A pathogenic non-coding RNA induces changes in dynamic DNA

A pathogenic non-coding RNA induces changes indynamic DNA methylation of ribosomal RNA genesin host plantsGerman Martinez Mayte Castellano Maria Tortosa Vicente Pallas and Gustavo Gomez

Instituto de Biologıa Molecular y Celular de Plantas (IBMCP) Consejo Superior de Investigaciones Cientıficas(CSIC)-UPV CPI Edificio 8 E Avenida de los Naranjos sn 46022 Valencia Spain

Received July 17 2013 Revised September 20 2013 Accepted October 1 2013

ABSTRACT

Viroids are plant-pathogenic non-coding RNAs ableto interfere with as yet poorly known host-regula-tory pathways and to cause alterations recognizedas diseases The way in which these RNAs coercethe host to express symptoms remains to be totallydeciphered In recent years diverse studies haveproposed a close interplay between viroid-inducedpathogenesis and RNA silencing supporting thebelief that viroid-derived small RNAs mediate thepost-transcriptional cleavage of endogenousmRNAs by acting as elicitors of symptoms expres-sion Although the evidence supporting the role ofviroid-derived small RNAs in pathogenesis is robustthe possibility that this phenomenon can be a morecomplex process also involving viroid-inducedalterations in plant gene expression at transcrip-tional levels has been considered Here we showthat plants infected with the lsquoHop stunt viroidrsquoaccumulate high levels of sRNAs derived from ribo-somal transcripts This effect was correlated with anincrease in the transcription of ribosomal RNA(rRNA) precursors during infection We observedthat the transcriptional reactivation of rRNA genescorrelates with a modification of DNA methylation intheir promoter region and revealed that somerRNA genes are demethylated and transcriptionallyreactivated during infection This study reports apreviously unknown mechanism associatedwith viroid (or any other pathogenic RNA) infectionin plants providing new insights into aspects ofhost alterations induced by the viroid infectiouscycle

INTRODUCTION

Impelled by their need to optimize a humble (250ndash400 nt)non-protein-coding genome to guarantee their infectiouscycle viroids have evolved into versatile molecular entitiescapable of interacting with the host-cell machinery atdiverse functional levels (1) In some cases this crosstalkcan affect key host-regulatory pathways and cause pheno-typic alterations recognized as plant diseases (23) Hostfactors andor mechanisms associated with basic aspectsof the life cycle of viroids such as sub-cellular compart-mentalization (45) replication (6ndash8) and movement(9ndash12) have been thoroughly studied in the past years togenerate a relatively clear picture of the plantndashviroid inter-action (13) However the way in which these tiny non-coding RNAs (ncRNAs) subvert the plant cell machineryby coercing the host to express symptoms remains aconundrum (3)As viroids lack mRNA activity it was initially assumed

that viroid-induced pathogenesis must result from a directinteraction between their genome and host factors(proteins or nucleic acids) Based on this notion earlypathogenesis models envisioned the plant-symptomexpression as a consequence of alterations in the endogen-ous RNA metabolism induced by base pair interactionsbetween viroid and host ncRNAs like U1 (14) or 7S (15)The identification of sequencestructural elements in theviroid genome (pathogenicity determinants) (1617)supports the notion that these specific viroid domainsmay interact with yet-to-be-identified host factorsinterfering in their physiological role and consequentlyincite disease (13) In recent years and enhanced by theadvent of RNA silencing diverse studies have providedevidence for the existence of close interplay betweenviroid-induced pathogenesis and this small RNA(sRNA)-dependent regulatory mechanism The idea thatviroid-derived sRNAs (vd-sRNAs) can mediate the

To whom correspondence should be addressed Tel +34 963877874 Fax +34 963877859 Email ggomezibmcpupvesPresent addressGerman Martinez Center for RNA Biology The Ohio State University Columbus OH 43210 USA

Published online 30 October 2013 Nucleic Acids Research 2014 Vol 42 No 3 1553ndash1562doi101093nargkt968

The Author(s) 2013 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby30) whichpermits unrestricted reuse distribution and reproduction in any medium provided the original work is properly cited

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post-transcriptional cleavage of endogenous mRNAsacting as elicitors of symptoms expression (18ndash20) wasreinforced by the observation that the expression of hostsymptoms is associated with post-transcriptional RNAsilencing in representative members of both Pospiviroidae(21) and Avsunviroidae (22) families Despite strongevidence supporting the role of vd-sRNAs as inductorsof host symptoms the possibility that this phenomenoncan be a more complex process also involving viroid-induced alterations in plant gene expression at transcrip-tional levels cannot be ruled out (313)Genome activity at the transcriptional level in plants is

regulated by epigenetic modifications of DNA andhistones Cytosine DNA methylation in plants is a stableepigenetic mark basic for the maintenance of genomestability including regulation of coding and non-codingregions (23) Methylation takes place in three differentsequence contexts CG and CHG (symmetric) andCHH (asymmetric where H refers to A T or C) (24)De novo DNA methylation is regulated by DOMAINSREARRANGED METHYLTRANSFERASE 2(DRM2) with three different pathways being involvedin its maintenance DNA METHYLTRANSFERASE 1(MET1) and DNA CHROMOMETHYLASE3 (CMT3)respectively maintain CG and CHG methylation throughDNA replication whereas CHH methylation requires denovo continuous maintenance by DRM2 (2425) sRNAsare important in de novo establishment and reinforcementof stable DNA methylation because they can target hom-ologous DNA sequences and induce cytosine DNAmethylation in all three contexts A specific RNA silencingpathway termed RNA-directed DNA methylation(RdDM) mediates the biosynthesis of these epigeneticallyactive sRNAs (26) In the ArabidopsisndashRdDM pathwaysingle-strand transcripts originated by RNA polymeraseIV (PolIV) are used by RNA-dependent RNA polymerase2 (RDR2) as substrates to generate dsRNAs which aresubsequently processed by Dicer-like 3 (DCL3) into23ndash24 nt siRNAs (25) These siRNAs guide Argonaute(AGO4AGO6) proteins (likely through an siRNA-nascent PolV transcript base pairing mechanism) totarget genomic regions (27) Next DRM2 is recruited todirect both symmetric and asymmetric de novo methyla-tion Conversely Arabidopsis encodes a family of DNAglycosylaseslyases that mediate the active demethylationof methylcytosines (28) Recently an alternative RDR62122-nt siRNA-dependent pathway has been describedto mediate the initiation of de novo DNA methylation oftransposable elements (29)The first evidence linking viroid infection and transcrip-

tional regulation in plants was provided by the demonstra-tion that Potato spindle tuber viroid (PSTVd) replicationdirects de novo methylation of cognate DNA sequences inviroid-expressing transgenic tobacco plants (30)Furthermore other studies have shown that diverse hostgenes exhibit a transcriptional alteration during PSTVd(3132) Citrus viroid III (33) Peach latent mosaic viroid(34) and Citrus exocortis viroid (35) infections Althoughall this experimental evidence may support the idea thatviroid infection incites alterations in plant gene expressionat transcriptional levels (13) viroid-induced methylation

of the host gene(s) or disruption of methylation pathways(which may eventually result in symptom expression) hasnot yet been reported in natural infections (3)

To address this issue we first analyzed the alteration inthe levels of endogenous sRNAs associated with lsquoHopstunt viroidrsquo (HSVd) infection in cucumber (Cucumissativus) plants Our results indicate that HSVd-infectedplants differentially accumulate high levels of thesRNAs derived from ribosomal transcripts (rb-sRNAs)associated with an increasing accumulation of ribosomalRNA precursors(pre-rRNAs) during infection Finally weobserved that deregulation in rRNA transcriptionalactivity correlates with a dynamic modification of theDNA methylation level during the infection process toprovide unprecedented insight into the potential endogen-ous regulatory pathways affected during the viroid lifecycle in infected plants

MATERIALS AND METHODS

Plant material

Cucumber (C sativus cv Suyo) plants were agroinoculatedwith Agrobacterium tumefaciens strain C58C1 transformedwith a binary pMOG800 vector carrying a head-to-tailinfectious dimeric HSVd cDNA (Y09352) (36) as previ-ously described (21) (Supplementary Materials) Mock-inoculated cucumber plants were used as a negativecontrol Plants were maintained in growing chambersand were analyzed at 10 (T1) 20 (T2) and 30 (T3) dayspost-infiltration (dpi) (Supplementary Figure S4A)

sRNA library information

The sRNA sequences used in this work were obtained froma library generated by starting from an sRNA populationrecovered from leaves and phloem exudates of healthy andHSVd-infected cucumber (C sativus) plants and sequencedby 454 Life Science Technology (Lifesequencing BranfordCT USA wwwlifesequencingcom) [(37) - NCBISRAaccession code SRP001408] The vd-sRNAs recoveredfrom infected plants were filtered out from this analysis

RNA isolation

Total RNA was extracted from the leaves (01 g) of dif-ferent infected and healthy cucumber plants using the TRIreagent (SIGMA St Louis MO USA) according to themanufacturerrsquos instructions The low-molecular-weightRNA (lt200 nt) fraction was enriched from totalRNA using MIRACLE (miRNA isolation KitSTRATAGENE) according to the manufacturerrsquos instruc-tions (Supplementary Materials)

Northern blot assays

Total RNA was analyzed by electrophoresis underdenaturing conditions in 1 agarose gels with 025Tris-Borate-EDTA (TBE) and 8 M urea (38) The RNAwas blotted on nylon membranes (ROCHE DiagnosticsGmbH Mannheim Germany) and was hybridized aspreviously described (11) Approximately 25 mg oflow-molecular-weight RNA was loaded onto 20

1554 Nucleic Acids Research 2014 Vol 42 No 3

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polyacrylamide gels with 025 TBE and 8M ureaRNA was transferred to a nylon membrane(ROCHE Diagnostics GmbH Mannheim Germany)Hybridization was performed as previously described (39)

Bisulfite conversion and sequencing

Total genomic DNA was extracted from the leaves(01 g) of different infected and healthy cucumberplants (Supplementary Figure S7A and B) (40) Bisulfitetreatment was performed using the EpiTec Bisulfite kit(Qiagen) (Supplementary Material) Modified DNA wasamplified by polymerase chain reaction (PCR) and wascloned (Supplementary Figure S7C) Between 13 and 24clones were sequenced for each analyzed time in both theinfected and mock-treated plants

RESULTS

rRNA-derived sRNAs are highly recovered fromHSVd-infected plants

To evaluate whether viroid infection induces alterations inthe general profiles of host endogenous sRNAs wecompared the size distribution of the total reads recoveredfrom HSVd-infected plants with the standard sRNA levelspreviously described for cucumber (41) As observed inFigure 1A (left) 21-nt sRNAs were the dominant sizeclass in infected plants whereas lengths of 22 and 24 ntwere the most abundant in non-inoculated plants The 23-and 25-nt-sized classes showed similar accumulation levelsin both the control and HSVd-infected sRNAs popula-tions This scenario was in general reproduced (exceptfor the 22-nt reads) when the unique sequences recoveredfrom both datasets were analyzed (Figure 1A right)indicating that HSVd infection is associated withchanges in the endogenous sRNA population levelsbeing the 21-and 24-nt species up- and downregulated ininfected plants respectively

When plant endogenous sRNAs recovered frominfected and healthy plants were analyzed by pairwisealignment against the cucumber transcript database weobserved that the sRNAs derived from 45S ribosomalRNA were the most differentially accumulated populationof sRNAs in viroid-infected plants (Figure 1B) The accu-mulation of 45S rRNA-derived sRNAs (rb-sRNAs) was atleast 4-fold higher in infected (274) compared withcontrol (68) plants This difference slightly increased(242 and 49 respectively) when considering onlysRNAs with expected DCL canonical sizes ranging from21 to 24 nt (Figure 1C) In contrast negligible differenceswere found when considering the levels of the unexpectedsize (lt21 to gt24 nt in length) reads (Figure 1D)Consistent with the global sRNA population(Figure 1A) the rb-sRNAs of 21 and 24 nt wererespectively up- and down recovered from the HSVd-infected dataset in comparison with control cucumberreads (Figure 1E) To examine the distribution of the rb-sRNA set all the potentially DCL-processed sequences(21ndash24 nt) recovered from the healthy and HSVd-infected cucumber plants were mapped on the 45SrRNA transcriptional unit (Figure 2A) Despite both

sRNA populations displaying a relatively heterogeneousdistribution pattern along the rRNA sequence(Figure 2B) the total number of sequences matchingthroughout the rRNA gene significantly increased ininfected plants as compared with the reads recoveredfrom mock-inoculated cucumbers (Figure 2C parametrict-test arithmetic means of 376 and 94 for infected andmock-inoculated plants respectively Plt 22 1016)Moreover we noticed two considerable differencesbetween rb-sRNA profiles in both analyzed samplesFirst in the infected plants the differentially recoveredrb-sRNAs are predominantly mapping to two homolo-gous regions of 300 nt located in the intergenic spacer(IGS) region and the 30-end of the 25s rRNA(Figure 2B) The northern blot analyses using a probecorresponding to this specific IGS region revealed a re-markable accumulation of rb-sRNAs in the infectedplants (Figure 2D) Second a major proportion of rb-sRNAs complementary to the rRNA transcripts fromboth polarities was recovered from the HSVd-infectedplants (Figure 2E and F Supplementary Figure S1) sug-gesting that the over-accumulation of rb-sRNAs observedin infected plants might originate from the rRNA-deriveddouble-stranded RNAs processed in sRNAsInterestingly the accumulation of sRNAs derived from

5S rRNA transcripts (5S sRNAs) was also increased inHSVd-infected (017) compared with control (006)plants (Supplementary Figure S2A) Consistent with ob-servations in the 25S sRNA population the 5S sRNAs of21 and 24 nt were up- and downregulated in the HSVd-infected plants respectively and a major proportion ofsRNAs complementary to 5S transcripts was recoveredfrom the infected cucumber plants (SupplementaryFigure S2B and C) When the 5S sRNAs were mappedonto their transcriptional unity we observed that in theinfected plants the 5S sRNAs are heterogeneouslydistributed along the 5S rRNA sequence (SupplementaryFigure S2D) However the 5S sRNAs recovered frommock control disproportionately (72 of the analyzedreads) match to a region adjacent to the transcriptionstart site Importantly the totality of these sRNAs was24 nt long All together these results support the beliefthat in cucumber HSVd infection is associated with adrastic alteration of rRNA-derived sRNA processingmainly characterized by an increase in the accumulationof 21-nt-long sRNAs (potential products of the processingof ds-rRNAs) and a significant decrease of ribosomal-specific 24-nt sRNAs (assumed to be involved in mainten-ance of methylation status)

rRNA precursors accumulate differentially during viroidinfection

In Arabidopsis the 45S rRNA genes (Figure 2A) aretandemly arrayed by hundreds at the chromosomal lociknown as nucleolus organizers (42) RNA polymerase I(PolI) transcribes primary transcripts (called pre-rRNAs)that are then extensively processed into 18S 58S and 25SrRNAs by the sequential deletion of external and internaltranscribed spacers (ETS and ITS respectively) (43) EachrRNA gene is separated from its adjacent one by an IGS

Nucleic Acids Research 2014 Vol 42 No 3 1555

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and its expression is regulated at several levels accordingto the physiological need for ribosomes with one suchlevel being the epigenetic onoff switch that controls thenumber of active rRNA genes (44) A deficiency in thisregulatory mechanism which controls rRNA transcrip-tional activity has been recently associated with overpro-duction of rb-sRNAs in Arabidopsis mutants (4546) Todetermine whether an alteration in the transcriptionalactivity of rRNA genes can be linked to the substantialaccumulation of rb-sRNAs in HSVd-infected cucumberplants we analyzed the accumulation of pre-rRNAs atspecific points (T1 10 dpi and T3 30 dpi) during viroidinfection The reverse transcriptase-polymerase chainreaction (RT-PCR) assay was used to compare the pre-RNA levels analyzing specifically two regions in the 27SrRNA transcription unit (Figure 3A) Two differentprimers complementary to the 30-end of 25S rRNA

(25s-A) and ITS2 (ITS2-A) were used to generate thecDNA template The pair 58s-Fw58s-Rv was used todifferentially amplify by PCR the unprocessed rRNAsSignificant differences in the pre-rRNA transcript accu-mulation levels were found in viroid-infected plants incomparison with mock-inoculated controls at infectiontime T3 when using both the 25s-A and the ITS2-Aprimers for RT (Figure 3B B1 and B2 respectively) Inthe analysis performed at T1 (10 dpi) we observed only aselective accumulation of pre-rRNAs when the PCR wasdone from the ITS2-A-generated cDNA This slight in-congruence however can be attributed to poorer effi-ciency in the generation of sufficiently long cDNAtemplates when using the 25 s-A primer instead of ITS2-A in the RT reaction The differential accumulation ofpre-rRNAs in infected plants at infection time T3 wascorroborated by northern blot assays of 27 S pre-RNA

Figure 1 Characterization of the sRNAs recovered from cucumber leaves by deep sequencing (A) Graphic representation of the differentialaccumulation and distribution of the total (left) and unique (right) reads of endogenous cucumber sRNAs ranging between 21 and 25 nt recoveredfrom both the control and HSVd-infected samples analyzed at 30 days post-inoculation (B) Differential recovering of rb-sRNAs from infected andhealthy tissues The accumulation of rb-sRNAs is expressed as the percentage of total rb-sRNAs from the overall sRNAs in the library (C) and(D) show the relative accumulation of recovered rb-sRNAs with canonical (21ndash24 nt) and non-canonical (lt21 and gt24 nt) predicted sizes respect-ively (E) Graphic representation of the distribution of the total reads of rb-sRNAs (ranging between 21 and 25 nt) recovered from both theHSVd-infected (left) and control (right) analyzed samples


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levels (Figure 3C) The finding that HSVd infection isassociated with an increase in the pre-rRNA levelssuggests that the regulation of the transcriptionalactivity of rRNA genes might be impaired in viroid-infected cucumber

Viroid infection modifies dynamic rDNA methylation

During diverse phases of the plantsrsquo life cycle rRNA genesare presumably in excess and are consequently silenced

At present it is well accepted that the silencing of rRNAgenes is a self-reinforcing regulatory phenomenonmediated by siRNA-directed cytosine methylation andheterochromatin formation (4447) To investigatewhether the transcriptional deregulation of rRNA genes(revealed as pre-rRNA accumulation) observed duringviroid infection can be associated with reduced cytosinemethylation we analyzed by bisulfite sequencing whichidentifies the position of methylated and unmethylatedcytosines a 131-bp region of genomic 45 S rDNA(Figure 4A) Within this sequence there were 9 symmetric(5 CG 4 CHG) and 14 asymmetric (CHH) potentialmethylation sites (Figure 4B)The DNA extracted from leaves of HSVd-infected and

mock-inoculated cucumber plants at 10 dpi (T1) and30 dpi (T3) was treated with bisulfite reagent to convertunmethylated cytosines into uracil followed by PCR toamplify a specific rDNA sequence of 131 nt comprisedbetween positions 115 and +15 PCR products werecloned and the sequences of 13ndash24 clones were compiledfor each time point Methylation analysis revealed that atT1 point HSVd infection resulted in a decrease in therelative number of methylated cytosine residues (83)when compared with control plants However compar-able relative methylation levels between infected andhealthy plants were observed at 30 dpi (T3) (Figure 4C)When dynamic methylation was differentially analyzed forboth symmetric and asymmetric pathways we saw twodifferent patterns Symmetric methylation (CGCHG)slightly decreased during infection and displayed loweredrelative methylation levels between 10 and 12 at T1 andT3 respectively (Figure 4D and F Supplementary FigureS3) These results indicate that HSVd infection is linked toCGCHG demethylation which may contribute to thetranscriptional activation of normally silenced rDNAunits and can consequently induce the accumulation ofpre-rRNA during pathogenesis Conversely in the asym-metric context (CHH) loss of relative methylation ininfected plants at T1 was more significant than in the sym-metric pathway and a drastic increase in the relativemethylation pattern of rDNA was observed at T3 of theHSVd-infected plants (Figure 4D and G SupplementaryFigure S4) To provide a more accurate picture of thechanges occurring in the CHH methylation during viroidinfection we next studied an intermediate point of theinfective cycle at 20 dpi (T2) by bisulfite sequencing(Supplementary Figure S3) As shown in Figure 4E thelevel of asymmetric methylation progressively increased atthe analyzed infection times thus explaining the equilib-rium in the total methylation level observed at point T3when comparing the HSVd-infected and mock-inoculatedplants (Figure 4C) The alteration in the methylation levelsof infected plants at T3 infection time was validated bycombined bisulfite and restriction analysis (SupplementaryFigure S5) Taken together these results indicate that inthe analyzed rDNA regions while CG and CHG sitesmaintain a constant hypomethylated status during infec-tion the CHH sites show a dual scenario beinghypomethylated at T1 and actively hypermethylatedduring HSVd infection development

Figure 2 Analysis of ribosomal-derived sRNAs differentially expressedin infected cucumber plants (A) Diagram (no scale) of the rRNA geneunit The rRNA gene repeats are arranged in long tandem arrays of45S rRNA genes each including the region for the 18S 58S and 25SrRNAs and separated from adjacent units by an IGS The transcrip-tion start site is indicated by+1 The ETS and ITS are removed duringrRNA processing (B) The rb-sRNAs recovered from the infected(above the X-axis) or the non-inoculated plants (below the X-axis)were plotted according to the position of their 50-end onto thecucumber rRNA sequence The values on the Y-axis represent thenumber of total reads in each library The nucleotide positions 115to+15 of the 45S rRNA-analyzed region are represented on the X-axis(C) Comparison of the accumulation of the rb-sRNAs that map alongthe rRNA transcriptional unit between both the infected and controlplants as shown in the box-plot (boxes represent the medians and thefirst and third quartiles of the dataset circles refer to the data whosevalues are beyond the quartiles Comparison of the means of the boxesshowed significant differences between both samples using a parametrict-test arithmetic means for mock and infected plants are 949 and3766 respectively t=2711 Plt 22 1016) (D) The hyper-accumu-lation in the infected samples of the rb-sRNAs derived from an 300-nt region of the IGS (IGS-R) was validated by northern blot assaysThe miR167 equally recovered from both analyzed sRNA datasets wasused as a load control Hybridization with a probe against the HSVd-derived sRNAs (vd-sRNAs) was used as a positive control (E) Therelative accumulation of total rb-sRNAs complementary to rRNA tran-scripts increased in the HSVd-infected plants (left) A similar result wasobtained when individually analyzing the expected canonical size of therb-sRNAs (right)


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To obtain additional evidence supporting the influenceof HSVd infection on host transcriptional activity we alsoanalyze by bisulfite sequencing a 149-bp region of 5 SrDNA (Supplementary Figure S6A and B) another well-established target transcriptionally regulated by DNAmethylation in plants (48) Consistent with observationsfor 45S rRNA sequence analysis revealed that HSVd in-fection is also associated with alterations in the methyla-tion status of the 5S rDNA Methylation analysis revealedthat at initial states of the infectious cycle (T1) the methy-lation levels observed in the analyzed region of the 5 SrDNA were comparable for both analyzed samples(Supplementary Figure S6C) However HSVd infectionresulted in a significant reduction in the relative numberof methylated cytosine residues in infected plants (536)compared with mock-inoculated controls (624) at theT3 analyzed infection time (Supplementary Figure S6D)The loss of relative methylation in infected plants at T3

was comparable for cytosine residues in both the symmet-ric and asymmetric sequence context (SupplementaryFigure S6D E and F) Collectively our results provideunprecedented insights into alterations in the dynamicsof the host DNA methylation status in viroid-inducedpathogenesis

DISCUSSION

Increasing evidence supports the notion that the study ofviroidndashhost interactions can shed light on the regulatorypathways directed by ncRNAs in plants (213) Within thisframework deciphering the molecular basis of the viroidpathogenesis processes emerges as a fundamental mile-stone to be fulfilled Since the first viroid-induced diseasewas reported diverse pathogenesis models have beenproposed (3) The actual prevailing notion linking viroidpathogenesis and RNA silencing essentially considers the

Figure 3 Precursor for rRNAs (pre-rRNAs) accumulates in infected plants (A) Diagram (no scale) of the pre-27S rRNA (ITS1-p and 30ETS-p referto partially processed transcribed spacers) The dotted arrows below depict the oligos used for the RT reaction starting from the 25S 30-end (25s-ART-1) and the ITS2 (ITS2-ART-2) regions of the pre-rRNA Solid arrows indicate the oligos used in the PCR amplification (B) The RT-PCRanalyses of the pre-rRNA expression in the HSVd-infected (+) and mock-inoculated () plants at 10 (T1) and 30 (T3) days post-inoculation Theinitial cDNAs were transcribed using the 25s-A (B1) and ITS2-A (B2) oligos respectively (B3) RT-PCR amplification of U6 Small nucleolar RNA(snoRNA) served as control for RNA load (C) The accumulation of the pre-rRNA (marked with arrows) in the infected plants was validated bynorthern blot assays in the total RNAs extracted from plants at T3 using a probe complementary to the 30-end of the 25S rRNA (D) The bandintensity was measured using the Image-J application httpwwwimagejensoftoniccom


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interference of vd-sRNAs in endogenous RNA metabol-ism at a post-transcriptional level (1920) However thepossibility that the expression of plant symptoms couldeventually be a consequence of specific alterations inhost regulatory mechanisms at a transcriptional level hasalso been envisioned (31331ndash35) Interestingly the data

obtained in this work reveal that viroid-inducedpathogenesis is also associated with transcriptional hostalterationsThe initial observation that HSVd-infected cucumber

plants show an unexpected hyper-accumulation of rb-sRNAs prompted us to speculate about the possibility

Figure 4 HSVd infection affects the methylation patterns in 45S rRNA genes (A) Diagram of the rRNA gene intergenic region highlighting thepromoter zone analyzed by bisulfite sequencing The arrows represent the oligos used in the PCR assay and their relative position in the rRNA gene(B) Graphic representation of the potential symmetric (CGndashred bars and CHGndashblue bars) and asymmetric (CHHndashgreen bars) positions predicted toexist within the analyzed region (C) Histogram documenting the relative (HSVdmock) total DNA methylation levels at infection times T1 and T3(D) Schematic representation of the differential analysis of both symmetric and asymmetric cytosine methylation at infection times T1 and T3 (pairedt-test values T1 means symmetric methylation (mock) 073 (infected) 067 t=1604 means CHH methylation (mock) 022 (infected) 013t=2180 T3 means symmetric methylation (mock) 072 (infected) 064 t=2481 means CHH methylation (mock) 011 (infected) 022t=3047 Plt 005 Plt 001) (E) Evolution of the CHH methylation during HSVd infection in comparison with the level observed in themock-inoculated plants (F) Positions of methylcytosines in the analyzed regions displayed in the symmetric (CG and CHG) context (G) Positions ofmethylcytosines in the analyzed regions displayed in the asymmetric context The height of the bar represents the frequency at which cytosine wasmethylated at the analyzed infection times T1 (left) and T3 (right)


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that during infection HSVd [as previously observed fora symptomatic variant of Peach latent mosaic viroid(49)] could interfere in a yet undetermined manner inthe rRNA maturation process and that the rb-sRNAshighly recovered from infected plants could be a prod-uct of the DCL-mediated degradation of aberrantlyprocessed rRNAs Nonetheless the observation thatmature forms of rRNAs such as 25 S accumulate atcomparable levels in both healthy and infected plants(Figure 3B and C) was incongruent with this suppositionConsequently we explored an alternative option thatrb-sRNA accumulation in infected plants could be theresult of the viroid-induced transcriptional deregulationof rRNAs resembling that recently observed inArabidopsis where rb-sRNAs overproduction has beenlinked to deficiencies in the onoff switch controlling thenumber of active rRNA genes (46) Increasedaccumulation of 27 S pre-rRNA in infected plants inparallel to the maintenance of equivalent levels of pro-cessed rRNA forms (ie 25 S) strongly suggests thatduring infection HSVd may induce the upregulation ofsome of the normally inoperative rRNA transcriptionalunitsBisulfite sequencing clearly correlated HSVd infection

with dynamic DNA methylation changes taking place inthe analyzed regulatory region (115+15) of 45 S- rDNAThe symmetrical cytosine methylation progressivelydecreased during infection whereas the asymmetric denovo cytosine methylation also decreased at the initial in-fection time but actively increased throughout infectiondevelopment Interestingly it has been previouslyreported in Arabidopsis that changes in the expression ofrRNA transcripts are related with the activation ofsilenced 45S rRNA genes and correlate with reduced CGand CHG methylation in the position flanking the rRNAgene transcription initiation site (4446) Accordingly wefavor the idea that 27 S pre-rRNA over-accumulation inHSVd-infected plants can result from activation of other-wise generally repressed rRNA genes by loss in mainten-ance of the methylation status in both symmetric andasymmetric motifs Additional bisulfite sequencingapproaches focused on analysis of the methylation statusof 5 S rDNA unities (region 143+5) revealed that HSVdinfections also induce hypomethylation in both a symmet-ric and asymmetric context in this rDNA family thusreinforcing the biological relevance of this HSVd-induced phenomenon At this point it is important toemphasize that our data were obtained from HSVd-agroinfected plants and not from transgenic plants consti-tutively expressing viroid RNAs Consequently we cannotexclude the possibility that the analyzed tissues couldrepresent a mix of infected and non-infected cells thusunderestimating the effects of viroid infection on hostDNA methylation Our novel and unexpected result forthe HSVd pathogenesis process is consistent with that pre-viously reported for plantndashbacteria interactions where 5 SrRNA repeats were demethylated at a significant level inresponse to infection (50) Furthermore dynamic changesin DNA methylation patterns have been recentlydescribed during antibacterial defense in rice (51)tobacco (52) and Arabidopsis (2350) Finally in a recent

study using transgenic Nicotiana benthamiana plants ex-pressing the replication-associated protein (Rep) of ageminivirus it was proposed that this type of plantDNA viruses can induce a substantial reduction in thelevels of host DNA methylation at CG sites in infectedplants (53) Interestingly the geminiviruses have as HSVddo a nuclear replication

Speculations on the nature of the molecular mechanismregulating this active change in rDNA methylation duringHSVd infection seem premature at this stage Intriguinglyhowever a similar scenario to that observed in infectedcucumber plants has been described in Arabidopsis when amutant for histone deacetylase 6 (HDA6) a key regulatorof gene silencing that displays a complex interrelationshipwith DNA methylation was analyzed (46) Earley et alreported that hda6 mutants lose the maintenance of sym-metric methylation in 45S rRNA promoter regions inparallel with a gain of de novo CHH methylationUnexpectedly and in concert with our results increasingCHH methylation in hda6 mutants a typically repressivephenomenon failed to suppress rRNA over-transcriptionIn addition the siRNAs derived from the 45S IGS regionhyper-accumulated in the hda6 mutant resembling atleast in part that found in viroid-infected cucumberplants (Figure 2B and C) Interestingly it was alsoshown that the loss of HDA6 activity induced a decreaseof the symmetrical methylation of 5 S rDNA and leads tothe release of 5 S rDNA silencing in mutant Arabidopsisplants (54) On the other hand it was also proposed thatspurious transcription of ribosomal genes by PolII can beassociated with the elimination of the repressive modifica-tion regulating the rRNA expression in Arabidopsis (46)By bearing this in mind it is important to consider thatduring infection HSVd reprograms PolII activity to tran-scribe viroid RNA instead of the DNA template (55)which perhaps favors the spurious transcription ofrRNAs in infected plants Further studies are requiredto explore whether there is some interrelation betweenthe observations herein and the rRNA silencing ofthe rRNA genes mediated by HDA6 and PolII inArabidopsis Moreover we cannot rule out the possibilitythat viroid infection may induce changes in widespreaddynamic DNA methylation resembling what wasobserved in other pathogenndashplant interactions(2350ndash52) Broader analyses of DNA methylome onviroid-infected plants would help define the impact ofviroid infection on DNA demethylation and host genetranscription

In summary the data shown here support the belief thatduring its pathogenesis process HSVd induces changes inthe DNA methylation of inactive rRNA genes a previ-ously non-described mechanism linked to viroid (or anyother pathogenic RNA) infection in plants Moreover ourfindings provide new insights into aspects of the hostalterations associated with the HSVd infectious cycleand constitute additional support for the emergingnotion that viroid pathogenesis can be a consequence ofa multilayered process involving diverse pathogenndashhostinteractions at both the post-transcriptional and transcrip-tional levels


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ACCESSION NUMBERS

NCBISRA accession code SRP001408

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Onlineincluding [56]

ACKNOWLEDGEMENTS

The authors thank Drs M Fares and J Forment(Bioinformatics Service of IBMCP) for their valuable con-tribution in the statistical analysis of the data and thesRNA sequence analysis respectively

FUNDING

The Spanish granting agency Direccion General deInvestigacion Cientifica [BIO2011-25018 to VP] andfrom the Prometeo program [2011003] from theGeneralitat Valenciana GM is the recipient of a MarieCurie IOF fellowship Funding for open access chargeDireccion General de Investigacion Cientifica [BIO2011-25018]

Conflict of interest statement None declared

REFERENCES

1 DingB (2009) The biology of viroid-host interactions Annu RevPhytopathol 47 105ndash131

2 DingB (2010) Viroids self-replicating mobile and fast-evolvingnoncoding regulatory RNAs Wiley Interdiscip Rev RNA 1362ndash375

3 NavarroB GiselA RodioME DelgadoS FloresR and DiSerioF (2012) Viroids how to infect a host and cause diseasewithout encoding proteins Biochimie 94 1474ndash1480

4 ZhaoY OwensRA and HammondRW (2001) Use of avector based on potato virus X in a whole plant assay todemonstrate nuclear targeting of potato spindle tuber viroidJ Gen Virol 82 1491ndash1497

5 GomezG and PallasV (2012) Studies on subcellularcompartmentalization of plant pathogenic noncoding RNAs givenew insights into the intracellular RNA-traffic mechanisms PlantPhysiol 159 558ndash564

6 NavarroJA VeraA and FloresR (2000) A chloroplastic RNApolymerase resistant to tagetitoxin is involved in replication ofavocado sunblotch viroid Virology 268 218ndash225

7 NohalesMA FloresR and DarosJA (2012) Viroid RNAredirects host DNA ligase 1 to act as an RNA ligase Proc NatlAcad Sci USA 109 13805ndash13810

8 NohalesMA Molina-SerranoD FloresR and DarosJA(2012) Involvement of the chloroplastic isoform of tRNA ligasein the replication of viroids belonging to the family AvsunviroidaeJ Virol 86 8269ndash8276

9 ZhongX and DingB (2008) Distinct RNA motifs mediatesystemic RNA trafficking Plant Signal Behav 3 58ndash59

10 GomezG and PallasV (2004) A long-distance translocatablephloem protein from cucumber forms a ribonucleoproteincomplex in vivo with Hop stunt viroid RNA J Virol 7810104ndash10110

11 GomezG and PallasV (2001) Identification of an in vitroribonucleoprotein complex between a viroid RNA and a phloemprotein from cucumber plants Mol Plant Microbe Interact 14910ndash913

12 OwensRA BlackburnM and DingB (2001) Possibleinvolvement of the phloem lectin in long-distance viroidmovement Mol Plant Microbe Interact 14 905ndash909

13 GomezG and PallasV (2013) Viroids a light in the darkness ofthe lncRNA-directed regulatory networks in plants New Phytol198 10ndash15

14 DienerTO (1981) Are viroids escaped introns Proc Natl AcadSci USA 78 5014ndash5015

15 HaasB KlannerA RammK and SangerHL (1988) The 7SRNA from tomato leaf tissue resembles a signal recognitionparticle RNA and exhibits a remarkable sequencecomplementarity to viroids EMBO J 7 4063ndash4074

16 VisvaderJE and SymonsRH (1985) Eleven new sequencevariants of citrus exocortis viroid and the correlation of sequencewith pathogenicity Nucleic Acids Res 13 2907ndash2920

17 SchnolzerM HaasB RaamK HofmannH and SangerHL(1985) Correlation between structure and pathogenicity of potatospindle tuber viroid (PSTV) EMBO J 4 2181ndash2190

18 PapaefthimiouI HamiltonA DentiM BaulcombeDTsagrisM and TablerM (2001) Replicating potato spindle tuberviroid RNA is accompanied by short RNA fragments that arecharacteristic of post-transcriptional gene silencing Nucleic AcidsRes 29 2395ndash2400

19 WangMB BianXY WuLM LiuLX SmithNAIseneggerD WuRM MasutaC VanceVB WatsonJMet al (2004) On the role of RNA silencing in the pathogenicityand evolution of viroids and viral satellites Proc Natl Acad SciUSA 101 3275ndash3280

20 GomezG MartinezG and PallasV (2009) Interplay betweenviroid-induced pathogenesis and RNA silencing pathways TrendsPlant Sci 14 264ndash269

21 GomezG MartinezG and PallasV (2008) Viroid-inducedsymptoms in Nicotiana benthamiana plants are dependent onRDR6 activity Plant Physiol 148 414ndash423

22 NavarroB GiselA RodioME DelgadoS FloresR and DiSerioF (2012) Small RNAs containing the pathogenicdeterminant of a chloroplast-replicating viroid guide thedegradation of a host mRNA as predicted by RNA silencingPlant J 70 991ndash1003

23 DowenRH PelizzolaM SchmitzRJ ListerR DowenJMNeryJR DixonJE and EckerJR (2012) Widespread dynamicDNA methylation in response to biotic stress Proc Natl AcadSci USA 109 E2183ndashE2191

24 LawJA and JacobsenSE (2010) Establishing maintaining andmodifying DNA methylation patterns in plants and animals NatRev Genet 11 204ndash220

25 MatzkeM KannoT DaxingerL HuettelB and MatzkeAJ(2009) RNA-mediated chromatin-based silencing in plants CurrOpin Cell Biol 21 367ndash376

26 ZhangX HendersonIR LuC GreenPJ and JacobsenSE(2007) Role of RNA polymerase IV in plant small RNAmetabolism Proc Natl Acad Sci USA 104 4536ndash4541

27 HaagJR and PikaardCS (2011) Multisubunit RNApolymerases IV and V purveyors of non-coding RNA for plantgene silencing Nat Rev Mol Cell Biol 12 483ndash492

28 GongZ Morales-RuizT ArizaRR Roldan-ArjonaTDavidL and ZhuJK (2002) ROS1 a repressor oftranscriptional gene silencing in Arabidopsis encodes a DNAglycosylaselyase Cell 111 803ndash814

29 NuthikattuS McCueAD PandaK FultzD DefraiaCThomasEN and SlotkinRK (2013) The initiation of epigeneticsilencing of active transposable elements is triggered by RDR6and 21-22 nucleotide small interfering RNAs Plant Physiol 162116ndash131

30 WasseneggerM HeimesS RiedelL and SangerHL (1994)RNA-directed de novo methylation of genomic sequences inplants Cell 76 567ndash576

31 ItayaA MatsudaY GonzalesRA NelsonRS and DingB(2002) Potato spindle tuber viroid strains of differentpathogenicity induces and suppresses expression of common andunique genes in infected tomato Mol Plant Microbe Interact 15990ndash999

32 OwensRA TechKB ShaoJY SanoT and BakerCJ (2012)Global analysis of tomato gene expression during Potato spindle


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tuber viroid infection reveals a complex array of changes affectinghormone signaling Mol Plant Microbe Interact 25 582ndash598

33 TessitoriM MariaG CapassoC CataraG RizzaS DeLucaV CataraA CapassoA and CarginaleV (2007)Differential display analysis of gene expression in Etrog citronleaves infected by Citrus viroid III Biochim Biophys Acta 1769228ndash235

34 HerranzMC NiehlA RosalesM FioreN ZamoranoAGranellA and PallasV (2013) A remarkable synergistic effect atthe transcriptomic level in peach fruits doubly infected by prunusnecrotic ringspot virus and peach latent mosaic viroid Virol J10 164

35 LisonP TarragaS Lopez-GresaP SauriA TorresCCamposL BellesJM ConejeroV and RodrigoI (2013) Anoncoding plant pathogen provokes both transcriptional andposttranscriptional alterations in tomato Proteomics 13 833ndash844

36 GomezG and PallasV (2006) Hop stunt viroid is processed andtranslocated in transgenic Nicotiana benthamiana plants MolPlant Pathol 7 511ndash517

37 MartinezG DonaireL LlaveC PallasV and GomezG (2010)High-throughput sequencing of Hop stunt viroid-derived smallRNAs from cucumber leaves and phloem Mol Plant Pathol 11347ndash359

38 PallasV NavarroA and FloresR (1987) Isolation of a viroid-like RNA from hop different from hop stunt viroid J GenVirol 68 3201ndash3205

39 GomezG and PallasV (2007) Mature monomeric forms of Hopstunt viroid resist RNA silencing in transgenic plants Plant J51 1041ndash1049

40 DellaportaSL WoodJ and HicksJB (1983) A plant DNAminipreparation version II Plant Mol Biol Rep 1 19ndash21

41 MartinezG FormentJ LlaveC PallasV and GomezG(2011) High-throughput sequencing characterization anddetection of new and conserved cucumber miRNAs PLoS One6 e19523

42 LongEO and DawidIB (1980) Repeated genes in eukaryotesAnnu Rev Biochem 49 727ndash764

43 HenrasAK SoudetJ GerusM LebaronS Caizergues-FerrerM MouginA and HenryY (2008) The post-transcriptional steps of eukaryotic ribosome biogenesis Cell MolLife Sci 65 2334ndash2359

44 PontvianneF BlevinsT ChandrasekharaC FengWStroudH JacobsenSE MichaelsSD and PikaardCS (2012)Histone methyltransferases regulating rRNA gene dose anddosage control in Arabidopsis Genes Dev 26 945ndash957

45 PreussS and PikaardCS (2007) rRNA gene silencing andnucleolar dominance insights into a chromosome-scale epigeneticonoff switch Biochim Biophys Acta 1769 383ndash392

46 EarleyKW PontvianneF WierzbickiAT BlevinsTTuckerS Costa-NunesP PontesO and PikaardCS (2010)Mechanisms of HDA6-mediated rRNA gene silencingsuppression of intergenic Pol II transcription and differentialeffects on maintenance versus siRNA-directed cytosinemethylation Genes Dev 24 1119ndash1132

47 TuckerS VitinsA and PikaardCS (2010) Nucleolar dominanceand ribosomal RNA gene silencing Curr Opin Cell Biol 22351ndash356

48 LayatE CotterellS VaillantI YukawaY TutoisS andTourmenteS (2012) Transcript levels alternative splicing andproteolytic cleavage of TFIIIA control 5S rRNA accumulationduring Arabidopsis thaliana development Plant J 71 35ndash44

49 RodioME DelgadoS De StradisA GomezMD FloresRand Di SerioF (2007) A viroid RNA with a specific structuralmotif inhibits chloroplast development Plant Cell 19 3610ndash3626

50 YuA LepereG JayF WangJ BapaumeL WangYAbrahamAL PentermanJ FischerRL VoinnetO et al(2013) Dynamics and biological relevance of DNA demethylationin Arabidopsis antibacterial defense Proc Natl Acad Sci USA110 2389ndash2394

51 ShaAH LinXH HuangJB and ZhangDP (2005) Analysisof DNA methylation related to rice adult plant resistance tobacterial blight based on methylation-sensitive AFLP (MSAP)analysis Mol Genet Genomics 273 484ndash490

52 BoykoA KathiriaP ZempFJ YaoY PogribnyI andKovalchukI (2007) Transgenerational changes in the genomestability and methylation in pathogen-infected plants (virus-induced plant genome instability) Nucleic Acids Res 351714ndash1725

53 Rodriguez-NegreteE Lozano-DuranR Piedra-AguileraACruzadoL BejaranoER and CastilloAG (2013) GeminivirusRep protein interferes with the plant DNA methylationmachinery and suppresses transcriptional gene silencing NewPhytol 199 464ndash475

54 VaillantI TutoisS CuvillierC SchubertI and TourmenteS(2007) Regulation of Arabidopsis thaliana 5S rRNA genes PlantCell Physiol 48 745ndash752

55 MuhlbachHP and SangerHL (1979) Viroid replication isinhibited by alpha-amanitin Nature 278 185ndash188

56 LiLC and DahiyaR (2002) MethPrimer designing primers formethylation PCRs Bioinformatics 18 1427ndash1431


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post-transcriptional cleavage of endogenous mRNAsacting as elicitors of symptoms expression (18ndash20) wasreinforced by the observation that the expression of hostsymptoms is associated with post-transcriptional RNAsilencing in representative members of both Pospiviroidae(21) and Avsunviroidae (22) families Despite strongevidence supporting the role of vd-sRNAs as inductorsof host symptoms the possibility that this phenomenoncan be a more complex process also involving viroid-induced alterations in plant gene expression at transcrip-tional levels cannot be ruled out (313)Genome activity at the transcriptional level in plants is

regulated by epigenetic modifications of DNA andhistones Cytosine DNA methylation in plants is a stableepigenetic mark basic for the maintenance of genomestability including regulation of coding and non-codingregions (23) Methylation takes place in three differentsequence contexts CG and CHG (symmetric) andCHH (asymmetric where H refers to A T or C) (24)De novo DNA methylation is regulated by DOMAINSREARRANGED METHYLTRANSFERASE 2(DRM2) with three different pathways being involvedin its maintenance DNA METHYLTRANSFERASE 1(MET1) and DNA CHROMOMETHYLASE3 (CMT3)respectively maintain CG and CHG methylation throughDNA replication whereas CHH methylation requires denovo continuous maintenance by DRM2 (2425) sRNAsare important in de novo establishment and reinforcementof stable DNA methylation because they can target hom-ologous DNA sequences and induce cytosine DNAmethylation in all three contexts A specific RNA silencingpathway termed RNA-directed DNA methylation(RdDM) mediates the biosynthesis of these epigeneticallyactive sRNAs (26) In the ArabidopsisndashRdDM pathwaysingle-strand transcripts originated by RNA polymeraseIV (PolIV) are used by RNA-dependent RNA polymerase2 (RDR2) as substrates to generate dsRNAs which aresubsequently processed by Dicer-like 3 (DCL3) into23ndash24 nt siRNAs (25) These siRNAs guide Argonaute(AGO4AGO6) proteins (likely through an siRNA-nascent PolV transcript base pairing mechanism) totarget genomic regions (27) Next DRM2 is recruited todirect both symmetric and asymmetric de novo methyla-tion Conversely Arabidopsis encodes a family of DNAglycosylaseslyases that mediate the active demethylationof methylcytosines (28) Recently an alternative RDR62122-nt siRNA-dependent pathway has been describedto mediate the initiation of de novo DNA methylation oftransposable elements (29)The first evidence linking viroid infection and transcrip-

tional regulation in plants was provided by the demonstra-tion that Potato spindle tuber viroid (PSTVd) replicationdirects de novo methylation of cognate DNA sequences inviroid-expressing transgenic tobacco plants (30)Furthermore other studies have shown that diverse hostgenes exhibit a transcriptional alteration during PSTVd(3132) Citrus viroid III (33) Peach latent mosaic viroid(34) and Citrus exocortis viroid (35) infections Althoughall this experimental evidence may support the idea thatviroid infection incites alterations in plant gene expressionat transcriptional levels (13) viroid-induced methylation

of the host gene(s) or disruption of methylation pathways(which may eventually result in symptom expression) hasnot yet been reported in natural infections (3)

To address this issue we first analyzed the alteration inthe levels of endogenous sRNAs associated with lsquoHopstunt viroidrsquo (HSVd) infection in cucumber (Cucumissativus) plants Our results indicate that HSVd-infectedplants differentially accumulate high levels of thesRNAs derived from ribosomal transcripts (rb-sRNAs)associated with an increasing accumulation of ribosomalRNA precursors(pre-rRNAs) during infection Finally weobserved that deregulation in rRNA transcriptionalactivity correlates with a dynamic modification of theDNA methylation level during the infection process toprovide unprecedented insight into the potential endogen-ous regulatory pathways affected during the viroid lifecycle in infected plants

MATERIALS AND METHODS

Plant material

Cucumber (C sativus cv Suyo) plants were agroinoculatedwith Agrobacterium tumefaciens strain C58C1 transformedwith a binary pMOG800 vector carrying a head-to-tailinfectious dimeric HSVd cDNA (Y09352) (36) as previ-ously described (21) (Supplementary Materials) Mock-inoculated cucumber plants were used as a negativecontrol Plants were maintained in growing chambersand were analyzed at 10 (T1) 20 (T2) and 30 (T3) dayspost-infiltration (dpi) (Supplementary Figure S4A)

sRNA library information

The sRNA sequences used in this work were obtained froma library generated by starting from an sRNA populationrecovered from leaves and phloem exudates of healthy andHSVd-infected cucumber (C sativus) plants and sequencedby 454 Life Science Technology (Lifesequencing BranfordCT USA wwwlifesequencingcom) [(37) - NCBISRAaccession code SRP001408] The vd-sRNAs recoveredfrom infected plants were filtered out from this analysis

RNA isolation

Total RNA was extracted from the leaves (01 g) of dif-ferent infected and healthy cucumber plants using the TRIreagent (SIGMA St Louis MO USA) according to themanufacturerrsquos instructions The low-molecular-weightRNA (lt200 nt) fraction was enriched from totalRNA using MIRACLE (miRNA isolation KitSTRATAGENE) according to the manufacturerrsquos instruc-tions (Supplementary Materials)

Northern blot assays

Total RNA was analyzed by electrophoresis underdenaturing conditions in 1 agarose gels with 025Tris-Borate-EDTA (TBE) and 8 M urea (38) The RNAwas blotted on nylon membranes (ROCHE DiagnosticsGmbH Mannheim Germany) and was hybridized aspreviously described (11) Approximately 25 mg oflow-molecular-weight RNA was loaded onto 20


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RESULTS









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DISCUSSION




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DISCUSSION




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DISCUSSION




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DISCUSSION




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A pathogenic non-coding RNA induces changes in dynamic DNA

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Transcript of A pathogenic non-coding RNA induces changes in dynamic DNA