercase letters represent the bases which weremodified LNAs. Other end-labeled oligos were used to detect miR168(cx0161 5¢-GTCCCGATCTGCACCAAGCGA-3¢) and U6 (cx32565¢-TGTATCGTTCCAATTTTATCGGATGT-3¢), respectively. The probecorresponding to CentO siRNA was prepared as previous described(Liu et al., 2005).

Quantification of the RNAs was carried by scanning the phosphorscreens with Typhoon Trio (GE Healthcare, http://www.gehealth-care.com/) and generating the density readings of RNAs bands withthe help of ImageQuant 5.2 software (GE Healthcare). The relativelevels were then determined by setting the value of Nipponbare as 1and calculate the values of other samples accordingly.

5¢-Phosphate-dependent exonuclease (Terminator) was pur-chased from Epicentre Biotechnologies (http://www.epibio.com/)and experiments were performed according to the manufacturer’sinstructions.

ACKNOWLEDGEMENTS

The authors would like to thank Shouhong Sun and Lianfeng Gufrom the Institute of Genetics and Developmental Biology for theirtechnical support. This work was supported by National BasicResearch Program of China (grant no. 2009CB941500 to XC), byNational Natural Science Foundation of China (grant nos. 30921061and 30870534 to XC), by Department of Agriculture of China (grantnos. 2008ZX08009-001 and 2011ZX08009-001 to CL), by 863 project(grant no. 2007AA10Z142 to CL) and by NSF PGRP award 0701745 toBCM and USDA grant no. 2007-01991 to PJG.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Flowchart for identification of phased small RNA clusters.Figure S2. Abundance and phasing scores for each position of TAS3in seedlings and panicles of 93-11 versus osdcl4-1.Figure S3. Distribution of 21- and 24-nucleotide phased small RNAclusters in 93-11.Figure S4. The knock down of OsDCL3a and OsDCL3b.Table S1. Summary of small RNA profiling.Table S2. Summary of phased small RNA clusters from 19- to25-nucleotide intervals in rice and Arabidopsis.Table S3. Profiles of phased small RNA clusters with intervals of 21and 24 nucleotides.Table S4. Primers of RNA ligase mediated-5¢ rapid amplification ofcDNA ends (RLM 5¢-RACE) for 21-nucleotide phased small RNAclusters.Table S5. The validation of motif-mediated cleavage for 21-nucle-otide phased small RNAs from the parallel analysis of RNA ends(PARE)/degradome data. (Please see separate Excel file for thistable).Table S6. Primers of RNA ligase mediated-5¢ rapid amplification ofcDNA ends (RLM 5¢-RACE) for 24-nucleotide phased small RNA clusters.Table S7. The validation of motif-mediated cleavage for 24-nucleo-tide phased small RNAs from the parallel analysis of RNA ends(PARE)/degradome data. (Please see separate Excel file for this table)Table S8. Location of miR2118 in riceTable S9. Location of miR2275 in ricePlease note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

REFERENCES

Allen, E., Xie, Z., Gustafson, A.M. and Carrington, J.C. (2005) microRNA-

directed phasing during trans-acting siRNA biogenesis in plants. Cell, 121,

207–221.

Axtell, M.J., Jan, C., Rajagopalan, R. and Bartel, D.P. (2006) A two-hit trigger

for siRNA biogenesis in plants. Cell, 127, 565–577.

Bailey, T.L. and Elkan, C. (1994) Fitting a mixture model by expectation

maximization to discover motifs in biopolymers. Proc. Int. Conf. Intell. Syst.

Mol. Biol. 2, 28–36.

Batista, P.J., Ruby, J.G., Claycomb, J.M. et al. (2008) PRG-1 and 21U-RNAs

interact to form the piRNA complex required for fertility in C. elegans. Mol.

Cell, 31, 67–78.

Baulcombe, D. (2004) RNA silencing in plants. Nature, 431, 356–363.

Carthew, R.W. and Sontheimer, E.J. (2009) Origins and Mechanisms of

miRNAs and siRNAs. Cell, 136, 642–655.

Chen, H.M., Li, Y.H. and Wu, S.H. (2007) Bioinformatic prediction and exper-

imental validation of a microRNA-directed tandem trans-acting siRNA

cascade in Arabidopsis. Proc. Natl. Acad. Sci. USA, 104, 3318–3323.

Chen, H.M., Chen, L.T., Patel, K., Li, Y.H., Baulcombe, D.C. and Wu, S.H. (2010)

22-Nucleotide RNAs trigger secondary siRNA biogenesis in plants. Proc.

Natl. Acad. Sci. USA, 107, 15269–15274.

Crooks, G.E., Hon, G., Chandonia, J.M. and Brenner, S.E. (2004) WebLogo: a

sequence logo generator. Genome Res, 14, 1188–1190.

Cuperus, J.T., Carbonell, A., Fahlgren, N., Garcia-Ruiz, H., Burke, R.T., Takeda,

A., Sullivan, C.M., Gilbert, S.D., Montgomery, T.A. and Carrington, J.C.

(2010) Unique functionality of 22-nt miRNAs in triggering RDR6-dependent

siRNA biogenesis from target transcripts in Arabidopsis. Nat. Struct. Mol.

Biol. 17, 997–1003.

De Paoli, E., Dorantes-Acosta, A., Zhai, J., Accerbi, M., Jeong, D.H., Park, S.,

Meyers, B.C., Jorgensen, R.A. and Green, P.J. (2009) Distinct extremely

abundant siRNAs associated with cosuppression in petunia. RNA, 15,

1965–1970.

12 Xianwei Song et al.

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04805.x

Dsouza, M., Larsen, N. and Overbeek, R. (1997) Searching for patterns in

genomic data. Trends Genet. 13, 497–498.

Dunoyer, P., Himber, C. and Voinnet, O. (2005) DICER-LIKE 4 is required for RNA

interference and produces the 21-nucleotide small interfering RNA com-

ponent of the plant cell-to-cell silencing signal. Nat. Genet. 37, 1356–1360.

Elbashir, S.M., Lendeckel, W. and Tuschl, T. (2001) RNA interference is

mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200.

Garcia-Ruiz, H., Takeda, A., Chapman, E.J., Sullivan, C.M., Fahlgren, N.,

Brempelis, K.J. and Carrington, J.C. (2010) Arabidopsis RNA-dependent

RNA polymerases and dicer-like proteins in antiviral defense and small

interfering RNA biogenesis during Turnip Mosaic Virus infection. Plant

Cell, 22, 481–496.

Gasciolli, V., Mallory, A.C., Bartel, D.P. and Vaucheret, H. (2005) Partially

redundant functions of Arabidopsis DICER-like enzymes and a role

for DCL4 in producing trans-acting siRNAs. Curr. Biol. 15, 1494–1500.

Girard, A., Sachidanandam, R., Hannon, G.J. and Carmell, M.A. (2006) A

germline-specific class of small RNAs binds mammalian Piwi proteins.

Nature, 442, 199–202.

He, G., Zhu, X., Elling, A.A. et al. (2010) Global epigenetic and transcriptional

trends among two rice subspecies and their reciprocal hybrids. Plant Cell,

22, 17–33.

Henderson, I.R., Zhang, X., Lu, C., Johnson, L., Meyers, B.C., Green, P.J. and

Jacobsen, S.E. (2006) Dissecting Arabidopsis thaliana DICER function in

small RNA processing, gene silencing and DNA methylation patterning.

Nat. Genet. 38, 721–725.

Hiei, Y., Ohta, S., Komari, T. and Kumashiro, T. (1994) Efficient transformation

of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis

of the boundaries of the T-DNA. Plant J. 6, 271–282.

Howell, M.D., Fahlgren, N., Chapman, E.J., Cumbie, J.S., Sullivan, C.M.,

Givan, S.A., Kasschau, K.D. and Carrington, J.C. (2007) Genome-wide

analysis of the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 path-

way in Arabidopsis reveals dependency on miRNA- and tasiRNA-directed

targeting. Plant Cell, 19, 926–942.

Itoh, J., Nonomura, K., Ikeda, K., Yamaki, S., Inukai, Y., Yamagishi, H., Kitano,

H. and Nagato, Y. (2005) Rice plant development: from zygote to spikelet.

Plant Cell Physiol. 46, 23–47.

Johnson, C., Kasprzewska, A., Tennessen, K., Fernandes, J., Nan, G.L., Wal-

bot, V., Sundaresan, V., Vance, V. and Bowman, L.H. (2009) Clusters and

superclusters of phased small RNAs in the developing inflorescence of rice.

Genome Res. 19, 1429–1440.

Jones-Rhoades, M.W. and Bartel, D.P. (2004) Computational identification of

plant microRNAs and their targets, including a stress-induced miRNA. Mol.

Cell, 14, 787–799.

Kapoor, M., Arora, R., Lama, T., Nijhawan, A., Khurana, J.P., Tyagi, A.K. and

Kapoor, S. (2008) Genome-wide identification, organization and phyloge-

netic analysis of Dicer-like, Argonaute and RNA-dependent RNA Polymer-

ase gene families and their expression analysis during reproductive

development and stress in rice. BMC Genomics, 9, 451.

Kurihara, Y., Takashi, Y. and Watanabe, Y. (2006) The interaction between

DCL1 and HYL1 is important for efficient and precise processing of pri-

miRNA in plant microRNA biogenesis. RNA, 12, 206–212.

Lagos-Quintana, M., Rauhut, R., Lendeckel, W. and Tuschl, T. (2001) Identi-

fication of novel genes coding for small expressed RNAs. Science, 294,

853–858.

Lau, N.C., Lim, L.P., Weinstein, E.G. and Bartel, D.P. (2001) An abundant class

of tiny RNAs with probable regulatory roles in Caenorhabditis elegans.

Science, 294, 858–862.

Lee, R.C. and Ambros, V. (2001) An extensive class of small RNAs in Ca-

enorhabditis elegans. Science, 294, 862–864.

Lister, R., O’Malley, R.C., Tonti-Filippini, J., Gregory, B.D., Berry, C.C., Millar,

A.H. and Ecker, J.R. (2008) Highly Integrated Single-Base Resolution Maps

of the Epigenome in Arabidopsis. Cell, 133, 523–536.

Liu, B., Li, P., Li, X., Liu, C., Cao, S., Chu, C. and Cao, X. (2005) Loss of function

of OsDCL1 affects microRNA accumulation and causes developmental

defects in rice. Plant Physiol. 139, 296–305.

Liu, B., Chen, Z., Song, X. et al. (2007) Oryza sativa dicer-like4 reveals a key

role for small interfering RNA silencing in plant development. Plant Cell, 19,

2705–2718.

Margis, R., Fusaro, A.F., Smith, N.A., Curtin, S.J., Watson, J.M., Finnegan, E.J.

and Waterhouse, P.M. (2006) The evolution and diversification of Dicers in

plants. FEBS Lett. 580, 2442–2450.

Meyers, B.C., Axtell, M.J., Bartel, B. et al. (2008) Criteria for Annotation of

Plant MicroRNAs. Plant Cell, 20, 3186–3190.

Mosher, R.A., Melnyk, C.W., Kelly, K.A., Dunn, R.M., Studholme, D.J. and

Baulcombe, D.C. (2009) Uniparental expression of PolIV-dependent siRNAs

in developing endosperm of Arabidopsis. Nature, 460, 283–286.

Nagasaki, H., Itoh, J., Hayashi, K. et al. (2007) The small interfering RNA

production pathway is required for shoot meristem initiation in rice. Proc.

Natl Acad. Sci. USA, 104, 14867–14871.

Nobuta, K., Venu, R.C., Lu, C. et al. (2007) An expression atlas of rice mRNAs

and small RNAs. Nat. Biotechnol. 25, 473–477.

Olmedo-Monfil, V., Duran-Figueroa, N., Arteaga-Vazquez, M., Demesa-Are-

valo, E., Autran, D., Grimanelli, D., Slotkin, R.K., Martienssen, R.A. and

Vielle-Calzada, J.P. (2010) Control of female gamete formation by a small

RNA pathway in Arabidopsis. Nature, 464, 628–632.

Park, W., Li, J., Song, R., Messing, J. and Chen, X. (2002) CARPEL FACTORY, a

Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in

Arabidopsis thaliana. Curr. Biol. 12, 1484–1495.

Rajagopalan, R., Vaucheret, H., Trejo, J. and Bartel, D.P. (2006) A diverse and

evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev.

20, 3407–3425.

Ramachandran, V. and Chen, X. (2008) Small RNA metabolism in Arabidopsis.

Trends Plant Sci. 13, 368–374.

Reinhart, B.J., Weinstein, E.G., Rhoades, M.W., Bartel, B. and Bartel, D.P.

(2002) MicroRNAs in plants. Genes Dev. 16, 1616–1626.

Ron, M., Saez, M.A., Williams, L.E., Fletcher, J.C. and McCormick, S. (2010)

Proper regulation of a sperm-specific cis-nat-siRNA is essential for double

fertilization in Arabidopsis. Genes Dev. 24, 1010–1021.

Schauer, S.E., Jacobsen, S.E., Meinke, D.W. and Ray, A. (2002) DICER-LIKE1:

blind men and elephants in Arabidopsis development. Trends Plant Sci. 7,

487–491.

Slotkin, R.K., Vaughn, M., Borges, F., Tanurdzic, M., Becker, J.D., Feijo, J.A.

and Martienssen, R.A. (2009) Epigenetic reprogramming and small RNA

silencing of transposable elements in pollen. Cell, 136, 461–472.

Urayama, S., Moriyama, H., Aoki, N., Nakazawa, Y., Okada, R., Kiyota, E.,

Miki, D., Shimamoto, K. and Fukuhara, T. (2010) Knock-down of OsDCL2 in

rice negatively affects maintenance of the endogenous dsRNA virus, Oryza

sativa endornavirus. Plant Cell Physiol. 51, 58–67.

Vazquez, F., Vaucheret, H., Rajagopalan, R., Lepers, C., Gasciolli, V., Mallory,

A.C., Hilbert, J.L., Bartel, D.P. and Crete, P. (2004) Endogenous trans-acting

siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol. Cell, 16, 69–

79.

Vogel, J. (2010) Genome sequencing and analysis of the model grass

Brachypodium distachyon. Nature, 463, 763–768.

Wu, L., Zhou, H., Zhang, Q., Zhang, J., Ni, F., Liu, C. and Qi, Y. (2010) DNA

methylation mediated by a microRNA pathway. Mol. Cell, 38, 465–475.

Xie, Z., Johansen, L.K., Gustafson, A.M., Kasschau, K.D., Lellis, A.D., Zilber-

man, D., Jacobsen, S.E. and Carrington, J.C. (2004) Genetic and functional

diversification of small RNA pathways in plants. PLoS Biol. 2, E104.

Xie, Z., Allen, E., Wilken, A. and Carrington, J.C. (2005) DICER-LIKE 4 functions

in trans-acting small interfering RNA biogenesis and vegetative phase

change in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA, 102, 12984–

12989.

Yoshikawa, M., Peragine, A., Park, M.Y. and Poethig, R.S. (2005) A pathway

for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev. 19,

2164–2175.

Zamore, P.D., Tuschl, T., Sharp, P.A. and Bartel, D.P. (2000) RNAi: double-

stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23

nucleotide intervals. Cell, 101, 25–33.

Zhang, W., Yi, C., Bao, W., Liu, B., Cui, J., Yu, H., Cao, X., Gu, M., Liu, M. and

Cheng, Z. (2005) The transcribed 165-bp CentO satellite is the major func-

tional centromeric element in the wild rice species Oryza punctata. Plant

Physiol. 139, 306–315.

Zhou, M., Gu, L., Li, P., Song, X., Wei, L., Chen, Z. and Cao, X. (2010) Degra-

dome sequencing reveals endogenous small RNA targets in rice (Oryza

sativa L. ssp. indica). Front. Biol. 5, 67–90.

Zhu, Q.H., Spriggs, A., Matthew, L., Fan, L., Kennedy, G., Gubler, F. and

Helliwell, C. (2008) A diverse set of microRNAs and microRNA-like small

RNAs in developing rice grains. Genome Res 18, 1456–1465.

Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization

prediction. Nucleic Acids Res. 31, 3406–3415.

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