Biogenesis of Plant MicroRNAs

March 2014

ScienceDirect

Vol. 21 No. 1 84-96

Journal of Northeast Agricultural University (English Edition)

Available online at www.sciencedirect.com

Biogenesis of Plant MicroRNAs Kong Wen-wen, Wang Hong-bo, and Li Jing* College of Life Sciences, Northeast Agricultural University, Harbin 150030, China

Abstract: microRNAs (miRNAs) play important regulatory roles in eukaryotic gene expression, predominantly at the posttranscriptional level. Elaborate and diverse biogenesis pathways have evolved to produce miRNAs. miRNA biogenesis is a multistep process including transcription, precursor slicing, methylation, nuclear export, and RNA-induced silencing complex assembly. In the decade, since the first discovery of plant miRNAs, many enzymes and regulatory proteins involved in miRNA biogenesis in plants have been uncovered and a basic picture of miRNA processing is emerging gradually. In this article, we summarized the current study of plant miRNA biogenesis and discussed the multiple integrated steps and diverse pathways of miRNA processing. Key words: miRNA, biogenesis, Arabidopsis thaliana, pathway CLC number: Q74

Document code: A

Article ID: 1006-8104(2014)-01-0084-13

themselves are generated and regulated.

Introduction

　The first miRNA was discovered in C. elegans in

MicroRNAs (miRNAs) are 18-25 nt long, single-

was reported in Arabidopsis in 2002 (Reinhart et al.

stranded, regulatory non-coding RNAs derived

2002). The miRNAs biogenesis pathways in animals

from hairpin-like precursors. These small molecules

and plants are now basically established after more

regulate gene expression through mRNA cleavage,

than twenty and ten years investigation, respectively.

translation repression, and DNA methylation (Llave et

Despite both animal and plant miRNAs perform

al., 2002; Mallory et al., 2008; Brodersen et al., 2008;

important roles during their life cycle, there are many

Wu et al., 2010; Sun, 2012). In plants, miRNAs play

different details in animal and plant miRNA biogenesis

important roles in diverse regulatory pathways and are

procedures. In animals, MIRs are transcribed by poly-

involved in almost all the developmental events such

merase II (POL II) similar to plant MIRs transcription,

as leaf shape, floral transition (Liu et al., 2006; Park

while some MIR genes are transcribed by polymerase

et al., 2002) as well as mediating responses to both

III (POL III) (Bartel, 2004; Chen et al. 2004). Then,

abiotic (drought, temperature, salinity) (Wang et al.,

the transcripts are sliced twice, and the first slicing

2012; Feng et al., 2013; Ni et al., 2013; Sunkar et al.,

in animals is the nuclear cleavage of the pri-miRNA,

2007) and biotic (bacteria, viruses) (Brotman et al.,

which liberates a stem loop intermediate, known as

2012; Fagard et al., 2007) environmental conditions.

the pre-miRNA by the Drosha RNase III endonuclease

To further dissect the miRNA regulation network, it

(Lee et al., 2003). By Ran-GTP and Exportin-5, this

is important to understand how miRNA genes (MIRs)

pre-miRNA is transported to the cytoplasm and sliced

1993 by Lee et al. (1993) and the first plant miRNA

Received 6 May 2013 Supported by the National Natural Science Foundation of China (31070265) Kong Wen-wen (1990-), male, Ph. D, engaged in the research of plant molecular biology. E-mail: [email protected] * Corresponding author. Li jing, professor, supervisor of Ph. D student, engaged in the research of plant molecular biology. E-mail: [email protected] E-mail: [email protected]

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Kong Wen-wen et al. Biogenesis of Plant MicroRNAs

by another RNase III endonuclease Dicer for the

(previously known as At-Negative on TATA-less2

second time and formed the miRNA/miRNA* duplex.

[NOT2] and VIRE2-INTERACTING PROTEIN2,

(Lund et al., 2004; Lee et al., 2003; Bernstein et al.,

respectively), was recently found to promote this trans-

2001). In plants, MIRs are only transcribed by RNA

cription through their interaction with Pol II (Fig. 1).

polymerase II (Pol II) to form stem-loop structured

NOT2s also interact with DCL1 and some other slic-

primary microRNA (pri-miRNA) (Xie et al., 2005).

ing factors and thus are functional in subsequent

The Dicer-like1(DCL1) complex then slices the pri-

processing steps (Wang et al., 2013). The transcripts

miRNA twice similar to in animals, while both of them

are modified by adding a 5' cap and a 3' polyadenylate

occurred in nucleus. The base of the stem is cut off in

tail like other coding genes (Xie et al., 2005; Lee

the first slicing and precursor miRNA (pre-miRNA)

et al., 2004; Jones-Rhoades et al., 2006).

is produced, while the loop is removed in the second

Some MIR genes contain introns, which have to

slicing and a duplex of miRNA/miRNA* is formed

be removed before the formation of the stem-loop

(Reinhart et al., 2002). After the miRNA/miRNA*

structure (Aukerman and Sakai, 2003; Nikovics et al.,

duplex formation, the miRNA strand (guide strand)

2006). STABILIZED1 (STA1), a protein functioning

is selected and incorporated into the ARGONAUTE1

in pre-mRNA processing in Arabidopsis, was identi-

(AGO1) component of the RNA-induced silencing

fied to be responsible for the intron splicing of

complex (RISC), which then mediates the repression

miRNA transcripts (Ben et al., 2013) (Fig. 1). A

of gene expressions (Song et al., 2004). Recently,

dramatic reduction in the number of mature miRNAs

increasing numbers of new proteins involved in plant

and an accumulation of unspliced pri-miRNAs was

miRNA biogenesis are being discovered. Here, we

detected in sta1. In addition, the transcript level of

focused on current advancements in plant miRNA

DCL1 was reduced in sta1, indicating that STA1 is

studies, and the integrated processes and diverse

responsible for splicing the pre-mRNAs of DCL1 (Ben

pathways of plant miRNA biogenesis were discussed

et al., 2013). These results suggested that STA1 was

in details.

involved in miRNA biogenesis directly by functioning in pri-miRNA splicing and indirectly by modulating

Transcription of MIR Genes

the DCL1 transcript level (Ben et al., 2013).

The majority of MIR genes are located in intergenic

cribed RNA will fold back into a stem-loop struc-

loci between protein-coding genes, while others are

tured pri-miRNA. The length of pri-miRNAs can be

found in intragenic loci, in either introns or exons

variable and ranges from hundreds to several kilo-

(Rajagopalan et al., 2006; Cui et al., 2009). MIR genes

bases (Szarzynska et al., 2009). Generally, one pri-

are transcribed by Pol II in plants (Xie et al., 2005)

miRNA produces one mature miRNA, but it has been

(Fig. 1). As the important gene expression regulator,

found that pri-miRNAs containing multiple stem-

MIR genes transcribed are under precision regulated.

loop structures can produce more than one miRNA

Recent study found that introns of plant pri-miRNAs

(Merchan et al., 2009; Zhang et al., 2010; Lacombe

can enhance miRNA biogenesis (Bielewicz et al.

et al., 2008).

Based on its own compliment sequence, the trans-

2013). Otherwise, the transcriptional co-activator mediator, a conserved protein known to promote trans-

Slicing of pri- and pre-miRNA

cription of protein-coding genes, is required to recruit

The pri-miRNA undergoes two slicing steps to cut

Pol II to transcript factor-binding sites in MIR pro-

off the loop and the stem separately. The slicing is

moters to regulate MIRs transcript (Kim et al., 2011)

a complicated process and a series of proteins are

(Fig. 1). A pair of NOT2 proteins, NOT2a and NOT2b

involved. http: //publish.neau.edu.cn

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Fig. 1　Biogenesis of plant miRNAs MIR genes are transcribed by Pol II. Their transcription is promoted by the co-activators mediator and NOT2s. For intron-containing MIR genes, introns are spliced by STA1. DDL stabilizes pri-miRNAs. The slicing of pri- and pre-miRNA is catalyzed by DCL1 and the proper slicing entails the concerted action and physical interaction of a set of proteins including DCL1, HYL1, SE, CPL1, CBC, DDL, SIC, TGH, and NOT2s. MOS2 is not localized in the D-body but can facilitate the recruitment of pri-miRNA to the dicing complex. The sliced miRNA/miRNA* duplex is methylated by HEN1. The methylated miRNA duplex (also presumably pre-miRNA or mature miRNA) is then exported to the cytoplasm, possibly dependent on HST or the nuclear pore. HYL1 itself or together with DCL1 directionally loads the methylated miRNA/miRNA* duplex into AGO1, which is associated with a complex containing the chaperone HSP90 and SQN. The guide strand is then selected and regulates gene expression, while the passenger strand is either degraded or in non-canonical conditions, loaded into another RISC. E-mail: [email protected]

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Kong Wen-wen et al. Biogenesis of Plant MicroRNAs

A few miRNAs like miR822, miR839, and miR869

Proteins Involved in Slicing

are sliced primarily by DCL4, possibly due to the

DCL and DRB family

(Rajagopalan et al., 2006; Ben et al., 2009).

The DCL and double-stranded RNA-binding (DRB)

　 In Arabidopsis, besides the canonical 21-nt

families are very important in plant miRNA produc-

miRNAs, a minor fraction of 23-25 nt miRNAs are

tion. Several members in each family are involved

generated independently from the same miRNA pre-

in miRNA biogenesis and the relationships of the

cursors by DCL3 for a large number of MIR genes

members within and between families result in the

(Vazquez et al., 2008). In rice, dozens of MIR genes

diversity and complexity of miRNA biogenesis.

producing only 24 nt long miRNAs or both canonical

　 In animals, the slicing of miRNA is catalyzed

and long miRNAs have been found (Wu et al., 2010;

by Drosha (Bartel, 2004; Lee et al., 2003), while in

Wu et al., 2009; Zhu et al., 2008). These DCL3-

plants, it is performed by a homolog of Drosha, DCL1,

dependent long miRNAs were demonstrated to be

which is a key protein in plant miRNA biogenesis

loaded into the AGO4-containing RISC complex

(Park et al., 2002). Loss of DCL1 function results in

and direct DNA methylation at their loci of origin as

severely defective phenotypes. In Arabidopsis, dcl1 is

well as in trans at their target genes (Wu et al., 2010)

embryo lethal (Golden et al., 2002). DCL1 belongs to

(Fig. 2c). The generation of long miRNAs is usually

a multidomain ribonuclease III (RNase III)-like family

dependent on the spatial or temporal expression of

that contains four members, DCL1, DCL2, DCL3, and

DCL3, implying possible competition among DCL

DCL4 in Arabidopsis (Voinnet, 2009). DCL proteins

proteins for miRNA precursor slicing under certain

contain a DExD/H-box RNA helicase domain, a

conditions (Vazquez et al., 2008).

DUF283 RNA-binding domain, a small RNA-binding

　DRB proteins have widespread functions in RNA

PAZ domain, two tandem RNase III domains, and two

metabolism (Eamens et al., 2012). In Arabidopsis, the

tandem dsRNA-binding domains (dsRBD) (Margis

DRB family contains five closely related members,

et al., 2006; Liu et al., 2006). The length of small

DRB1-DRB5, all of which are involved in small RNA

RNAs sliced by DCLs is based on the distance between

biogenesis. DRB1, also called HYL1 (HYPONASTIC

the PAZ and RNase III domains (Macrae et al., 2006),

LEAVES1), facilitates pri-miRNA processing and was

consequently DCL enzymes themselves are mole-

verified to associate with DCL1 both in vitro (Lu and

cular rulers controlling the length of the small RNAs

Fedoroff, 2000) and in vivo (Kurihara et al., 2006)

they produce. DCL1 can produce 18-21 nt long RNAs

(Fig. 1, 2a). In hyl1, both spliced and intron-containing

while DCL2, DCL3, and DCL4 mainly produce 22,

pri-miRNAs are accumulated (Vazquez et al., 2004;

24, and 21 nt products, respectively (Voinnet, 2009;

Han et al., 2004) and DCL1 cleavage sites are mis-

Takanashi et al., 2011).

placed, resulting in a lower level of functional mature

　DCL1 is believed to be the slicing enzyme for most

miRNA (Vazquez et al., 2004). Therefore, HYL1

plant miRNAs (Park et al., 2002; Reinhart et al.,

is required for efficient and precise cleavage of pri-

2002) (Figs. 1 and 2a), while the other DCLs were

miRNA. However, there are still some functional

originally characterized as proteins that function in

mature miRNAs in the hyl1 null mutant, suggesting

other small RNA biogenesis pathways (Xie et al.,

that additional factors or alternative DRB proteins

2004; Gasciolli et al., 2005). However, DCL4, pre-

are involved in accurate DCL slicing (Kurihara et al.,

viously identified as an enzyme for biogenesis of

2006; Han et al., 2004; Yang et al., 2010).

trans-acting small-interfering RNAs (ta-siRNAs), was

　DRB2 was reported to be involved in the maturation

found to be involved in miRNA processing (Fig. 2d).

of subsets of miRNAs. Therefore, another DRB2-medi-

highly complementary fold-backs in these pri-miRNAs

http: //publish.neau.edu.cn

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ated non-canonical miRNA pathway was proposed

or with an alternate RISC complex containing an un-

(Eamens et al., 2012) (Fig. 2b). In this pathway, the

known AGO protein, DRB3 and DRB5 instead (Fig. 2b).

pri-miRNA transcript is recognized alternatively by a

In the subsequent step, AGO1-catalyzed RISC

DCL1/DRB2 pairing complex and is sliced to form a

mediates mRNA cleavage and the DRB3 and DRB5-

miRNA/miRNA* duplex. The selected miRNA strand

containing RISC causes translational repression of the

is associated either with an AGO1-catalyzed RISC

target mRNA (Eamens et al., 2012).

Fig. 2　Biogenesis pathways of plant miRNAs a, The DCL1- and DRB1-dependent miRNA biogenesis pathway. In this canonical miRNA pathway, the MIR gene is transcribed by Pol II to form a stem-loop structured pri-miRNA. The step-loop structure is recognized by the DCL1/DRB1-containing complex and sliced twice to generate a miRNA/miRNA* duplex. The mature miRNA strand is associated with the AGO1 component of the RISC to regulate target gene expression by mRNA cleavage; b, The DCL1- and DRB2-dependent miRNA biogenesis pathway. In this non-canonical pathway, the pri-miRNA transcript is recognized alternatively by a DCL1/DRB2 pairing complex and sliced to form a miRNA/miRNA* duplex. The mature miRNA is associated either with an AGO1-catalyzed RISC to mediate mRNA cleavage or an alternate RISC complex containing DRB3 and DRB5 to repress translation of target mRNA; c, The DCL3-dependent miRNA biogenesis pathway. In this non-canonical pathway, the pri-miRNA transcript is recognized by a DCL3containing complex and sliced to form a miRNA/miRNA* duplex. The 23-25 nt long mature miRNA is loaded into an AGO4-containing RISC to direct DNA methylation at its loci of origin as well as in trans at its target gene; d, The DCL4-dependent miRNA biogenesis pathway. The pri-miRNA transcript is recognized by a DCL4-containing complex and sliced to form a miRNA/miRNA* duplex. In this non-canonical pathway, both DRB2 and DRB4 are required. The mature miRNA is loaded into an AGO1-containing RISC to guide mRNA cleavage. Images a and b are from Eamens et al. (2012).

　 DRB4 was previously identified to play an im-

by associating with DCL4 (Hiraguri et al., 2005;

portant role in small interfering RNA biogenesis

Nakazawa et al., 2007) (Fig. 2d). DCL4-dependent

E-mail: [email protected]

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Kong Wen-wen et al. Biogenesis of Plant MicroRNAs

miRNA accumulation is strongly reduced in drb4,

selection of the miRNA duplex (Manavella et al.,

indicating that DRB4 is required in DCL4-dependent

2012).

miRNA biogenesis (Fukudome et al., 2011; Pélissier

　DAWDLE (DDL), a nuclear RNA-binding protein,

et al., 2011). Furthermore, northern blot hybridization

was shown to stabilize pri-miRNA in miRNA bio-

showed that accumulation of miR839 was reduced in

synthesis (Yu et al., 2008) (Fig. 1). In Arabidopsis,

drb2 and drb4, indicating that DRB2 is also required

mature miRNA accumulation is greatly reduced in the

for DCL4-mediated miRNA accumulation (Vazquez

ddl mutant. DDL is capable of binding pri-miRNA

et al., 2010) (Fig. 2d).

and interacting with DCL1 in vitro. Therefore, it was suggested that DDL could improve the stability of pri-

Other slicing factors

miRNA either by binding to the end of the stem-loop

Serrate (SE) is a C2H2-zinc finger protein localized in

or by the indirect effect of DCL1 recruitment (Yu et

the nucleus (Laubinger et al., 2008). The phenotypes

al., 2008; Rogers and Chen, 2013). DDL binding is

of se show a lot of similarities to other miRNA-

general to RNAs and not specific to pri-miRNA; thus,

processing enzyme mutants (Lu and Fedoroff, 2000;

it is involved in other RNA metabolism, which might

Prigge and Wagner, 2001; Bezerra et al., 2004; Clarke

explain why the ddl mutant showed more serious

et al., 1999; Bollman et al., 2003). It was revealed

developmental deficiencies than those of the dcl1

that most pri-miRNAs are accumulated while mature

mutants (Yu et al., 2008).

miRNAs are reduced in se, and the decreased levels of

　CAP BINDING Complex (CBC) is a heterodimer

mature miRNAs are correlated with increased levels

that contains two subunits, CBP80 and CBP20

of their target gene transcripts (Lobbes et al., 2006).

(Laubinger et al., 2008; Gregory et al., 2008), and

SE enhances the accuracy of pri-miRNA cleavage by

binds to the 5' cap structure of Pol II transcripts. Pri-

DCL1 both in vitro (Dong et al., 2008) and in vivo

miRNA is accumulated while mature miRNA is

(Manavella et al., 2012). Thus, SE is required for

reduced in cbp80 and cbp20, indicating that CBC

proper slicing of pri-miRNA (Fig. 1). SE was found

plays a role in the pri-miRNA slicing process (Gregory

to be able to react with most of the pri-miRNA slicing

et al., 2008) (Fig. 1). CBP20 binds to SE in vivo and

proteins like DCL1 and HYL1 (Lobbes et al., 2006;

in vitro, suggesting that CBC might be required for

Machida et al., 2011), and was therefore suggested to

the formation of a pri-miRNA processing center by

work as a scaffold-like protein capable of binding both

interacting with SE (Wang et al., 2013). Like DDL,

proteins and RNA to guide the positioning of miRNA

CBC does not function only in miRNA biogenesis,

precursors toward the DCL1 catalytic site (Machida

as mRNA intron slicing is also suppressed in cbp80

et al., 2011).

and cbp20, suggesting that CBC is required in intron

　 C-TERMINAL DOMAIN PHOSPHATASE-

slicing (Laubinger et al., 2008; Gregory et al., 2008;

LIKE1 (CPL1), containing TFIIF-interacting CTD

Kim et al., 2008).

phosphatase1-like phosphatase and dsRBD domains,

　NOT2s are required in pri-miRNA slicing in addi-

is a recently identified protein that affects the accuracy

tion to promoting MIR gene transcription by inter-

of miRNA maturation (Fig. 1). Loss of CPL1 function

acting with Pol II. NOT2s associate with some key

results in the accumulation of miRNA target mRNAs

miRNA processing factors, including DCL1, CBC,

and inaccurate miRNAs without changing the total

and SE (Fig. 1). Impairment of NOT2s leads to mislo-

miRNA level. It was discovered that CPL1 is recruited

calization of DCL1, suggesting that NOT2 proteins

by SE to the DCL1 complex and dephosphorylates

facilitate efficient recruitment of DCL1 and other

HYL1. Dephosphorylation of HYL1 was found to be

processing factors in miRNA biogenesis. These results

important for accurate miRNA processing and strand

explain why inactivation of NOT2s decreases the http: //publish.neau.edu.cn

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accumulation of both pri-miRNA and mature miRNA

its role in regulating the DRB4 and DCL4-containing

(Wang et al., 2013).

dicing complex (Wu et al., 2013).

　TOUGH (TGH) contains G-patch and SWAP domains (Suppressor-of-White-APricot), which often

Direction of slicing

exist within RNA metabolism-related proteins

In animals, the rules for miRNA excision from the

(Calderon-Villalobos et al., 2005), and was found to

precursor are simple because of the uniform size

be a component of the DCL1 slicing complex (Fig. 1).

and shape of the pri-miRNA. In contrast, the stem-

In tgh, the accumulation of both DCL1- and DCL4-

loops of pri-miRNAs in plants are quite variable in

mediated miRNAs is reduced and the transcript levels

both length and secondary structure. Accordingly,

of their pri-miRNAs are increased (Ren et al., 2012).

the mechanism for miRNA precursor recognition by

TGH binds ssRNA but not dsRNA in vitro, so it

the DCL1 complex is more complicated and less well

possibly functions by binding to the loop or the bulge

understood. Most plant pri-miRNAs are sliced in the

of pri- or pre-miRNAs. Unlike HYL1 and SE, TGH

stem-to-loop direction like in animals (Breakfield

does not affect miRNA precision, as evidenced by the

et al., 2012). Analysis of pre-miR172 processing

low ratio of imprecise miRNAs in tgh.

showed that the initial cut by the DCL1 complex is

　Sickle (SIC), a hydroxyproline-rich glycoprotein,

located –15 nt from the base of the stem, while the

was recently discovered to be involved in miRNA

loop is removed in the second slicing (Mateos et al.,

biogenesis (Fig. 1). Immunolocalization revealed that

2010; Song et al., 2010; Werner et al., 2010).

SIC and HYL1 were colocalized in nuclear bodies.

　Unlike most plant miRNAs, miR159 and miR319

sic exhibited some common phenotypes of mutants

are sliced in the loop-to-base direction (Bologna et al.,

defective in miRNA biogenesis and accumulated

2013). Both miR159 and miR319 have long transcripts

lower levels of a subset of miRNAs and higher levels

and can fold-back into a long double-stranded

of corresponding pri-miRNAs than the wild type (Zhan

backbone. Evidence from detailed mutagenesis

et al., 2012). It remains unknown how SIC functions

experiments showed that pre-miR159 and pre-miR319

in pri-miRNA processing. SIC has a function in sliced

processing begins with a cleavage near the loop

intron decay (Zhan et al., 2012), suggesting a possible

(Bologna et al., 2009; Naqvi et al., 2012). DCL1 then

common mechanism for the cleavage of pri-miRNA

continues to cut the precursor three more times at

hairpins and the decay of sliced introns.

20-22 nt intervals until the miRNA is finally released

　 MOS2 encodes a G-patch and KOW domain-

(Bologna et al., 2009; Addo-Quaye et al., 2009).

containing RNA-binding protein and was previously

Interestingly, the long precursors of miR319 and

identified to be essential for innate immunity in

miR159 are highly conserved in plants, indicating that

Arabidopsis (Zhang et al., 2005). MOS2 is required for

this processing mechanism is ancient (Bologna et al.,

efficient processing of pri-miRNA through facilitating

2009).

the recruitment of pri-miRNA by the dicing complex, as evidenced by the greatly reduced association

Methylation of miRNA/miRNA* duplex

between HYL1 and pri-miRNA in mos2. Interestingly,

HUA ENHANCER1 (HEN1) is a protein contain-

MOS2 binds pri-miRNA both in vitro and in vivo, but

ing a dsRNA-binding motif and a C-terminal methyl-

does not interact with HYL1, SE, or DCL1 and is not

transferase domain and therefore is able to bind

localized in the D-body which is nuclear processing

dsRNA and deposit a methyl group on to the 2'-OH of

center discussed in the following (Fig. 1). Furthermore,

the 3'-terminal nucleotide (Yang et al., 2006). HEN1

MOS2 is also involved in the biogenesis of ta-siRNA,

plays an important role in miRNA maturation because

which is dependent on DRB4 and DCL4, indicating

of its ability to stabilize the miRNA/miRNA* duplex

E-mail: [email protected]

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Kong Wen-wen et al. Biogenesis of Plant MicroRNAs

(Chen, 2005; Li et al., 2005; Yu et al., 2005) (Fig. 1).

be selected as miRNA (guide strand) while the other

After being sliced from the pre-miRNA, the miRNA/

strand (passenger strand/miRNA*) undergoes degrada-

miRNA* duplex is methylated by HEN1 and thus is

tion. The determinant for this selection was discover-

protected from degradation. The hen1 mutant shows a

ed by bioinformatic analysis and confirmed by arti-

similar phenotype to dcl1 and the 3'-ends of miRNAs

ficial miRNA experiments (Rajagopalan et al., 2006).

are found to have additional nucleotides, primarily

The majority of Arabidopsis miRNAs are preferen-

uridines instead of methylated ends. It was discovered

tially selected over their miRNA* strands because of

that the uridylation of unmethylated miRNAs in

the asymmetric thermodynamic stability of the miRNA/

hen1 is performed by a DNA polymerase β family

miRNA* duplex terminus, and the strand with a wea-

protein, HEN1 SUPPRESSOR1 (HESO1), and that

ker 5'-terminus is preferentially chosen as the miRNA

the uridylation leads to degradation (Ren et al., 2012;

guide strand (Rajagopalan et al., 2006; Eamens et al.,

Zhao et al., 2012). Furthermore, the methylation of

2009).

miRNA duplexes may also act as an export signal to

　 In addition, DRB1 (HY1) is required for strand

the cytoplasm (Park et al., 2005).

selection as evidenced by the higher level of miRNA* in hyl1 mutants (Eamens et al., 2009). Consistently,

Transportation and selection of mature miRNA

cpl1 mutants accumulate higher levels of miRNA*,

In mammals, Exportin 5 (Exp5) exports pre-miRNAs

indicating that the dephosphorylation of HYL1 by

to the cytoplasm before the mature miRNA is gene-

CPL1 is also required for proper guide strand selection

rated (Lund et al., 2004). In plants, the corresponding

(Manavella et al., 2012). DRB1 may bind to or

transportation process is still not clear and both the

interact with the more thermodynamically stable

transporter and the transported molecules remain

end of the miRNA duplex, and then DRB1 alone,

unknown. HASTY (HST), a nuclear shuttle protein

or in a heterodimer with DCL1, directionally loads

that is homologous to Exp5, was suggested to function

the miRNA duplex to the critical miRNA machinery

by exporting methylated miRNA/miRNA* duplexes

protein AGO1 (Eamens et al., 2009; Matranga et al.,

or single-stranded miRNA to the cytoplasm (Bollman

2005; Tomari et al., 2004; Baumberger et al., 2005)

et al., 2003; Park et al., 2005) (Fig. 1). Reduced accu-

where the guide strand is selected (Fig. 1).

mulation of many miRNAs in hst mutants indicates a role for HST in miRNA biogenesis. However, the

RISC assembly

similarly reduced levels of these miRNAs in both the

The assembly of RISC involves the loading of the

nucleus and cytoplasm do not seem to support the

miRNA/miRNA* duplex into AGO complex and

hypothesis that HST functions in miRNA export (Park

subsequent removal of the miRNA* strand. HEAT

et al., 2005). Several species of miRNA are exported

SHOCK PROTEIN 90 (HSP90) was found to be

to the cytoplasm by an HST-independent mechanism

involved in the assembly process as a molecular

and it was suggested that mRNA transport proteins are

chaperone that binds to AGO1 and facilitates the

possibly involved in this process (Park et al., 2005).

loading of the dsRNA duplex (Iki et al., 2010) (Fig. 1).

In addition, the nuclear pore complex might play a

HSP90 mediates substrate peptide association in

role in miRNA transportation as evidenced by the

response to ATP binding and releases the substrate

reduced accumulation of some miRNAs in mutants

on ATP hydrolysis by changing its conformation (Iki

of the nuclear pore component TRANSLOCATED

et al., 2010).

PROMOTER REGION (TPR) (Jacob et al., 2007)

　SQUINT (SQN), a member of the immunophilin

(Fig. 1). Before mature miRNA is generated, one strand

family, facilitates RISC assembly and is able to form

of the methylated miRNA/miRNA* duplex has to

a complex with HSP90, AGO1, and small RNA http: //publish.neau.edu.cn

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duplexes (Fig. 1). Expression of SQN can rescue the

Interestingly, recent studies indicate that miRNA* can

phenotype of sqn null alleles, while expression of

be active in gene silencing in Drosophila melanogaster,

a mutated SQN showing reduced interaction with

humans, and plants (Ghildiyal et al., 2010; Manavella

HSP90 was unable to rescue the phenotype, indicat-

et al., 2013; Meng et al., 2011) (Fig. 1). Large-scale

ing that interaction with HSP90 is required for

sequencing research demonstrates that some miRNA*s

proper SQN function (Iki et al., 2010; Earley et al.,

are quite abundant and enriched in AGO1 complexes

2011; Smith et al., 2009). ATP hydrolysis induces

and miRNA* guided mRNA cleavage products

dissociation of HSP90 and SQN from the complex,

are detected (Devers et al., 2011). In Arabidopsis

followed by unwinding and release of the miRNA*.

miR171a* was verified to be abundant and trigger the

Finally, a mature RISC that contains AGO1 and

silencing of SU (VAR) 3-9 HOMOLOG8 by associa-

the guide strand miRNA is formed (Fig. 1). Once

tion with the AGO1 complex possibly under tissue-

the mature RISC is formed, the miRNA can guide

specific control (Manavella et al., 2013). Meng et al.

the RISC to repress the expression of target genes

(2011) predicted all of the potential miRNA* target

(Voinnet, 2009).

pairs in rice and Arabidopsis based on degradome sequencing data. Their results suggested widespread

Subcellular organization of miRNA biogenesis

miRNA*-mediated gene regulation in plants, but little

The subcellular organization of miRNA biogenesis is

is known about the exact mechanism.

not very clear so far. Fang and Spector (2007) showed that DCL1 and HYL1 colocalize in discrete nuclear bodies, which are refered as nuclear dicing bodies

Conclusions

(D-bodies). Since several proteins essential for miRNA

In the decade since the first report of plant miRNAs,

processing and pri-miRNAs are also associated with

considerable advances have been made in our under-

D-bodies, they suggested that D-bodies are the nuclear

standing of the biogenesis of miRNAs in plants and

sites for the dicing reaction of DCL1 and protein-

a general picture of plant miRNA biogenesis has

protein interaction of the respective proteins (Fig. 1).

emerged. However, there are still some hazy parts. The

HEN1 and AGO1 localize both in the nucleus and the

nuclear export of miRNA in plants is not as clear as in

cytoplasm, and in nucleus a large nucleoplasmic signal

their metazoan counterparts, with both the transporter

is observed in additional to a low level localization

and transported molecules remaining unknown; the

to D-bodies (Fang and Spector, 2007). Therefore,

mechanisms of some processing factors are also not

the methylation of miRNA/miRNA* duplexes and

yet clear; the generation of functional miRNA* is

the loading of miRNA can be either in cytoplasm

waiting to be discovered.

or in nucleus, with the possibility in nucleoplasm.

　 Some key protein family (e.g., DCL and DRB)

The molecules that are transported through the

mem-bers that were originally identified as being

nuclear membrane and the proteins responsible for

involved in other small RNA pathways have been

the transporting are still kept unknown. It is of great

found to take part in miRNA processing and can

interest to further discover the detailed cellular basis of

cooperate or compete with their miRNA-producing

miRNA biogenesis.

relatives. The intertwined relationships among these processing proteins have revealed the diversity and

miRNA*

complexity of miRNA biogenesis in plants.

MiRNA* strand is originally considered to be non-

　Considering its complexity and the large number

functional and simply removed and degraded

of participating proteins, understanding the regulation

(Rajagopalan et al., 2006; Eamens et al., 2009).

of miRNA biogenesis, especially for spatially and

E-mail: [email protected]

·93·

Kong Wen-wen et al. Biogenesis of Plant MicroRNAs

temporally specific miRNAs, can be quite challenging. However, deep sequencing (Friedländer et al., 2008)

Bologna N G, Schapire AL, Palatnik J F. 2013. Processing of plant microRNA precursors. Brief Funct Genomics, 12(1): 37-45.

and tiling array (Liu et al., 2008) technologies have

Breakfield N W, Corcoran D L, Petricka J J, et al. 2012. High-resolution

allowed the examination of precursor, mature, and

experimental and computational profiling of tissue-specific known

intermediate miRNA molecules at an unprecedented

and novel miRNAs in Arabidopsis. Genome Res, 22(1): 163-76.

global level and provide unlimited potential for further study of plant miRNA.

Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, et al. 2008. Widespread translational inhibition by plant miRNAs and siRNAs. Science, 320(5880): 1185-90.

References

Brotman Y, Lisec J, Méret M, et al. 2012. Transcript and metabolite

Addo-Quaye C, Snyder J A, Park Y B, et al. 2009. Sliced microRNA

analysis of the Trichoderma induced systemic resistance response

targets and precise loop-first processing of MIR319 hairpins revealed

to Pseudomonas syringae in Arabidopsis thaliana. Microbiology,

by analysis of the Physcomitrella patens degradome. RNA, 15(12):

158(pt1): 139-46.

2112-21. Aukerman M J, Sakai H. 2003. Regulation of flowering time and floral organ identity by a MicroRNA and its APETALA2-like target genes. Plant Cell, 15(11): 2730-41. Bartel D P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116(2): 281-97. Baumberger N, Baulcombe D C. 2005. Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci USA, 102(33): 11928-33. Ben Amor B, Wirth S, Merchan F, et al. 2009. Novel long non-protein coding RNAs involved in Arabidopsis differentiation and stress responses. Genome Res, 19(1): 57-69.

Calderon-Villalobos L I, Kuhnle C, Dohmann E M, et al. 2005. The evolutionarily conserved TOUGH protein is required for proper development of Arabidopsis thaliana. Plant Cell, 17(9): 2473-85. Chen CZ, Li L, Lodish H F, et al. 2004. MicroRNAs modulate hematopoietic lineage differentiation. Science, 303(5654): 83-6. Chen X. 2005. MicroRNA biogenesis and function in plants. FEBS Lett, 579(26): 5923-31. Clarke JH, Tack D, Findlay K, et al. 1999. The SERRATE locus controls the formation of the early juvenile leaves and phase length in Arabidopsis. Plant J, 20(4): 493-501. Cui X, Xu S M, Mu D S, et al. 2009. Genomic analysis of rice microRNA promoters and clusters. Gene, 431(1-2): 61-6.

Ben Chaabane S, Liu R, Chinnusamy V, et al. 2013. STA1, an

Devers EA, Branscheid A, May P, et al. 2011. Stars and symbiosis:

Arabidopsis pre-mRNA processing factor 6 homolog, is a new player

microRNA- and microRNA*-mediated transcript cleavage involved

involved in miRNA biogenesis. Nucleic Acids Res, 41(3): 1984-97.

in arbuscular mycorrhizal symbiosis. Plant Physiol, 156(4):

Bernstein E, Caudy A A, Hammond S M, et al. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 409(6818): 295-296. Bezerra I C, Michaels S D, Schomburg F M, et al. 2004. Lesions in

1990-2010. Dong Z, Han M H, Fedoroff N. 2008. The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1. Proc Natl Acad Sci USA, 105(29): 9970-5.

the mRNA cap-binding gene ABA HYPERSENSITIVE 1 suppress

Eamens A L, Smith N A, Curtin S J, et al. 2009. The Arabidopsis

FRIGIDA-mediated delayed flowering in Arabidopsis. Plant J, 40(1):

thaliana double-stranded RNA binding protein DRB1 directs guide

112-119.

strand selection from microRNA duplexes. RNA, 15(12): 2219-35.

Bielewicz D, Kalak M, Kalyna M, et al. 2013. Introns of plant primiRNAs enhance miRNA biogenesis. EMBO Rep, 14(7): 622-8. Bollman K M, Aukerman M J, Park M Y, et al. 2003. HASTY, the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis. Development, 130(8): 1493-504. Bologna N G, Mateos J L, Bresso E G, et al. 2009. A loop-to-base processing mechanism underlies the biogenesis of plant microRNAs miR319 and miR159. EMBO J, 28(23): 3646-56.

Eamens A L, Wook Kim K, Waterhouse P M. 2012. DRB2, DRB3 and DRB5 function in a non-canonical microRNA pathway in Arabidopsis thaliana. Plant Signal Behav, 7(10): 1224-9. Earley K W, Poethig R S. 2011. Binding of the cyclophilin 40 ortholog SQUINT to Hsp90 protein is required for SQUINT function in Arabidopsis. J Biol Chem, 286(44): 38184-9. Fagard M, Dellagi A, Roux C, et al. 2007. Arabidopsis thaliana expresses multiple lines of defense to counterattack Erwinia

http: //publish.neau.edu.cn

·94·

Journal of Northeast Agricultural University (English Edition)

chrysanthemi. Mol Plant Microbe Interact, 20(7): 794-805. Fang Y, Spector D L. 2007. Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants. Curr Biol, 17(9): 818-823. Feng H, Zhang Q, Wang Q, et al. 2013. Target of tae-miR408, a

Vol. 21　No. 1　2014

and CBP80 are involved in processing primary MicroRNAs. Plant Cell Physiol, 49(11): 1634-1644. Kim Y J, Zheng B, Yu Y, et al. 2011. The role of Mediator in small and long noncoding RNA production in Arabidopsis thaliana. EMBO J, 30(5): 814-822.

chemocyanin-like protein gene (TaCLP1), plays positive roles in

Kurihara Y, Takashi Y, Watanabe Y. 2006. The interaction between

wheat response to high-salinity, heavy cupric stress and stripe rust.

DCL1 and HYL1 is important for efficient and precise processing of

Plant Mol Biol [Epub ahead of print].

pri-miRNA in plant microRNA biogenesis. RNA, 12(2): 206-212.

Friedländer M R, Chen W, Adamidi C, et al. 2008. Discovering

Lacombe S, Nagasaki H, Santi C, et al. 2008. Identification of pre-

microRNAs from deep sequencing data using miRDeep. Nat

cursor transcripts for 6 novel miRNAs expands the diversity on the

Biotechnol, 26(4): 407-415.

genomic organization and expression of miRNA genes in rice. BMC

Fukudome A, Kanaya A, Egami M, et al. 2011. Specific requirement of DRB4, a dsRNA-binding protein, for the in vitro dsRNA-cleaving activity of Arabidopsis Dicer-like 4. RNA, 17(4): 750-760. Gasciolli V, Mallory A C, Bartel D P, et al. 2005. Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Curr Biol, 15(16): 1494-1500. Ghildiyal M, Xu J, Seitz H, et al. 2010. Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway. RNA, 16(1): 43-56.

Plant Biol, 8(7): 123-142. Laubinger S, Sachsenberg T, Zeller G, et al. 2008. Dual roles of the nuclear cap-binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis thaliana. Proc Natl Acad Sci USA, 105(25): 8795-800. Lee Y, Ahn C, Han J, et al. 2003. The nuclear RNase III Drosha initiates microRNA processing. Nature, 425(6956): 415-419. Lee Y, Kim M, Han J, et al. 2004. MicroRNA genes are transcribed by RNA polymerase II. EMBO J, 23(20): 4051-4060.

Golden TA, Schauer S E, Lang J D, et al. 2002. SHORT INTEGU-

Li J, Yang Z, Yu B, et al. 2005. Methylation protects miRNAs and

MENTS1/SUSPENSOR1/CARPEL FACTORY, a dicer homolog,

siRNAs from a 3'-end uridylation activity in Arabidopsis. Curr Biol,

is a maternal effect gene required for embryo development in

15(16): 1501-1507.

Arabidopsis. Plant Physiol, 130(2): 808-822. Gregory B D, O'Malley R C, Lister R, et al. 2008. A link between RNA metabolism and silencing affecting Arabidopsis development. Dev Cell, 14(6): 854-866. Han M H, Goud S, Song L, et al. 2004. The Arabidopsis doublestranded RNA-binding protein HYL1 plays a role in microRNAmediated gene regulation. Proc Natl Acad Sci USA, 101(4): 1093-8. Hiraguri A, Itoh R, Kondo N, et al. 2005. Specific interactions between dicer-like proteins and HYL1/DRB-family dsRNA-binding proteins in Arabidopsis thaliana. Plant Mol Biol, 57(2): 173-188. Iki T, Yoshikawa M, Nishikiori M, et al. 2010. In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90. Mol Cell, 39(2): 282-291. Jacob Y, Mongkolsiriwatana C, Veley K M, et al. 2007. The nuclear pore protein AtTPR is required for RNA homeostasis, flowering time, and auxin signaling. Plant Physiol, 144(3): 1383-1390. Jones-Rhoades M W, Bartel D P, Bartel B. 2006. MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol, 57(10): 19-53. Kim S, Yang J Y, Xu J, et al. 2008. Two cap-binding proteins CBP20

E-mail: [email protected]

Liu B, Li P C, Li X, et al. 2005. Loss of function of OsDCL1 affects microRNA accumulation and causes developmental defects in rice. Plant Physiol, 139(1): 296-305. Liu C, Axtell M J, Fedoroff N V. 2012. The helicase and RNaseIIIa domains of Arabidopsis dicer-Like1 modulate catalytic parameters during microRNA biogenesis. Plant Physiol, 159(2): 748-758. Liu H H, Tian X, Li Y J, et al. 2008. Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana. RNA, 14(5): 836-843. Llave C, Xie Z, Kasschau K D, et al. 2002. Cleavage of scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science, 297(5589): 2053-2056. Lobbes D, Rallapalli G, Schmidt D D, et al. 2006. SERRATE: a new player on the plant microRNA scene. EMBO Rep, 7(10): 1052-1058. Lu C, Fedoroff N. 2000. A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin. Plant Cell, 12(12): 2351-2366. Lund E, Güttinger S, Calado A, et al. 2004. Nuclear export of microRNA precursors. Science, 303(5654): 95-98.

·95·

Kong Wen-wen et al. Biogenesis of Plant MicroRNAs Machida S, Chen HY, Adam Yuan Y. 2011. Molecular insights into

Park W, Li J, Song R, et al. 2002. CARPEL FACTORY, a dicer

miRNA processing by Arabidopsis thaliana SERRATE. Nucleic

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

Acids Res, 39(17): 7828-7836.

in Arabidopsis thaliana. Curr Biol, 12(17): 1484-1495.

Macrae I J, Zhou K, Li F, et al. 2006. Structural basis for doublestranded RNA processing by dicer. Science, 311(5758): 195-8. Mallory AC, Bouché N. 2008. MicroRNA-directed regulation: to cleave or not to cleave. Trends Plant Sci, 13(7): 359-367.

Pélissier T, Clavel M, Chaparro C, et al. 2011. Double-stranded RNA binding proteins DRB2 and DRB4 have an antagonistic impact on polymerase IV-dependent siRNA levels in Arabidopsis. RNA, 17(8): 1502-1510.

Manavella PA, Hagmann J, Ott F, et al. 2012. Fast-forward genetics

Prigge M J, Wagner D R. 2001. The Arabidopsis SERRATE gene

identifies plant CPL phosphatases as regulators of miRNA processing

encodes a zinc-finger protein required for normal shoot development.

factor HYL1. Cell, 151(4): 859-870.

Plant Cell, 13(6): 1263-1279.

Manavella PA, Koenig D, Rubio-Somoza I, et al. 2013. Tissue-specific

Rajagopalan R, Vaucheret H, Trejo J, et al. 2006. A diverse and

silencing of Arabidopsis SU(VAR)3-9 HOMOLOG8 by miR171a.

evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes

Plant Physiol, 161(2): 805-812.

Dev, 20(24): 3407-3425.

Margis R, Fusaro A F, Smith N A, et al. 2006. The evolution and diversification of Dicers in plants. FEBS Lett, 580: 2442-2450.

Reinhart B J, Weinstein E G, Rhoades M W, et al. 2002. MicroRNAs in plants. Genes Dev, 16(13): 1616-1626.

Mateos J L, Bologna N G, Chorostecki U, et al. 2010. Identification

Ren G, Xie M, Dou Y, et al. 2012. Regulation of miRNA abundance by

of microRNA processing determinants by random mutagenesis of

RNA binding protein TOUGH in Arabidopsis. Proc Natl Acad Sci

Arabidopsis MIR172a precursor. Curr Biol, 20(1): 49-54.

USA, 109(31): 12817-12821.

Matranga C, Tomari Y, Shin C, et al. 2005. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell, 123(4): 607-620. Meng Y, Shao C, Gou L, et al. 2011. Construction of microRNA- and microRNA*-mediated regulatory networks in plants. RNA Biol, 8(6): 1124-1148. Merchan F, Boualem A, Crespi M, et al. 2009. Plant polycistronic precursors containing non-homologous microRNAs target transcripts encoding functionally related proteins. Genome Biol, 10(12): R136. Nakazawa Y, Hiraguri A, Moriyama H, et al. 2007. The dsRNA-binding protein DRB4 interacts with the dicer-like protein DCL4 in vivo and functions in the trans-acting siRNA pathway. Plant Mol Biol, 63(6): 777-785. Naqvi AR, Sarwat M, Hasan S, et al. 2012. Biogenesis, functions and fate of plant microRNAs. J Cell Physiol, 227(9): 3163-3168. Nikovics K, Blein T, Peaucelle A, et al. 2006. The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis. Plant Cell, 18(11): 2929-2945. Ni Z, Hu Z, Jiang Q, et al. 2013. GmNFYA3, a target gene of miR169, is a positive regulator of plant tolerance to drought stress. Plant Mol Biol, 82(1-2): 113-129. Park MY, Wu G, Gonzalez-Sulser A, et al. 2005. Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci USA, 102(10): 3691-3696.

Rogers K, Chen X. 2013. microRNA Biogenesis and Turnover in Plants. Plant Cell [Epub ahead of print]. Smith MR, Willmann MR, Wu G, et al. 2009. Cyclophilin 40 is required for microRNA activity in Arabidopsis. Proc Natl Acad Sci USA, 106(13): 5424-5429. Song J J, Smith S K, Hannon G J, et al. 2004. Crystal structure of Argonaute and its implications for RISC slicer activity. Science, 305(5689): 1434-1437. Song L, Axtell M J, Fedoroff N V. 2010. RNA secondary structural determinants of miRNA precursor processing in Arabidopsis. Curr Biol, 20(1): 37-41. Sun G. 2012. MicroRNAs and their diverse functions in plants. Plant Mol Biol, 80(1): 17-36. Sunkar R, Chinnusamy V, Zhu J, et al. 2007. Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci, 12(7): 301-309. Szarzynska B, Sobkowiak L, Pant B D, et al. 2009. Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs. Nucleic Acids Res, 37(9): 3083-3093. Takanashi H, Ohnishi T, Mogi M, et al. 2011. DCL2 is highly expressed in the egg cell in both rice and Arabidopsis. Plant Signal Behav, 6(4): 604-606. Tomari Y, Matranga C, Haley B, et al. 2004. A protein sensor for siRNA asymmetry. Science, 306(5700): 1377-1380.

http: //publish.neau.edu.cn

·96·

Journal of Northeast Agricultural University (English Edition)

Vol. 21　No. 1　2014

Vazquez F, Blevins T, Ailhas J, et al. 2008. Evolution of Arabidopsis

Xie Z, Johansen L K, Gustafson A M, et al. 2004. Genetic and

MIR genes generates novel microRNA classes. Nucleic Acids Res,

functional diversification of small RNA pathways in plants. PLoS

36(20): 6429-6438.

Biol, 2(5): E104.

Vazquez F, Gasciolli V, Crété P, et al. 2004. The nuclear dsRNA

Yang SW, Chen HY, Yang J, et al. 2010. Structure of Arabidopsis

binding protein HYL1 is required for microRNA accumulation and

HYPONASTIC LEAVES1 and its molecular implications for

plant development, but not posttranscriptional transgene silencing.

miRNA processing. Structure, 18(5): 594-605.

Curr Biol, 14(4): 346-351. Vazquez F, Legrand S, Windels D. 2010. The biosynthetic pathways and biological scopes of plant small RNAs. Trends Plant Sci, 15(6): 337-345. Voinnet O. 2009. Origin, biogenesis, and activity of plant microRNAs. Cell, 136(4): 669-87. Wang L, Song X, Gu L, et al. 2013. NOT2 proteins promote polymerase II-dependent transcription and interact with multiple MicroRNA biogenesis factors in Arabidopsis. Plant Cell, 25(2): 715-727. Werner S, Wollmann H, Schneeberger K, et al. 2010. Structure determinants for accurate processing of miR172a in Arabidopsis thaliana. Curr Biol, 20(1): 42-48. Wang Y, Sun F, Cao H, et al. 2012. TamiR159 directed wheat TaGAMYB cleavage and its involvement in anther development and heat response. PLoS One, 7(11): e48445. Wu L, Zhang Q, Zhou H, et al. 2009. Rice MicroRNA effector complexes and targets. Plant Cell, 21(11): 3421-3435. Wu L, Zhou H, Zhang Q, et al. 2010. DNA methylation mediated by a microRNA pathway. Mol Cell, 38(3): 465-475. Wu X, Shi Y, Li J, et al. 2013. A role for the RNA-binding protein MOS2 in microRNA maturation in Arabidopsis. Cell Res, 23(5): 645-657. Xie Z, Allen E, Fahlgren N, et al. 2005. Expression of Arabidopsis MIRNA genes. Plant Physiol, 138(4): 2145-2154.

E-mail: [email protected]

Yang Z, Ebright YW, Yu B, et al. 2006. HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2' OH of the 3' terminal nucleotide. Nucleic Acids Res, 34(2): 667-675. Yu B, Bi L, Zheng B, et al. 2008. The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis. Proc Natl Acad Sci USA, 105(29): 10073-10078. Yu B, Yang Z, Li J, et al. 2005. Methylation as a crucial step in plant microRNA biogenesis. Science, 307(5711): 932-935. Zhang W, Gao S, Zhou X, et al. 2010. Multiple distinct small RNAs originate from the same microRNA precursors. Genome Biol, 11(8): R81. Zhang Y, Cheng Y T, Bi D, et al. 2005. MOS2, a protein containing G-patch and KOW motifs, is essential for innate immunity in Arabidopsis thaliana. Curr Biol, 15(21): 1936-1942. Zhan X, Wang B, Li H, et al. 2012. Arabidopsis proline-rich protein important for development and abiotic stress tolerance is involved in microRNA biogenesis. Proc Natl Acad Sci USA, 109(44): 1819818203. Zhao Y, Yu Y, Zhai J, et al. 2012. The Arabidopsis nucleotidyl transferase HESO1 uridylates unmethylated small RNAs to trigger their degradation. Curr Biol, 22(8): 689-694. Zhu Q H, Spriggs A, Matthew L, et al. 2008. A diverse set of microRNAs and microRNA-like small RNAs in developing rice grains. Genome Res, 18(9): 1456-1465.