Journal of Virological Methods 136 (2006) 217–223

A simplified method for cloning of short interfering RNAs fromBrassica juncea infected with Turnip mosaic potyvirus

and Turnip crinkle carmovirus

Thien Ho a,b, Denise Pallett a, Rachel Rusholme c, Tamas Dalmay c, Hui Wang a,∗a NERC/Centre for Ecology and Hydrology (CEH) Oxford, Mansfield Road, Oxford OX1 3SR, UK

b Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UKc School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK

Received 14 February 2006; received in revised form 15 May 2006; accepted 16 May 2006Available online 3 July 2006

Abstract

RNA silencing is a plant defense mechanism in which virus infected plants produce short interfering RNAs (siRNAs) derived from viral RNA,tbtwmws(o©

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hat attack the virus at the post-transcriptional level. In a previous study on Cymbidium ringspot tombusvirus (CymRSV) infection in Nicotianaenthamiana, siRNAs (determined by cloning and sequencing) predominantly originated from the sense (+) strand of the viral RNA, suggestinghat the majority of siRNAs are produced through the direct cleavage of the virus single strand (ss) RNA by the plant Dicer-like enzyme. To testhether this asymmetry in strand polarity is a generic rule for all plant viruses, siRNAs from Brassica juncea, either singly infected by Turniposaic potyvirus (TuMV, the family Potyviridae), or doubly infected with TuMV and Turnip crinkle carmovirus (TCV, the family Tombusviridae)ere investigated. A simplified siRNA cloning method was developed, using a single ligation reaction to attach both 5′ and 3′ adapters to the target

hort RNAs followed by one-step RT-PCR amplification. In the TCV infection, as for the CymRSV infection, siRNAs were produced predominantly97.6%) from the +ss RNA. However, for TuMV infections, siRNAs were derived from both strands (+/−, 58.1–41.9%), indicating the presencef alternative siRNA production mechanisms.

2006 Elsevier B.V. All rights reserved.

eywords: Short interfering RNA; siRNA cloning; Brassica juncea; Turnip mosaic potyvirus; Turnip crinkle carmovirus

. Introduction

RNA silencing is an ancient defense mechanism found inany eukaryotic organisms and triggered by the intracellular

resence of double stranded RNA (dsRNA). This leads to theomology-dependent degradation of dsRNA sequences to pro-ect the host genome against aberrant endogenous or exogenousNA sequences. This phenomenon was discovered by Fire et al.

1998) and termed RNA interference (RNAi) in animals, post-ranscriptional gene silencing (PTGS) in plants, and quelling inungi (Fire et al., 1998; Napoli et al., 1990; Romano and Macino,992; van der Krol et al., 1990a). RNA silencing involves theecognition and cleavage of dsRNA by the ribonuclease III

∗ Corresponding author. Tel.: +44 1865 281630; fax: +44 1865 281696.E-mail addresses: thien.ho@pembroke.oxford.ac.uk (T. Ho),

wpa@ceh.ac.uk (D. Pallett), r.rusholme@uea.ac.uk (R. Rusholme),.dalmay@uea.ac.uk (T. Dalmay), huw@ceh.ac.uk (H. Wang).

like enzyme, Dicer, into 19–25 nt duplexes, termed short inter-fering RNAs (siRNAs) (Bernstein et al., 2001; Hamilton andBaulcombe, 1999; Zamore et al., 2000). This siRNA duplexrepresents both polarities and has two nucleotide 3′ overhangswith 5′ phosphate and 3′ hydroxyl groups (Elbashir et al.,2001a,b). One strand of the siRNA duplex is then selected to beincorporated into an RNA-induced silencing complex (RISC)(Hammond et al., 2000). The single stranded siRNA guidesthe complex to search for and degrade perfectly complemen-tary RNA sequences, whilst mediating translation repressionof partially complementary ones (Aukerman and Sakai, 2003;Bernstein et al., 2001; Chen, 2004; Doench et al., 2003; Elbashiret al., 2001a; Hammond et al., 2000, 2001; Hutvagner andZamore, 2002; Zamore et al., 2000).

Several studies have successfully ‘switched off’ a wide rangeof genes, e.g. genes for leaf color development (van der Krol etal., 1990b), or even genes involved in the RNA silencing processitself (Westhof, 2004). RNA silencing can also be used against

166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2006.05.016

218 T. Ho et al. / Journal of Virological Methods 136 (2006) 217–223

viruses infecting plants and many other organisms (Angell andBaulcombe, 1997; Dougherty, 1994; Kumagai, 1995; Ruiz etal., 1998). However, there are still many problems which needto be addressed, such as the ‘off target effect’, where a designedsiRNA not only silences a desired gene, but also interferes withthe expression of other genes (Jackson et al., 2003), and non-systemic signalling (Hannon and Rossi, 2004).

In order to determine rules for artificial siRNA design,researchers have synthesized artificial siRNA molecules andchecked their silencing efficiency (Elbashir et al., 2001b;Harborth et al., 2003; Reynolds et al., 2004). To date, there areseveral computer programs available for artificial siRNA designproduced by different companies, e.g. MWG (http://www.mwg-biotech.com), Ambion (http://www.ambion.com), Invitrogen(http://www.invitrogen.com), GenScript (http://www.genscript.com), Promega (http://www.promega.com), and Chang Bio-science (http://www.changbioscience.com). However, as it hasnot been easy to test computer programs with any real siRNAprofile because a significant number of siRNA sequences areneeded, it is not known if these designs, based on artificialsiRNA experiments, reflect true in vivo siRNA. So far one reli-able approach for obtaining an siRNA profile for a virus infectedplant is by the cloning and sequencing of small RNAs isolatedfrom the total RNA (Molnar et al., 2005). An efficient siRNAcloning and sequencing method would accelerate the generationof siRNA profiles and thus be of significant benefit to currentri

gBaaArt5srcftaamtbteaatatgs–

(5′ adapter–3′ adapter) and multimers may be produced by RT-PCR. These trimers (∼65 bp) can subsequently be gel isolatedfrom the dimers (∼45 bp) and multimers (>80 bp), and purifiedfor cloning by conventional methods.

In this study, the possibility of using one-step adapter lig-ation for siRNA cloning was tested. This method enabled thesuccessful cloning of siRNAs from Brassica juncea cv. tendergreen (mustard) infected by the Turnip mosaic potyvirus (TuMV,the Potyviridae) and the Turnip crinkle carmovirus (TCV, theTombusviridae), demonstrating that the initial 5′ dephospho-rylation (Elbashir et al., 2001a; Tschudi et al., 2003) is notnecessary in siRNA cloning from virus infected plants. The newmethod significantly simplified the siRNA cloning technique sothat siRNA profiles may now be obtained with relative ease.Using the new method, siRNAs of the Tombusviridae viruses(TCV) were found to be produced predominantly (97.6%) fromthe positive strand of viral RNA (Molnar et al., 2005). However,the siRNA species of the potyvirus (TuMV), was derived fromboth strands (positive:negative = 58.1%:41.9%) implying that arange of mechanisms of siRNA production may exist in virusinfected plants.

2. Materials and methods

2.1. Virus infection

clDRpgbvsw(

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esearch on the mechanisms of siRNA biosynthesis in virusnfected organisms.

There are several methods for siRNA cloning. Two distin-uishable approaches for siRNA cloning are described in Fig. 1.oth require RNA or DNA ligation steps to attach a pair of 5′nd 3′ adapters to the siRNA molecules for subsequent PCRmplification (Elbashir et al., 2001a; Tschudi et al., 2003).fter short RNA isolation, the cloning begins with dephospho-

ylation of the short RNA 5′-end followed by RNA ligationo the 3′ adapter. In one method (Elbashir et al., 2001a), the′-end of small RNAs is re-phosphorylated, thus enabling sub-equent RNA ligation to the 5′ adapter. After gel purification,everse transcription followed by PCR amplification is thenarried out to obtain double stranded DNA (dsDNA) productor cloning. In the alternative method (Tschudi et al., 2003),he reverse transcription is performed immediately after the 3′dapter ligation, and the resulting cDNA is ligated to the 5′dapter to enable subsequent PCR amplification. The secondethod bypasses the 5′-rephosphorylation reaction (and at least

wo RNA extraction/precipitation steps), so it may achieve aetter yield in less time than the first method. Exclusion ofhe 5′-dephosphorylation procedure may have been used. How-ver, in theory, adapters need to be ligated in two separate stepss it is unlikely that a single step ligation (attaching both 5′nd 3′ adapters to the target RNA molecule in a single reac-ion) could work satisfactorily. It would be expected that thedapters would ligate to each other to form dimers while thearget small RNAs would form multimers. However, as the tar-et RNA molecules are small in size (19–25 nt) and similar inize to the adapters, a reasonable amount of trimers of 5′ adaptersmall RNA – 3′ adapter, together with a mixture of dimers

B. juncea (Mustard cv. tendergreen) sown in universal extraompost (Levington Horticulture Ltd.) was manually inocu-ated, 2 wk after sowing, with TuMV (GBR98, provided byr. John Walsh, HRI Warwick, UK) and TCV (UK isolate,S2), or TuMV only as described in Thurston et al. (2001). Thelants were maintained in a temperature controlled, insect prooflasshouse and were serologically tested (ELISA) as describedy Thurston et al. (2001) to confirm systemic infections of bothiruses at 3 wk after inoculation. The full length TuMV genomeequence was provided by Dr. John Walsh, HRI Warwick, UK,hile the sequence of the TCV isolate is available in GenBank

AY312063).

.2. Small RNA isolation

.2.1. Total RNA isolationShortly after infection had been confirmed by ELISA testing,

oung leaves with viral symptoms were sampled for RNA isola-ion. Total RNA was extracted by homogenizing 2 g of infectedeaf tissue in liquid nitrogen and 40 ml pre-chilled Tri ReagentSigma–Aldrich) at room temperature (RT). The homogenateas centrifuged at 12,000 × g for 10 min at 4 ◦C and the super-atant was transferred to a fresh centrifuge tube. The sample waseft to settle for 5 min at RT, 8 ml of chloroform was then addednd the mixture was shaken vigorously for 15 s. The mixtureas left to stand for 15 min at RT and centrifuged at 12,000 × g

or 15 min at 4 ◦C. The colorless upper aqueous phase wasransferred to a fresh centrifuge tube and mixed with 20 ml iso-ropanol. The tube was briefly vortexed, then allowed to standor 10 min at RT and centrifuged at 12,000 × g for 10 min at◦C. After removal of the supernatant, the RNA precipitate was

T. Ho et al. / Journal of Virological Methods 136 (2006) 217–223 219

Fig. 1. Comparison of three different strategies for PCR amplification of siRNA from total RNA. The new strategy differs from these by the ligation of 5′ and 3′adapters in one reaction.

immediately dissolved in 150 �l of RNase-free water. The RNAsolution was measured for quality and quantity by calculatingthe ratio of OD260/OD280 and 300 �g of total RNA was usedfor small RNA isolation.

2.2.2. Isolation of 19–25 nt small RNA from total RNAThe total RNA (300 �g) was diluted to 150 �l in RNA sample

buffer (5 mM EDTA, 0.1% bromophenol blue, 95% formamide)and denatured at 90 ◦C for 30 s before being loaded onto a 15%polyacrylamide gel [4.2 g urea, 3 ml RNase-free water, 3.74 ml40% polyacrylamide, 0.5 ml 5× TBE (Tris Borate EDTA buffer,MP Biomedicals Inc.), 100 �l 10% ammonium persulphate, 5 �lTEMED]. The gel was run in 1× TBE for 2–3 h at 120 V untilthe bromophenol blue dye reached the bottom of the gel, with amix of 19 and 24 nt synthesized single stranded RNAs as molec-ular markers. The gel was stained in ethidium bromide solution(200 ml 1× TBE, 10 �l 10 mg/ml ethidium bromide) for 5 minat RT, and observed in a UV transilluminator. The region con-taining the 19–25 nt RNA band was excised and RNA eluted in300–500 �l of 0.3 M NaCl in a 1.5 ml eppendorf tube at 4 ◦Covernight. The small RNA was ethanol precipitated and dis-solved in 7 �l RNase-free water.

2.3. Amplification of small RNA sequences

2

p

5′ adapter (5′-N6accctcttggcacccactAAA-3′: uppercase: RNA;lowercase: DNA; N6: amino 6-carbon spacer, for blocking the 5′end), 1 �l 3′ adapter (5′-UUUaccaggcacccagcaatgN3-3′: upper-case: RNA; lowercase: DNA; N3′: amino modifier, for blockingthe 3′ end), 2 �l 10×RNA ligation buffer, 6 �l 50% dimethyl sul-foxide (DMSO), and 2 �l bovine serum albumin (BSA, 0.1%).All the components of the reaction were mixed and given aheat shock at 90 ◦C for 30 s and immediately chilled on icefor 20 s. About 1 �l of T4 RNA ligase (Amersham) was added,and the mixture was incubated at 37 ◦C for 2 h. After 2 h, 37 �lRNase-free water and 3 �l 5 M NaCl were added to the reaction,followed by one phenol:chloroform:isoamyl alcohol extraction.The adapter-linked small RNA product was ethanol precipitatedand dissolved in 5 �l RNase-free water.

2.3.2. RT-PCR amplification of the adapter-linked shortRNA

OneStep RT-PCR Kit (Qiagen) was used for reverse tran-scription and small scale PCR amplification. About 5 �l ofthe adapter-linked RNA was given a heat shock at 90 ◦Cfor 30 s. The RT-PCR reaction was set up as follows: 5 �lRNA sample, 29 �l RNase-free water, 10 �l 5× QiagenOneStep RT-PCR buffer, 2 �l dNTP mix (5 mM each), 1 �lBG3a (forward primer: 5′-ACCCTCTTGGCACCCACTAAA-3′, 100 pmol, underline indicates BanI site), 1 �l BG4a (reversepuE

.3.1. Ligation of the 5′ and 3′ adapters to short RNAThe reaction mix was prepared as follows: 7 �l small RNA

reparation (from the step described in Section 2.2.2), 1 �l

rimer: 5′-CATTGCTGGGTGCCTGGTAAA-3′, 100 pmol,nderline indicates BanI site), and 2 �l Qiagen OneStep RT-PCRnzyme Mix. The reaction was performed at 50 ◦C for 30 min,

220 T. Ho et al. / Journal of Virological Methods 136 (2006) 217–223

95 ◦C for 15 min, followed by 30 cycles of 94 ◦C for 1 min, 50 ◦Cfor 1 min, 72 ◦C for 1 min and a final extension period of 72 ◦Cfor 10 min. The PCR product was run on a 3.5% agarose gel withHyperladder V (Bioline) as molecular weight marker. The band,containing the single short RNA insert, at ∼65 bp was excised,electro-eluted in a molecular porous membrane-tubing (Spec-trum Laboratories Inc., Spectra/Por Membrane MWCO: 3500)for 1 h at 110 V and ethanol precipitated, it was then dissolvedin 5 �l distilled water.

2.3.3. Secondary large scale PCR amplification of the65 bp product

The secondary PCR reaction was set up essentially asdescribed by Elbashir et al. (2001a) and Tschudi et al. (2003).The mass amplified 65 bp PCR product was gel purified asdescribed above and dissolved in 87 �l distilled water.

2.4. Concatemerization of the PCR products

2.4.1. BanI digestionThe digestion reaction was set up as follows: 87 �l of the

∼65 bp PCR product was digested by 3 �l BanI (New EnglandBiolabs, 20,000 U/ml) in a 100 �l reaction at 37 ◦C for 18 h. Theresulting ∼40 bp DNA fragment was gel purified as describedabove and dissolved in 87 �l of distilled water.

2

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3

3

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Fig. 2. Polyacrylamide electrophoresis of total RNA from a TuMV infectedplant. The short RNA band is visible within 19–24 nt region.

3.2. Small RNA isolation

A small RNA band was clearly visible from 300 �g of totalRNA extracted from TuMV (Fig. 2), and TuMV + TCV infectedB. juncea cv. tendergreen. The absence of smears with lowmolecular weights indicated that the RNA was good quality,therefore the band flanked by the molecular markers of 19 and24 nt was excised for cloning.

3.3. Amplification of small RNA sequences

After the first round amplification by one-step RT-PCR (Sec-tion 2.3.2), two bands were clearly separated in the 3.5% agarosegel (Fig. 3A). The upper band had a molecular weight ∼65 bpsuggesting that this was dsDNA amplified from the RNA tem-plates of 5′ adapter – short RNA – 3′adapter trimer. The lower

FaHTiamplification of the 65 bp product. M: Hyperladder V molecular weight marker;lane 1: PCR product of TuMV infected sample.

.4.2. Concatemerization of the BanI digested productTo increase sequencing efficiency, the BanI digested DNA

ragments were concatemerized by T4DNA ligase (NEB,00,000 U/ml) in a 100 �l reaction at RT overnight. The con-atemers were run on a TBE 2.5% agarose gel. A DNA smearf 400–1000 bp was excised and extracted using Qiagen Gelxtraction kit and dissolved in 100 �l of distilled water.

.5. Cloning the DNA concatemers to pGEM-T vector anddentification of siRNA

The DNA concatemers were treated with REDTaq DNA poly-erase (Sigma) in a 500 �l reaction at 72 ◦C for 30 min, and the

nd-filled DNA was purified using the Qiagen PCR Purificationit. The DNA concatemers were then cloned into the pGEM-plasmid using the pGEMT-Easy system (Promega). Positive

lones were selected and sequenced using T7 and SP6 primers.BLASTN search using BioEdit program (Hall, 1999) was

erformed against the TuMV and TCV genome sequences todentify siRNAs with 100% sequence homology. The polarityf the siRNA was also determined.

. Results

.1. Virus infection

ELISA tests (Thurston et al., 2001) confirmed that all plantssed in this study were systemically infected with TuMV and/orCV.

ig. 3. Amplification of TuMV siRNAs. (A) RT-PCR one-step amplification ofdapter-linked siRNAs. The product siRNA insert was approximately 65 bp. M:yperladder V molecular weight marker; lane 1: one-step RT-PCR products ofuMV infected short RNA; lane 2: one-step RT-PCR products of TuMV + TCV

nfected sample; lane 3: negative control (adapters only). (B) Secondary PCR

T. Ho et al. / Journal of Virological Methods 136 (2006) 217–223 221

Fig. 4. Concatemerization of the PCR products. (A) BanI digested PCR productto reveal the sticky ends. Lane 1: undigested DNA product (approx. 60–65 bp);lane 2: BanI digested DNA (approx. 40–45 bp) and M: Hyperladder V molecularweight marker. (B) Concatermerization of the PCR products. There was a smearbetween 100 and 1000 bp region indicating successful concatemerization. Theregion between 400 and 1000 bp was excised for cloning and sequencing. Lane1: BanI digested DNA (monomer); M: Hyperladder IV molecular weight markerand lane 2: DNA concatemers.

band had a molecular weight of ∼45 bp suggesting that it wasamplified from the 5′ adapter–3′ adapter dimer. When the upperband was gel purified and amplified by the second PCR (Section2.3.3), it produced a single DNA band of ∼65 bp (Fig. 3B), indi-cating that the dimers (Fig. 3A) had been successfully removed.

3.4. Concatemerization of the PCR product

The BanI digestion (Section 2.4.1) reduced the molecularweight of the ∼65 bp PCR product to ∼40 bp (Fig. 4A) indicat-ing complete digestions of the BanI sites located at the 5′ and3′ adapters, respectively. The digested PCR product, with theBanI overhangs at both ends, was concatemerized successfully,resulting in a DNA smear which ranged from 100 to 1000 bp(Fig. 4B). The fraction between 400 and 1000 bp was excisedand purified for cloning and sequencing.

3.5. siRNA of TuMV and TCV

The efficiency of cloning the concatemers was high. Forthe doubly infected plant sample, 96 positive bacterial clones

Table 1Origin of polarities of siRNA from singly (TuMV) or doubly (TuMV + TCV)infected Brassica juncea cv. tendergreen

Single infection, TuMV Double infection

TuMV TCV

siRNAa 92 31 42Sense siRNA 59 (64.1%) 18 (58.1%) 41 (97.6%)Anti-sense siRNA 33 (35.9%) 13 (41.9%) 1 (2.4%)

a siRNAs were identified by 100% match to the virus genome sequences.

(containing small RNA inserts) were randomly selected andsequenced. In total, 523 small RNA sequences were obtained. Alocal BLASTN search was performed against the viral genomes.Both 31 TuMV and 42 TCV siRNA sequences (16–24 nt inlength) were identified with 100% homology to the known viralsequences. Interestingly, from the same PCR product, (Fig. 3B)obtained from plants doubly infected with TuMV and TCV,siRNAs were derived for both viruses, from different strand ori-gins (Table 1). The siRNAs were produced from both the sense(58.1%) and antisense strands (41.9%) of TuMV, while in TCV asignificantly greater proportion of siRNAs were produced fromthe sense strand (97.6%) (χ2

[1] = 18.003, P < 0.001).In plant infected with only TuMV, 50 bacterial clones were

sequenced to generate 190 small RNA sequences. In this library,96 TuMV siRNAs were identified which were also producedfrom both strands, in similar proportions to those for in thedoubly infected plant (χ2

[1] = 0.364, P = 0.546) (Table 1). Thissuggests that the production of TuMV siRNA is not significantlyaltered by co-infection with TCV. The siRNAs were 16–24 nt inlength and the majorities had the size of 21–22 nt (Fig. 5), simi-lar to that of CymRSV (Molnar et al., 2005) and suggesting localsilencing activities (Hamilton et al., 2002). Fig. 5 also showedthat the new procedure did not generate a size bias of smallerRNAs, as in previous methods (Fig. 1) (Elbashir et al., 2001a;Tschudi et al., 2003).

Adapter–adapter ligation without small RNA insert wasiNedb

Fidt

ndeed observed, but the percentage was extremely low (<1%).early 100% of ∼40mers contained small RNAs, indicating

fficient gel isolation. TuMV siRNA species were repeatedlyetected at particular genome locations in both single and dou-ly infected samples, suggesting existence of siRNA hotspots

ig. 5. Size distribution of siRNAs. Size distribution of the TuMV siRNAsdentified (Table 1) from TuMV single infection (filled bars) and TuMV/TCVouble infection (open bars) are shown together with that of TCV siRNAs fromhe TuMV/TCV double infection (open bars with dashed line).

222 T. Ho et al. / Journal of Virological Methods 136 (2006) 217–223

for TuMV as previously reported for CymRSV (Molnar et al.,2005). These hotspot siRNA species were not all identical butmany were overlapping, indicating that they are derived fromgenuine hotspots and not generated by PCR bias.

4. Discussion

In CymRSV infected N. benthamiana, siRNAs are producedpredominantly from the sense viral RNA. This siRNA asym-metry in strand polarity suggests that CymRSV siRNAs maybe generated mainly from the highly folded regions of singlestrand sense (ss+) RNA, where long ss+ RNA forms imper-fectly paired duplexes, thus triggering the cleavage by Dicer-like protein(s) (Molnar et al., 2005). As with CymRSV, bothTuMV and TCV are subjected to host PTGS whilst infectingthe host plants (Xie et al., 2004). In this study, strong CymRSV(Tombusvirus, Tombusviridae) siRNA asymmetry observed ininfected N. benthamiana also occurred in B. juncea, as a resultof TCV (Carmovirus, Tombusviridae) infection. This supportsthe idea that for some plant viruses, particularly members ofthe Tombusviridae, the ss+ RNA is the predominant source ofsiRNA. However, although significance of a slight bias ver-sus random +/− strand polarity was not tested, TuMV siRNAs(Table 1) showed that the antisense strand RNA also significantlycontributed to siRNA production. A significant proportion ofT(ipivs

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Acknowledgements

The authors are grateful to Mrs. Delia McCall for the mainte-nance and propagation of plants used in this study. Thien Ho isa recipient of a postgraduate scholarship sponsored by the Min-istry of Education and Training, Vietnam. This work is supportedby CEH Biodiversity Programme, and NERC (UK) researchgrants NER/T/S/2002/902 and NER/A/S/2003/00548.

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