The Dictyostelium discoideum RNA-dependent RNApolymerase RrpC silences the centromericretrotransposon DIRS-1 post-transcriptionally and isrequired for the spreading of RNA silencing signalsStephan Wiegand1 Doreen Meier2 Carsten Seehafer1 Marek Malicki1

Patrick Hofmann1 Anika Schmith3 Thomas Winckler3 Balint Foldesi2

Benjamin Boesler2 Wolfgang Nellen2 Johan Reimegard4 Max Kaller4

Jimmie Hallman4 Olof Emanuelsson4 Lotta Avesson5 Fredrik Soderbom67 and

Christian Hammann1

1RibogeneticsBiochemistry Lab School of Engineering and Science Molecular Life Sciences ResearchCenter Jacobs University Bremen Campus Ring 1 DE-28759 Bremen Germany 2Abteilung GenetikUniversitat Kassel Heinrich-Plett-Strasse 40 DE-34132 Kassel Germany 3Friedrich-Schiller-Universitat JenaInstitut fur Pharmazie Lehrstuhl fur Pharmazeutische Biologie Semmelweisstraszlige 10 DE-07743 Jena Germany4Division of Gene Technology KTH Royal Institute of Technology Science for Life Laboratory (SciLifeLabStockholm) School of Biotechnology SE-171 65 Solna Sweden 5Garvan Institute of Medical Research 384Victoria St Darlinghurst NSW 2010 Australia 6Department of Cell and Molecular Biology Biomedical CenterUppsala University PO Box 596 S-75124 Uppsala Sweden and 7Science for Life Laboratory SE-75124Uppsala Sweden

Received August 6 2013 Revised November 29 2013 Accepted November 30 2013

ABSTRACT

Dictyostelium intermediate repeat sequence 1(DIRS-1) is the founding member of a poorlycharacterized class of retrotransposable elementsthat contain inverse long terminal repeats andtyrosine recombinase instead of DDE-type integraseenzymes In Dictyostelium discoideum DIRS-1forms clusters that adopt the function of centro-meres rendering tight retrotransposition controlcritical to maintaining chromosome integrity Wereport that in deletion strains of the RNA-dependentRNA polymerase RrpC full-length and shorter DIRS-1 messenger RNAs are strongly enriched Shorterversions of a hitherto unknown long non-codingRNA in DIRS-1 antisense orientation are alsoenriched in rrpCndash strains Concurrent with the accu-mulation of long transcripts the vast majority ofsmall (21 mer) DIRS-1 RNAs vanish in rrpCndash strainsRNASeq reveals an asymmetric distribution of theDIRS-1 small RNAs both along DIRS-1 and withrespect to sense and antisense orientation Weshow that RrpC is required for post-transcriptional

DIRS-1 silencing and also for spreading of RNAsilencing signals Finally DIRS-1 mis-regulation inthe absence of RrpC leads to retrotransposon mo-bilization In summary our data reveal RrpC as a keyplayer in the silencing of centromeric retrotrans-poson DIRS-1 RrpC acts at the post-transcriptionallevel and is involved in spreading of RNA silencingsignals both in the 50 and 30 directions

INTRODUCTION

Cellular RNA-dependent RNA Ppolymerases (RdRPs)are present in many eukaryotic organisms includingmammals (1) are involved in mechanisms of RNA-mediated gene regulation including RNA interference(RNAi) (23) RdRPs synthesize an RNA strand from acomplementary RNA template leading to either long orsmall antisense transcripts Based on in vivo and in vitrostudies including deep sequencing analyses severalprimer-dependent and -independent modes of actionhave been inferred for RdRPs from different organismsThese frequently lead to an amplification of a primarysilencing signal such as double-strand-derived smallinterfering RNA (siRNA) An amplifying component

To whom correspondence should be addressed Tel +49 4212 003 247 Fax +49 4212 003 249 Email chammannjacobs-universityde

3330ndash3345 Nucleic Acids Research 2014 Vol 42 No 5 Published online 24 December 2013doi101093nargkt1337

The Author(s) 2013 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (httpcreativecommonsorglicensesby-nc30) which permits non-commercial re-use distribution and reproduction in any medium provided the original work is properly cited For commercial re-use please contact journalspermissionsoupcom

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was postulated by Fire and Mello when they firstdescribed RNAi in Caenorhabditis elegans (4) leading tothe identification of RdRP EGO-1 as the responsibleenzyme (5) When RNA was injected against a reportergene secondary siRNAs were observed that predomin-antly localize 50 of the original trigger and this 50

spreading was shown to be RdRP-dependent (6) Thisphenomenon known as lsquotransitivityrsquo is diagnostic forRdRP activity in vivo (6ndash8) We recently observed transi-tivity in the amoeba Dictyostelium discoideum and haveshown that it depends on the presence of the RdRP RrpCand the dicer-related nuclease DrnB as inferred from anartificial reporter system (9) RrpC is one of three RdRPsin D discoideum all of which show a similar domain struc-ture (Supplementary Figure S1) (10)

Dictyostelium intermediate repeat sequence 1 (DIRS-1)is the founding member of a poorly characterized class oflong terminal repeat (LTR) retrotransposons DIRSelements differ from other LTR retrotransposons byhaving inverted instead of direct terminal repeatslacking an aspartic protease domain and using a tyrosinerecombinase instead of a DDE-type integrase protein forintegration Thus DIRS elements use a retrotranspositionmechanism fundamentally different from that of otherLTR retrotransposons [reviewed in (11)] This may favorthe recombinatorial integration into preexisting copies ofthe same element as seen in the D discoideum genome Thegenome of D discoideum features 40 intact copies and200ndash300 fragments of DIRS-1 which thus represents themost frequently occurring LTR retrotransposon in theamoeba (12) DIRS-1 sequences accumulate at one endof each chromosome (12) and these clusters have beensuggested to represent the centromeres of the chromo-somes in D discoideum (1314) Although observed clus-tering of DIRS-1 elements may result from preferentialintegration of mobilized DIRS-1 copies into preexistingDIRS-1 clusters (15) it is not known whether theapparent clustering of these elements is the result of dele-terious integration into coding regions of the haploidD discoideum genome that removes affected cells fromthe population (16) Nonetheless uncontrolled amplifica-tion of DIRS-1 elements and integration into centromericregions may seriously compromise centromere stabilityand thus genome integrity

DIRS-1 is transcribed as a 45-kb-long messenger RNA(mRNA) with three overlapping open reading frames(ORFs) (Figure 1A) (17) The left LTR possessespromoter activity to drive the transcription of the DIRS-1 mRNA (18) These DIRS-1 mRNA transcripts werefound to accumulate during D discoideum developmentand contain parts of the two LTRs (18) Additionallyheat shock or other stress conditions can trigger the ex-pression of a 900-nt-long antisense transcript (Figure 1A)which however is not expressed under the axenic growthconditions applied in this study (1920)

For retrotransposition to occur the mRNA is thoughtto be reverse transcribed by the enzyme activity of ORFIII (17) The ends of the resulting linear complementaryDNA (cDNA) feature the LTR fragments which exhibitsequence complementarity to a DIRS-1 region termed theinternal complementary region (ICR) According to a

model put forward by Lodish et al the ICR serves tobring the 50- and the 30-end of the reverse transcripttogether (11) to generate a single-stranded circular DNAreplication intermediate that serves as template for thegeneration of a double-stranded circular DNA to beinserted in the genome (17)We have shown earlier that the LTR of DIRS-1 is

methylated by the sole DNA methyltransferase DnmAidentified in D discoideum (21) Surprisingly no transcrip-tional activation was observed in a dnmA gene deletionstrain indicating that DIRS-1 is not under transcriptionalcontrol at least not by DnmA However in that study weobserved that regulation of DIRS-1 expression may becontrolled at the post-transcriptional level involvingsmall RNAs (21) Here we show that RrpC controlsDIRS-1 post-transcriptionally and its activity isrequired to prevent DIRS-1 retrotransposition Wefurther report RrpC-dependent spreading of an RNAsilencing signal in the 30 direction in D discoideum

MATERIALS AND METHODS

Oligonucleotides

DNA oligonucleotides (Sigma) used in this study are listedin Supplementary Table S1

Genes and strains

All experiments were carried out with AX2 and deriva-tives All D discoideum strains were grown axenically inHL5 medium The rrp genes are listed in the onlineresource dictybaseorg with their accession numbersDDB_G0289659 (rrpA) DDB_G0291249 (rrpB) andDDB_G0280963 (rrpC) and the generation of theirsingle and multiple deletion strains was describedrecently (9) The trx-1 gene has the accession numberDDB_G0294447

RNA isolation

Total RNA was isolated from 5 107 cells of axenicallygrown D discoideum strains using TRIzol (Invitrogen)Cells were pelleted for 5min at 500 rcf4C and washed in20 ml pre-cooled Sorenson phosphate buffer (2 mMNa2HPO4 15 mM KH2PO4 pH 60 with H3PO4) Onre-centrifugation of cells under the same conditions theywere lysed in 1 ml of TRIzol reagent for 5min Onaddition of 200 ml of chloroform the material wasvortexed and incubated for 3min at room temperaturePhases were separated by centrifugation at 16 000 rcf4C for 20min RNA was precipitated by addition ofequal amounts of 100 isopropanol incubation for10min at room temperature and centrifugation at 16 000rcf4C for 15min On washing twice with 70 ethanolthe pellet was dried and re-suspended in 150 ml dH2ORNA concentration was determined using a NanoDropspectrophotometer (Peqlab)

Northern blotting

For total RNA blots 10 mg was separated by gel electro-phoresis in a 12 GTC-agarose gel in 1Tris

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borate+ethylenediaminetetraacetic acid (pH 82) On ca-pillary transfer to nylon membranes (Roti-Nylon plus)ultraviolet cross-linking was carried out (05 Jcm2) Pre-hybridization and hybridization were carried out inChurch buffer [500mM sodium phosphate (pH 72)1mM ethylenediaminetetraacetic acid 7 wv sodiumdodecyl sulphate (SDS) 1 wv bovine serum albumin]using radioactively labeled in vitro transcripts orpolymerase chain reaction (PCR) productsHybridization conditions are summarized inSupplementary Tables S2 and S3 All blots were washedtwice for 15min in 2 saline sodium citrate (SSC) 01wv SDS twice for 10min in 1 SSC 01 wv SDS andtwice for 5min in 05SSC 01 wv SDS As controlblots were hybridized on stripping to probes against actin6mRNAFor small RNA blotting 20mg total RNA was

separated by gel electrophoresis in a 11 polyacrylamidegel in 20mM MOPS (pH 70) On electroblotting onto anylon membrane (Amersham HybondTM-NX) for 10minat 20V in dH2O (semi-dry) RNA was chemically cross-linked as described recently (22) Small RNAs wereprobed with 50 radioactively labeled DNA oligonucleo-tides as summarized in Supplementary Table S4 Allother procedures were carried out as described for ultra-violet cross-linking except for the use of the smallnucleolar RNA (snoRNA) DdR-6 as loading control

Southern blotting

Isolation of genomic DNA from 1ndash2 108 Dictyosteliumcells for Southern blotting (23) was performed asdescribed (24) For DNA digestion with restrictionenzymes aliquots containing 20 mg of DNA wereincubated with Sac I Kpn I or Eco RI (20U10 mg ofDNA) After precipitation samples were separated on12 agarose gels for 14 h at a field strength of15Vcm and fragments were visualized by ethidiumbromide staining (05 mgml) for 30min DNA wasdenatured by soaking the gel in 10 volumes of denatur-ation buffer (15M NaCl 05M NaOH) for 45minfollowed by 45min soaking in neutralization buffer (3MNaCl 05M Tris-HCl pH 7) DNA fragments weretransferred to a nylon membrane as described (25)Cross-linking and hybridization were carried out asdescribed for northern blotting using the right elementsequence labeled with [a-32P]dATP by random primingOn overnight incubation the membrane was washed with3SSC 001 SDS and then with 01 x SSC 01 SDSeach at 60C and for 1 h

Generation of expression constructs

pTX-DIRS-1rLTRfw-GFP was generated by initially di-gesting the original vector pTX-GFP (26) with Sal I andligating the oligonucleotides pTX-MCS-fw and pTX-MCS-rev for generating a multiple cloning site (MCS)Depending on the orientation of the MCS the resultingvectors were termed pTX-MCSfw-A15-GFP and pTX-MCSrev-A15-GFP To insert the DIRS-1 rLTR-sequence plasmid pGEM-T Easy DIRS-1 rLTR (21)was digested with Sac II and Sal I Ligation into the

MCS of pTX-MCSfw-A15-GFP and pTX-MCSrev-A15-GFP yielded the plasmids pTX-DIRS-1rLTRfw-A15-GFP and pTX-DIRS-1rLTRrev-A15-GFP The formerwas digested with Sac II and Xho I to isolate the expres-sion cassette which was ligated into the vector pLPBLP(27) that had been digested with the same enzymes be-forehand From this the Actin15-promoter was removedby a restriction digest with Hind III and Xho I The 50

overhangs were filled by Klenow fragment beforeligation and the expression cassettes without Actin15-promoter were ligated back in the original vector using aSac II and Xho I which yielded the plasmid pTX-DIRS-1rLTRfw-GFP From pTX-DIRS-1rLTRrev-A15-GFPthe expression cassette was removed with Sal I andBamH I and ligated into the vector pJET1-H2Bv1 fromwhich the H2Bv1 fragment had been removed with thesame enzymes The actin15-promoter was removed by adigest with Hind III and Sac II and the vector was re-ligated on removal of 50 overhangs using Klenowfragment The expression cassette was back-cloned intothe original vector using Sal I and BamH I yieldingplasmid pTX-DIRS-1rLTRrev-GFP

The DIRS-1 ORFs were amplified by PCR usingprimers listed in Supplementary Table S1 PCR productswere ligated into the cloning pJET12blunt and the re-sulting plasmids were sequenced The ORF DNAs wereexcised by means of BamH I and Spe I digests and ligatedinto pDM323 (28) that was previously linearized withBgl II and Spe I The resulting plasmids were transformedin AX2 and rrpC knockout strains and transformationpopulations were used for further experiments For theGFP-alone control plasmid pDM317 (28) was used thatfeatures an ATG in front of the GFP coding sequenceallowing to monitor GFP expression by western blotting

Fluorescence in situ hybridization and microscopy

Fluorescence in situ hybridization (FISH) usingdigoxigenin-labeled dNTPs and 46-diamidino-2-phenylindole (DAPI) staining were performed asdescribed recently (29) For the generation of probes forFISH oligonucleotides DIRS1 forward and DIRS1reverse were used resulting in a 503-nt-long single-stranded DNA of DIRS-1 sense or antisense orientationdepending on their relative concentration in asymmetricPCRs The fragment begins at the left end (LE) of ORFIII (Figure 1) and is extended by 200 nt Pictures weregenerated using an Alexa Fluor 488-coupled secondaryantibody

Quantitative real-time PCR

Expression analysis by quantitative real-time PCR (qRT-PCR) was carried out as described recently (3031) Inbrief total RNA was isolated using the Qiagen RNeasyMini kit according to the provided protocol from 2 107

cells that were collected from exponentially growing cellsand frozen at 80C until further use cDNA wassynthesized by reverse transcription of 500 ng of totalRNA using an oligodeoxythymidine primer and theQiagen Omniscript RT kit The Q1 and Q2 fragments ofDIRS-1 were amplified from oligo(dT)-primed cDNA

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Figure 1 DIRS-1 expression (A) Schematic representation of the DIRS-1 retrotransposon with the left and right inverted LTRs (l LTR and r LTRrespectively) The three ORFs encoding the GAG protein (ORF I) the tyrosine recombinase (ORF II) and reverse transcriptaseRNase H domain and amethyltransferase (ORF III) are displayed together with the 900-nt E1 antisense transcript Positions for expression analysis are Q1 and Q2 for qRT-PCR and LE and RE for northern blots (B) Expression of DIRS-1 sequences in the indicated gene deletion strains relative to the AX2 wild-type asmonitored by qRT-PCR using primer pairs at positions Q1 and Q2 and normalized to GAPDH expression (C) Northern blot analysis of RNA fromthe indicated strains using DIRS-1 sense strand-specific probes at positions LE (left) and RE (right) Ethidium bromide-stained rRNA served as loadingcontrol while in northern blots an actin6 probe acted to monitor transfer efficiency (D) Northern blots using DIRS-1 antisense strand-specific probes atpositions LE (left) and RE (right) Other details are as in (C) Size ranges of signals for shorter antisense RNAs are indicated to the right of each blotNorthern blot conditions are listed in Supplementary Tables S2 and S3 See also Figures 2 and 3 and Supplementary Figure S2

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with primer pairs Q-DIRS-01-02 and Q-DIRS-03-04 re-spectively Expression of DIRS-1 was standardizedagainst expression of the GAPDH gene which wasamplified using primers Q-gpdA-01 and Q-gpdA-02Real-time amplification was carried out on a StratageneMx3000P instrument in 25 ml reaction mixes containing1Taq buffer 02mM deoxynucleoside triphosphates04mM primers 1ROX reference dye (Jena BioscienceGermany) 1EvaGreen solution (Jena BioscienceGermany) varying amount of cDNA and 125U Taqpolymerase (Jena Bioscience Germany) After an initialdenaturing step at 95C for 10min the PCR was per-formed for 40 cycles at 95C for 30 s 58C for 30 s and72C for 30 s Regulation was calculated by the method ofPfaffl (32)

Deep sequencing and computational analysis

RNA was prepared and converted to cDNA from whichsequencing libraries were prepared and sequenced usingthe Illumina platform as described recently (33)Endogenous siRNA populations in the AX2 wild-typeand rrpC knockout strains each in two biological repli-cates were sequenced after ligation of Illumina TruSeqsmall RNA adapters that allow cluster amplification onthe flow cell surface Adapter sequences were trimmedfrom the sequences using Cutadapt (34) with at least3-nt overlap allowing for 10 mismatches The trimmedreads were mapped against a reference using Bowtie2 (35)The reference was either the genome of D discoideumstrain AX4 (downloaded from httpdictybaseorg) orour own repeat sequence library containing simplerepeats from Repbase (36) and consensus sequences ofinterspersed repeats including DIRS-1 The defaultsetting was used when mapping to the genome referencewhile a more sensitive setting was used when mapping tothe repeat reference (-M 500 -N 1 -L 20 -R 3 -D 20 -iS1050) Read count per annotated region (httpdictybaseorgdownloadgff3) was extracted with HTSeq(httpwww-huberembldeusersandersHTSeqdocoverviewhtml) The read distribution was visualized withthe integrative genomics viewer (37)

RESULTS

Accumulation of DIRS-1 sense transcripts in the absenceof the rrpC gene

In a previous study we observed the accumulation oftranscripts of the retrotransposon DIRS-1 in a gene dis-ruption strain of the RdRP RrpC (21) To study theimpact of RdRPs on DIRS-1 expression in D discoideumin detail we made use of a recently introduced series ofstrains (9) with the genes of the RdRPs in all possiblecombinations deleted by a one-step cloning strategy (38)that is based on the Cre-lox system (27) Unlike in theoriginally used gene disruption strains (21) both func-tional domains of each rrp gene (Supplementary FigureS1) were removed completely (9) resulting in threesingle three double and one triple rrp gene deletionstrain In addition DIRS-1 expression was monitored ina gene deletion strain of the dicer-related nuclease DrnB

(drnBmdash) (39) Analyses by qRT-PCR using the two primersets Q1 and Q2 (Figure 1A) showed a 50ndash100-fold increaseof DIRS-1 RNA in all rrpC gene deletion strainscompared with the isogenic wild-type strain (Figure 1B)In the drnBmdash strain a 5ndash10-fold increase in RNA levelswas observed depending on which primer set was usedTo assess how these RNA molecules correspond to full-length DIRS-1 RNA we next carried out northern blotanalyses with riboprobes against the left-end (LE) andright-end (RE) positions in the DIRS-1 sense transcriptthat localize to the 50-end or the 30 part of ORF III re-spectively (Figure 1A) As shown in Figure 1C theseanalyses revealed a considerable increase of a 45-kbfragment corresponding to the size of the DIRS-1 full-length mRNA (18) for each rrpC gene deletion strainwhile the transcript levels in the drnBmdash strain appearsimilar to that observed in the wild-type strain(Figure 1C) In the rrpCmdash strains the DIRS-1 transcriptsdisplay strong size heterogeneity as indicated by the smearin the respective lanes of the northern blots (summarizedin Table 1)

A long DIRS-1 antisense transcript

Considering that antisense transcripts are prevalent par-ticularly in mobile genetic elements (4041) we also usedriboprobes that bind to the same LE and RE positions(Figure 1A) on an antisense transcript We observed anRNA transcript of 4 kb by either riboprobe (Figure 1D)The existence of this hitherto unknown antisense RNAwas also confirmed independently by strand-specificqRT-PCR (Supplementary Figure S2) We termed thislong non-coding RNA (lncRNA) long antisense DIRS-1(lasDIRS-1) to discriminate it from the 900-nt E1 anti-sense transcript that is produced on heat shock (1920)but not under the axenic growth conditions used in thisstudy Unlike the situation seen for the DIRS-1 mRNAthe lasDIRS-1 lncRNA displays a discrete size and itsRNA levels appear to be identical in either gene deletionstrain investigated here Only in the rrpCmdash strains was alight smear below the full-length lasDIRS-1 RNAobserved reproducibly indicating size heterogeneity(Figure 1D and summarized in Table 1)

Promoter activity of the DIRS-1 right LTR

Because the sense transcript is derived from the promoteractivity of the left LTR (18) it seemed plausible that theinversely oriented right LTR sequence might contain thepromoter to drive lasDIRS-1 transcription To test thishypothesis we generated two constructs which containedthe right LTR in forward or reverse orientation in front ofa GFP expression cassette but lacked any furtherpromoter sequence (Figure 2A) Fluorescence microscopyof strains transformed with these two constructs revealedGFP expression only for the construct with the forward-oriented right LTR (Figure 2B) The promoter activity ofthe right LTR of DIRS-1 was also independently con-firmed by western blotting (Figure 2C) These datastrongly suggest that the lasDIRS-1 transcript derivesfrom the right LTR of the retrotransposon

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DIRS-1 transcripts cluster in the nucleus of rrpCmdash cells

To determine the localization of DIRS-1 transcripts inamoeba cells we used a recently established protocol forRNA-FISH in D discoideum (29) First we generateddigoxigenin-labeled strand-specific 500-nt-long probesstarting at the LE position of DIRS-1 (Figure 1A)

which we used in hybridization experiments of fixed andpermeabilized AX2 wild-type and rrpCmdash cellsFluorescence microscopy revealed for either probediffuse signals that predominantly localized to the cyto-plasm in AX2 wild-type cells as judged from the DAPIstaining of the nucleus (Figure 3A) In the rrpCmdash strain

A

pTX-DIRS-1rLTRfw-GFP pTX-DIRS-1rLTRrev-GFPDIRS-1rLTR

DIRS-1rLTR

GFP Terminatorgtgtgt GFP Terminatorltltlt

PFG-verRTLr1-SRIDPFG-wfRTLr1-SRIDB C

12377

coronin42

30

bright field bright field

30 GFP30

25 4255555 444444254

Figure 2 Analysis of DIRS-1 right LTR promoter activity (A) Constructs with forward and reverse right LTR a GFP ORF and terminator sequence(B) Fluorescence (top) and bright field (bottom) microscopy of AX2 cells transformed with constructs shown in (A) (C) Western blot of these transformedcells using antibodies against GFP and Coronin (loading control) The molecular weights of a size marker (in kDa) are indicated to the left

Table 1 Characteristics of rrp gene deletion strains

rrp gene deletion Change in DIRS-1 transcripts levels Spreading of small RNA signal

mRNAa lasDIRS1b Small RNAsc In 50 directiond In 30 directione

rrpAmdash Unchanged Unchanged Unchanged ı ırrpBmdash Unchanged Unchanged Unchanged ı ırrpCmdash Strongly increased Shorter versions increased Strongly decreased Not observed Not observedrrpAmdashrrpBmdash Unchanged Unchanged Unchanged ı ndf

rrpAmdashrrpCmdash Strongly increased Shorter versions increased Strongly decreased Not observed ndf

rrpBmdashrrpCmdash Strongly increased Shorter versions increased Strongly decreased Not observed ndf

rrpAmdashrrpBmdashrrpCmdash

(Triplemdash)Strongly increased Shorter versions increased Decreased Not observed ndf

aRelative to AX2 wild-type as determined by qRT-PCR and northern blotting (Figure 1) bRelative to AX2 wild-type as determined by qRT-PCRand northern blotting (Figure 1) cRelative to AX2 wild-type as determined by northern blotting (Figure 4) dShown in an earlier study using ab-galactosidase reporter assay (9) eAs determined by northern blotting (Figure 6)f nd not determined

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A

10 microm

sense

10 microm

anti-sense

B

sense

10 microm

10 microm

anti-sense

10 microm

Figure 3 Localization of DIRS-1 transcripts in D discoideum cells by FISH Sense and antisense DIRS-1 transcripts in AX2 (A) and rrpCmdash

cells (B) Shown are microscopic images of fixed and permeabilized cells First column phase contrast second column DAPI stainingthird column Alexa Fluor 488-coupled secondary antibody staining of DIRS-1 transcripts and fourth column merge of DAPI and AlexaFluor 488-coupled secondary antibody staining Arrows mark accumulated transcripts All pictures were taken at a 100-fold magnification and ascale bar is indicated for each row in the phase contrast panel Pictures were taken with exposure times of 260ndash350ms for DAPI and 700ndash1000msfor Alexa

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additional strong signals were observed (Figure 3B) whichappeared to cluster in one or more spots in the nucleusThese clusters of DIRS-1 transcripts were observed byusing either the sense or the antisense probe in therrpCmdash strain (Figure 3B)

As summarized in Table 1 our data indicate that full-length as well as shorter sense DIRS-1 RNAs accumulatesin rrpCmdash strains A long antisense RNA lasDIRS-1 isalso expressed and likely driven by the promoter activityof the right LTR Shorter fragments of lasDIRS-1 werefound to accumulate in the rrpCmdash strains Compared withthe AX2 wild-type strain additional signals for DIRS-1sense and antisense transcripts appeared to localize innuclear spots in the rrpCmdash strain

DIRS-1-specific small RNAs

Because gene silencing by RdRPs is commonly associatedwith the appearance of small RNAs we next analyzed thepresence of DIRS-1-specific small RNAs by northernblotting Using a radioactively labeled oligonucleotidecorresponding to the LE position of the retrotransposonwe observed a significant reduction of DIRS-1-specificsmall RNAs in all strains lacking the rrpC gene ascompared with the wild-type (Figure 4A) The reductionis also apparent albeit to a lesser extent in a northern blotusing a probe against the RE position of DIRS-1(Figure 4B) The individual deletions of the other rrpgenes rrpA or rrpB appeared to have no significanteffect on the accumulation of these DIRS-1 smallRNAs and the same is observed for DrnB (Figure 4)

However with both probes the decrease appeared lesspronounced in the triple knockout strain (see alsoTable 1) an observation that we cannot explain atpresent Yet these northern blot data indicate that theRdRP RrpC is required for the generation of DIRS-1small RNAs at least at the positions LE and RE(Figure 1A) In our previous study we had observedsmall RNAs distributed over the entire sequence of theretrotransposon (21) To analyze whether all theseDIRS-1 small RNAs depend on RrpC we next carriedout deep sequencing studies on RNA from D discoideumAX2 wild-type and rrpCmdash cells

Asymmetric distribution of small RNAs over theDIRS-1 sequence

The small RNA profiles of AX2 wild-type and rrpCmdash

strains were determined by deep sequencing on anIllumina platform using two biological replicates perstrain This resulted in 28 and 16 million sequencereads of 21 nt for AX2 and rrpCmdash respectively Thesereads were mapped against the D discoideum genomeand a library of repeat sequences (manuscript inpreparation)In the wild-type AX2 strain a substantial fraction of

small RNA sequencing reads (195) corresponded toDIRS-1 sequences confirming our earlier studies thatused 50-ligation-dependent cloning and SOLiD sequencingof small RNAs from the AX4 strain (43) However in therrpCmdash strain the number of DIRS-1 sequencing reads isstrongly (65) reduced from 55 to 19 M indicatingthat RrpC might be involved in their generation Wenext mapped these small RNA reads on the DIRS-1sequence (Figure 5A) The comparison of the smallRNA distribution patterns in the two biological replicatesof the wild-type AX2 strain (Figure 5B) indicates that thedata are highly reproducible and the same holds for thetwo biological replicates of the rrpCmdash strain (Figure 5C)The distribution of small RNAs over the DIRS-1

sequence appears uneven in the AX2 strain Themajority of reads coincide with ORF II while the 50-endof GAG appears to be devoid of any small RNAs(Figure 5B) This analysis also showed the reduction ofthe small DIRS-1 RNAs in the rrpCmdash strain but addition-ally indicated that not all positions within the retrotrans-poson are equally affected (Figure 5C) For examplealthough reduced reads are observed for the majority ofDIRS-1 positions in the rrpCmdash strain this is not the casefor the relatively small number of reads that map to theLTRs of the retrotransposon (box I Figure 5B and C)These LTR-specific small RNAs appear to be unchangedin number and position and thus likely are independent ofRrpC A small number of reads corresponding to the50-end of GAG was surprisingly observed only in therrpCmdash strain but not in the AX2 wild-type (box IIFigure 5B and C) These small RNAs thus seem to dis-appear when RrpC is present Reads mapping around the30-end of ORF I and the 50-end of ORF II appear to beparticularly reduced in the rrpCmdash strain (box IIIFigure 4B and C) while the distribution of the majorityof small RNAs in the rrpCmdash strain has an overall similar

Figure 4 DIRS-1-specific small RNAs expression Northern blotsmonitoring the small RNAs of DIRS-1 in the indicated strains usingoligoprobes LE (A) or RE (B) which are localized to DIRS-1 asindicated in Figure 1A Quantification of small RNA levels (indicatedbelow the blots) is relative to that of the AX2 wild-type and normalizedto the DdR-6 snoRNA sequence (42) The 26-bp band of the sizemarker pUC19 DNAMsp I is indicated Northern blot conditions arelisted in Supplementary Table S4

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pattern to that observed in the AX2 wild-type strainalbeit at drastically reduced numbers (box IV Figure 5Band C) These observations suggest that both of theseclasses of DIRS-1 small RNAs depend on RrpC

Asymmetric distribution of DIRS-1 small RNAs in senseand antisense orientation

In view of the presence of the lasDIRS-1 transcript(Figures 1D and 3) we next analyzed whether theDIRS-1-specific small RNAs mapped equally in senseand antisense orientation to the retrotransposon Theresults showed uneven distribution of the small RNAswith the sense small RNAs mapping predominantly toORF II and the antisense RNAs appearing to peak in

the region box III (Figure 5D and E) The asymmetry ofsense and antisense read distribution indicates that themajority of small DIRS-1 RNAs might not be generatedas classical double-stranded siRNAs

Post-transcriptional silencing of DIRS-1 mRNAs

Because the small DIRS-1 RNAs appear to be unevenlydistributed along the three ORFs of the retrotransposonwe wondered what effect they might have on these threeORFs To investigate this question we generated over-expression constructs in which GFP was C-terminallyfused to the full mRNA of either ORF (SupplementaryFigure S3) The resulting extrachromosomal vector con-structs were transformed in the AX2 wild-type and the

Figure 5 Distribution of DIRS-1-specific small RNAs (A) Schematic representation of the DIRS-1 sequence (for details see Figure 1A) Small RNAreads from the wild-type AX2 strain (B) and the rrpCndash strain (C) are shown for their biological duplicates (1 or 2 respectively) The DIRS-1 smallRNAs of sample (1) are shown separately for sense (D) and antisense (E) orientations All reads are mapped onto the DIRS-1 sequence in (A) Thesignal height corresponds to the number of small RNAs and reads are displayed in the same scale ranging from 0 to 400000 reads Regions withparticularly apparent deviations between the AX2 and rrpCndash strains are boxed (I II III IV)

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rrpCmdash strain Expression of the fusion proteins was moni-tored by western blotting using a GFP antibody TheORF I-GFP fusion protein was readily expressed inboth strains while expression of the other two ORFs

was not detectable in the AX2 wild-type but notablyobserved in the rrpCmdash strain (Figure 6A and B) This in-dicates that the RrpC-dependent small RNAs that arepresent in the AX2 wild-type strain are necessary and

A B

100 kDa

ORFI-GFP70 kDa

55 kDa

M

ORFII-GFP

ORFIII-GFP100 kDa

130 kDa

M

Severin35 kDa 70 kDa

C

24 nt

21 nt17 nt

GFP siRNAs(sense )

M

sno6 RNA

rRNA

ED

M

GFP siRNAs(sense )

6 RNA

Discoidin

70 kDa

55 kDa

35 kDa

M

sno6 RNAGFP25 kDa

Figure 6 Overexpression of DIRS-1 ORFs C-terminal GFP fusions of DIRS-1 ORF I (A) and ORF II or ORF III (B) were expressed in the AX2wild-type and in the rrpCmdash strain The expression of the fusion proteins was monitored by western blotting using a GFP antibody (see alsoSupplementary Figure S3) (C) Small GFP RNAs from the indicated strains were analyzed by northern blot using a strand-specific radioactivelylabeled probe The sizes of a radioactively labeled RNA size marker are shown to the left Ethidium bromide-stained rRNA served as loadingcontrol and northern blots using a sno6 riboprobe acted to monitor transfer efficiency (D) Expression of GFP in AX2 wild-type cells (E) SmallGFP RNAs from the indicated strains In (A B and D) severin or discoidin were used as loading controls and the sizes of a protein marker areshown M in (C and E) denotes small RNA size markers Northern blot conditions are listed in Supplementary Table S4 Note that boxed lanes arespliced together from the same blot deleting irrelevant samples

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sufficient to silence post-transcriptionally ORF II andORF III but not ORF I of the retrotransposon eventhough siRNAs in the 30 half of ORF I are mostly anti-sense to the coding transcript and could readily abolish it

Spreading of the RNA silencing signal in the 30 directionon the target mRNA

We have recently shown that RrpC is required for theappearance of small RNAs 50 of the original silencingsignal with respect to the target RNA (9) This phenom-enon is generally referred to as transitive silencing (6ndash8)and it is thought that the target RNA serves as thetemplate from which an RdRP synthesizes a complemen-tary RNA strand Because of the directionality of allnucleic acid polymerases such a secondary silencingsignal is expected to localize 50 of the original triggerHowever Pak and Fire reported that a subset of second-ary RNAs could also localize 30 of the original trigger inC elegans (44) an observation that otherwise is mainlyseen in the plant kingdom (784546) To test whether 30

spreading might also be observed in the case of the DIRS-1 ORF-GFP fusion constructs we isolated small RNAsfrom these strains and used northern blot analysis toidentify small RNAs derived from GFP (Figure 6C)Substantial amounts of small GFP-specific sense RNAswere detected in the ORF II-GFP and ORF III-GFP ex-pressing cells in the AX2 wild-type background but not inthe rrpCmdash strain To show that GFP itself does not causethe generation of siRNAs we transformed the AX2 wild-type with a GFP expression construct alone whichresulted in GFP expressing cells (Figure 6D) and did notcause generation of small GFP RNAs as monitored bynorthern blotting (Figure 6E) The inclusion of RNAfrom the C-terminal GFP fusions of the DIRS-1 ORFsserves here as control for the functionality of the blot asit otherwise would appear empty Because the GFP wasfused C-terminally to the DIRS-1 ORFs this result indi-cates that spreading of an RNA silencing signal in the 30

direction exists in D discoideum and depends on thepresence of RrpC (summarized in Table 1) We notethat a weak signal for small GFP RNAs is also presentin strains where the fusion protein is not silenced but notin the untransformed AX2 wild-type strain (Figure 6)

Mobilization of DIRS-1 in the genome of the rrpCmdash

strain

Having shown with the ORFndashGFP fusions that DIRS-1mRNAs are translated in the rrpCmdash strain we next set outto analyze what effects this has on the genome of therrpCmdash strain However the genome-wide analysisof DIRS-1 elements is hampered by the large number ofDIRS-1 elements and fragments (12) and the fact thatDIRS-1 retrotransposition takes place preferentially inother DIRS-1 elements or fragments (15)To investigate whether new DIRS-1 integrations can be

observed in the genome of the rrpCmdash strain we subjectedrrpCmdash and AX2 wild-type strains to extensive growth for48 days in shaking cultures kept in the exponential growthphase by regular dilution Under such conditions we hadearlier observed increased genomic copy numbers of

Skipper when that retrotransposon was dysregulated ina gene deletion strain of the putative DNAmethyltransferase DnmA (21) From the AX2 andrrpCmdash long-term cultures genomic DNA was isolatedand digested with three different restriction enzymes ofwhich Eco RI and Kpn I cleave inside the DIRS-1 DNA(Figure 7A) whereas Sac I cleaves outside The restrictionpatterns of genomic DNA from these two strains appearedto be highly similar (Figure 7B) In the subsequently per-formed Southern blot (Figure 7C) the overall picture isalso similar as might be expected given the many genomicoccurrences of DIRS-1 elements For the Eco RI and theSac I digests the most prominent signals from the AX2strain appear to be more intense in the rrpCmdash straingenomic DNA which may indicate an increase in DIRS-1 copy number More importantly we observe an add-itional signal at 5 kb in the rrpCmdash strain in the Kpn Idigest clearly indicating a DIRS-1 retrotranspositionevent (Figure 7C) To quantify these differences inDIRS-1 copy number Southern blotting was repeatedand signals for DIRS-1 and the thioredoxin gene asloading control were quantified for two digests ofgenomic DNA from AX2 wild-type and the rrpCmdash

strain (Supplementary Table S5) This analysis confirmedan increase in DIRS-1 copy numbers by gt50 in therrpCmdash strain compared with the AX2 wild-type asshown by loading-corrected signal increases of 52 and62 for the two restriction enzymes used (SupplementaryTable S5) Together the data indicate that additionalgenomic copies of the retrotransposon accumulate in thegenome of the rrpCmdash strain This is further supported by aprevious analysis of DIRS-1 copy numbers in the origin-ally generated gene disruption strain of the rrpC gene (21)where different DIRS-1 patterns were observed in clonallong-term cultures (M Kuhlmann and W Nellenunpublished)

DISCUSSION

In the present study we show that the RdRP RrpC acts inthe regulation of the centromeric retrotransposon DIRS-1 We report that sense transcripts accumulate in strainslacking the rrpC gene (Figure 1 summarized in Figure 8)and concurrent with this accumulation DIRS-1-specificsmall RNAs are dramatically reduced (Figures 4 and 5summarized in Table 1) As a consequence of theirabsence it is likely that the DIRS-1 ORFs II and III areno longer silenced leading to the accumulation of DIRS-1proteins as monitored by GFP fusions (Figure 6) andDIRS-1 retrotransposition (Figure 7) in the rrpCmdash cellsFrom these data we propose that DIRS-1 is constitutivelytranscribed in wild-type cells and these transcripts aretargeted by RrpC which thus exerts it control predomin-antly on the post-transcriptional level

For the full-length DIRS-1 elements transcription isdriven by the inherent promoter activity of the twoLTRs as reported earlier for the left LTR (18) and inthis study for the right LTR (Figure 2) resulting in thenewly discovered lncRNA antisense transcript lasDIRS-1(Figure 1) The observation that the full-length lasDIRS-1

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transcript is also present in the triple rrp gene deletionstrain excludes the possibility that it might be an RdRPproduct The sense and antisense transcripts that resultfrom the LTR promoter activities appear to have differentfates in the investigated cell lines The levels of thelncRNA lasDIRS-1 are stable whereas those of thesense RNA vary significantly (Figure 1 summarized inFigure 8) In particular we observe a dramatic accumula-tion of full-length and shorter DIRS-1 sense transcriptsnext to shorter versions of the lasDIRS-1 lncRNA in alldeletion strains of the rrpC gene (Figure 1) As shown byRNA FISH additional DIRS-1 signals can be observed inthe nucleus of rrpCmdash cells (Figure 3) and it appears plaus-ible that the accumulating shorter sense and antisensetranscripts that we observe by northern blotting are con-tained in these spots Bidirectional transcription has alsobeen observed for retrotransposons in other organismsincluding LINE-1 in humans (47) LINE elementsSINE elements and LTR retrotransposons in mice(4849) Tc1 in C elegans (50) and transposons inDrosophila (5152)

Concurrent with the accumulation of long transcriptswe observe by northern blot analyses and by deepsequencing (Figures 4 and 5) a drastic reduction ofsmall DIRS-1 RNAs in strains lacking the rrpC gene Inthe deep sequencing analysis we find the majority of smallRNAs to be asymmetrically distributed between the senseand antisense orientations of the retroelement in AX2cells This result indicates that these small RNAs are notreaction products of one of the two Dicer-like proteins inD discoideum (213943) because Dicers are expected togive rise to double-stranded siRNAs (53ndash55) whichshould cover DIRS-1 symmetrically in sense and antisenseorientationBecause the majority of small DIRS-1 RNAs disappear

in the absence of RrpC the simplest explanation is thatthese are directly synthesized by this RdRP The synthesisof small RNAs by RdRPs has also been described in otherspecies including C elegans (4456) Neurospora crassa(57) and Arabidopsis thaliana (58) Our conclusion isalso in line with our recently reported discovery thatRrpC is required for the 50 spreading of an RNA silencing

Figure 7 Southern blot analysis for genomic mobilization of DIRS-1 in the rrpCmdash strain (A) Schematic representation of DIRS-1 with recognitionsites of Eco RI and Kpn I that cleave within the retrotransposon sequence while Sac I (not shown) does not (B) Agarose gel electrophoresis of 20 mggenomic DNA from AX2 wild-type and rrpCmdash strains digested with the indicated restriction endonucleases As controls for hybridization 1 or 10 ngundigested plasmid DNA featuring the DIRS-1 sequence was applied The sizes of the 1-kb marker fragments (ThermoScientific) are indicated (C)Southern blot using a 32P-labeled DNA probe corresponding to the RE position Note that the positions with an additional or stronger signalsobserved for the rrpCmdash genomic DNA are indicated by arrows using the same colors for Eco RI and Kpn I as in (A) Changes in patterns of DNAdigested with Sac I which cleaves outside DIRS-1 are denoted by black arrows

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signal (9) Changes in the distribution of small DIRS-1RNAs in the rrpCmdash strain compared with AX2 appearto cluster in four regions (boxes I-IV in Figure 5summarized in Figure 8) which correspond to the LTRwith unchanged levels (I) to the 50-end of GAG with smallRNAs absent in AX2 (II) to a region with barely anysmall RNAs left (III) and to a region with reducednumbers but a similar distribution pattern (IV) For thelatter RrpC might serve as the amplifying component of aprimary silencing signal like a double-stranded RNASuch an entity has been proposed earlier by Fire andMello when they discovered RNAi in C elegans (4)The responsible enzyme was characterized subsequentlyas the RdRP EGO-1 (5) The small RNAs in region IIIappear to strongly accumulate in antisense orientation(Figure 5E) We propose that RrpC synthesizes thesesmall RNAs using the DIRS-1 sense transcript as atemplate Why these RNA appear to be non-functional

in the post-transcriptional silencing of the ORF I-GFPfusion protein (Figure 6A) is currently unclear but onepossible reason is that their concentration in that regionmight be too low (Figure 5)

The small RNAs of the LTR region on the other handappear to be unaffected by the deletion of rrpC and inview of their symmetric sense and antisense distributionthey might represent Dicer products In this respect it isworth noting that the sense transcript has been shown tocontain little of the left LTR but nearly the entire rightLTR (18) Of these expressed right and left LTR se-quences 58 nt and 33 nt respectively are complementaryto a DIRS-1 region termed the ICR During DIRS-1retrotransposition the ICR is proposed to bring the50-end and the 30-end of a reverse transcript together(11) to allow for the formation of a single-strandedcircular DNA replication intermediate (17) It seemsplausible to suggest that such base pairing also takes

Figure 8 Summary of DIRS-1 RNAs in AX2 and rrpCmdash strains (A) Schematic representation of the DIRS-1 sequence (for details see Figure 1A)Relative levels of DIRS-1 sense transcripts (B) antisense transcripts (C) and small RNAs (D) are shown for the AX2 wild-type (purple) and for therrpCmdash (light blue) strains Arrows in (B) and (C) represent the transcripts mapped onto the DIRS-1 element in (A) The thickness of the arrowsindicates relative changes in RNA quantity Note that RNA levels are comparable only within panels and that the positions of the shorter fragments(dotted) are not exactly determined In (D) the distribution of small RNAs is also mapped onto the element in (A) and the height of the peaksindicates abundance using the same relative scaling (for details see Figure 5)

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place on the RNA level and the resulting RNA doublestrands would be long enough to allow for Dicer activitygiving rise to the RrpC-independent DIRS-1 LTR siRNAs(box I in Figures 5 and 8)

Although the small DIRS-1 RNAs that are present onlyin the absence of RrpC (box II Figures 5 and 8) are low innumber they appear reproducibly Similarly elevatedsmall RNA levels in strains lacking the functional rrpCgene have also been shown for endogenous microRNAs(3943) the retrotransposon Skipper (43) and recentlyalso in transgene silencing (9) Given that RdRPs areenzymes that synthesize RNA small RNA accumulationin their absence is counter-intuitive However someRdRPs in other organisms (5758) have been proposedto act in a small RNA primer-dependent mode in whichthe RNA primer is extended resulting in the formation oflong double-stranded RNAs which act as bona fide Dicersubstrates If primer extension does not take place theprimers might accumulate In view of small RNAs fromvarious genetic backgrounds accumulating in rrpCmdash

strains a primer-dependent activity of this RdRP onthese sequences appears to be a plausible interpretationAlbeit indirectly this interpretation is also supported bythe observation that classical (30ndash50) transitive silencing inD discoideum depends on both the dicer-related nucleaseDrnB and RrpC (9) Also for several other RdRPs bothprimer-dependent and -independent modes of action havebeen inferred like QDE-1 in N crassa (57) or theA thaliana enzyme RDR6 (5859) which act in theprimer-dependent mode also in the biosynthesis oftasiRNAs (5860)

To our surprise we observed significant levels of smallRNAs 30 to the endogenous DIRS-1 small RNA triggerduring the analysis of the C-terminal GFP fusion con-structs of the DIRS-1 ORFs (Figure 6) Substantialamounts of GFP-specific small RNAs were only detect-able for the ORF II and ORF III fusion constructs butnot for ORF I-GFP Early work on the activity of RdRPshas shown that unprimed activity is preferentiallyobserved at the 30-end of mRNAs (6162) Because thesmall GFP RNAs are not observed in the GAGndashGFPconstruct it appears unlikely that the GFP mRNAsequence itself is recognized as aberrant and is causativefor the observed spreading in the 30 direction Because thepresence of GFP alone does not lead to the generation ofsmall GFP RNAs (Figures 2 and 6D and E) this possi-bility can be excluded From the use of probes monitoringsmall GFP RNAs in the sense orientation we concludethat these are not direct products of RrpC on the mRNAof the respective ORFndashGFP Rather they are likely pro-cessing products of an RrpC-dependant double-strandthat might be targeted by one of the D discoideumArgonaute or Dicer proteins

In a previous study we had investigated transcriptionalsilencing of DIRS-1 by DNA methylation Although theDIRS-1 right LTR was found to be methylated by the soleDNA methyltransferase in D discoideum DnmA (21) adeletion of its gene did not result in a transcriptionalupregulation of DIRS-1 From this we concluded thatDIRS-1 is not under transcriptional control by DnmAunlike the retrotransposon Skipper (21) However we

cannot exclude the possibility that other components ofthe heterochromatization machineries in the amoebamight add a transcriptional control of DIRS-1 inaddition to the post-transcriptional control by RrpCthat we report hereControl at the post-transcriptional level appears uneco-

nomic as DIRS-1 transcripts are first generated in orderto then be degraded However this control might be aconsequence of the integration behavior of DIRS-1Because DIRS-1 retrotransposition takes place preferen-tially into other DIRS-1 elements or fragments thereof(15) highly diverse DIRS-1 sequences are expected to beproduced In these diverse DIRS-1 sequences motifs otherthan the LTRs might serve as promoters which thenmight render the mobile genetic elements refractory totranscriptional control at least by using LTR promotermethylation

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online

ACKNOWLEDGEMENTS

The authors thank Dianna K Bautista and TimothyJ Wilson for most helpful comments on the manuscriptThe authors acknowledge support from Science for LifeLaboratory the National Genomics Infrastructure (NGI)and Uppmax for providing assistance in massively parallelsequencing and computational infrastructure They alsothank Markus Maniak for providing antibodies againstGFP Severin and Coronin

FUNDING

The Deutsche Forschungsgemeinschaft [Heisenbergstipend HA34595-2 HA34597-1 to CH Wi11426-1to TW] by the European Molecular BiologyOrganisation [ASTF497-2011 to CS] and by theSwedish Research Council [to OE and FS] Supportedby a stipend from the Land Hessen (to DM and BB)Funding for open access charge DeutscheForschungsgemeinschaft

Conflict of interest statement None declared

REFERENCES

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2 MaidaY and MasutomiK (2011) RNA-dependent RNApolymerases in RNA silencing Biol Chem 392 299ndash304

3 WasseneggerM and KrczalG (2006) Nomenclature andfunctions of RNA-directed RNA polymerases Trends Plant Sci11 142ndash151

4 FireA XuS MontgomeryMK KostasSA DriverSE andMelloCC (1998) Potent and specific genetic interference bydouble-stranded RNA in Caenorhabditis elegans Nature 391806ndash811

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8 VoinnetO VainP AngellS and BaulcombeDC (1998)Systemic spread of sequence-specific transgene RNA degradationin plants is initiated by localized introduction of ectopicpromoterless DNA Cell 95 177ndash187

9 WiegandS and HammannC (2013) The 50 spreading of smallRNAs in Dictyostelium discoideum depends on the RNA-dependent RNA polymerase RrpC and on the dicer-relatednuclease DrnB PLoS One 8 e64804

10 MartensH NovotnyJ OberstrassJ SteckTL PostlethwaitPand NellenW (2002) RNAi in Dictyostelium the role of RNA-directed RNA polymerases and double-stranded RNase MolBiol Cell 13 445ndash453

11 PoulterRT and GoodwinTJ (2005) DIRS-1 and the othertyrosine recombinase retrotransposons Cytogenet Genome Res110 575ndash588

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13 DubinM FuchsJ GrafR SchubertI and NellenW (2010)Dynamics of a novel centromeric histone variant CenH3 revealsthe evolutionary ancestral timing of centromere biogenesisNucleic Acids Res 38 7526ndash7537

14 GlocknerG and HeidelAJ (2009) Centromere sequence anddynamics in Dictyostelium discoideum Nucleic Acids Res 371809ndash1816

15 CappelloJ CohenSM and LodishHF (1984) Dictyosteliumtransposable element DIRS-1 preferentially inserts into DIRS-1sequences Mol Cell Biol 4 2207ndash2213

16 WincklerT SchiefnerJ SpallerT and SiolO (2011)Dictyostelium transfer RNA gene-targeting retrotransposonsstudying mobile element-host interactions in a compact genomeMob Genet Elements 1 145ndash150

17 CappelloJ HandelsmanK and LodishHF (1985) Sequence ofDictyostelium DIRS-1 an apparent retrotransposon with invertedterminal repeats and an internal circle junction sequence Cell 43105ndash115

18 CohenSM CappelloJ and LodishHF (1984) Transcription ofDictyostelium discoideum transposable element DIRS-1 Mol CellBiol 4 2332ndash2340

19 RosenE SivertsenA and FirtelRA (1983) An unusualtransposon encoding heat shock inducible anddevelopmentally regulated transcripts in Dictyostelium Cell 35243ndash251

20 ZukerC CappelloJ ChisholmRL and LodishHF (1983)A repetitive Dictyostelium gene family that is induced duringdifferentiation and by heat shock Cell 34 997ndash1005

21 KuhlmannM BorisovaBE KallerM LarssonP StachDNaJ EichingerL LykoF AmbrosV SoderbomF et al(2005) Silencing of retrotransposons in Dictyosteliumby DNA methylation and RNAi Nucleic Acids Res 336405ndash6417

22 PallGS Codony-ServatC ByrneJ RitchieL and HamiltonA(2007) Carbodiimide-mediated cross-linking of RNA to nylonmembranes improves the detection of siRNA miRNA andpiRNA by northern blot Nucleic Acids Res 35 e60

23 SouthernEM (1975) Detection of specific sequences amongDNA fragments separated by gel electrophoresis J Mol Biol98 503ndash517

24 HughesJE and WelkerDL (1988) A mini-screen technique foranalyzing nuclear DNA from a single Dictyostelium colonyNucleic Acids Res 16 2338

25 GreenMR and SambrookJ (2012) Molecular Cloning aLaboratory Manual 4th edn Cold Spring Harbor LaboratoryPress Cold Spring Harbor NY

26 LeviS PolyakovM and EgelhoffTT (2000) Green fluorescentprotein and epitope tag fusion vectors for Dictyosteliumdiscoideum Plasmid 44 231ndash238

27 FaixJ KreppelL ShaulskyG SchleicherM and KimmelAR(2004) A rapid and efficient method to generate multiplegene disruptions in Dictyostelium discoideum using a singleselectable marker and the Cre-loxP system Nucleic Acids Res32 e143

28 VeltmanDM AkarG BosgraafL and Van HaastertPJ(2009) A new set of small extrachromosomal expression vectorsfor Dictyostelium discoideum Plasmid 61 110ndash118

29 HofmannP KruseJ and HammannC (2013) Transcriptlocalization in D discoideum cells by RNA FISH Methods MolBiol 983 311ndash323

30 BilzerA DolzH ReinhardtA SchmithA SiolO andWincklerT (2011) The C-module-binding factor supportsamplification of TRE5-A retrotransposons in the Dictyosteliumdiscoideum genome Eukaryot Cell 10 81ndash86

31 LucasJ BilzerA MollL ZundorfI DingermannTEichingerL SiolO and WincklerT (2009) The carboxy-terminaldomain of Dictyostelium C-module-binding factor is anindependent gene regulatory entity PLoS One 4 e5012

32 PfafflMW (2001) A new mathematical model for relativequantification in real-time RT-PCR Nucleic Acids Res 29 e45

33 HallmanJ AvessonL ReimegardJ KallerM andSoderbomF (2013) Identificaton and verification of microRNAsby high-throughput sequencing Methods Mol Biol 983 125ndash138

34 MartinM (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads EMBnet J 17 10ndash12

35 LangmeadB TrapnellC PopM and SalzbergSL (2009)Ultrafast and memory-efficient alignment of short DNAsequences to the human genome Genome Biol 10 R25

36 JurkaJ KapitonovVV PavlicekA KlonowskiP KohanyOand WalichiewiczJ (2005) Repbase Update a database ofeukaryotic repetitive elements Cytogenet Genome Res 110462ndash467

37 RobinsonJT ThorvaldsdottirH WincklerW GuttmanMLanderES GetzG and MesirovJP (2011) Integrativegenomics viewer Nat Biotechnol 29 24ndash26

38 WiegandS KruseJ GronemannS and HammannC (2011)Efficient generation of gene knockout plasmids for Dictyosteliumdiscoideum using one-step cloning Genomics 97 321ndash325

39 AvessonL ReimegardJ WagnerEG and SoderbomF (2012)MicroRNAs in Amoebozoa deep sequencing of the small RNApopulation in the social amoeba Dictyostelium discoideum revealsdevelopmentally regulated microRNAs RNA 18 1771ndash1782

40 WernerA (2005) Natural antisense transcripts RNA Biol 253ndash62

41 FaghihiMA and WahlestedtC (2009) Regulatory roles ofnatural antisense transcripts Nat Rev Mol Cell Biol 10637ndash643

42 AspegrenA HinasA LarssonP LarssonA and SoderbomF(2004) Novel non-coding RNAs in Dictyostelium discoideum andtheir expression during development Nucleic Acids Res 324646ndash4656

43 HinasA ReimegardJ WagnerEG NellenW AmbrosVRand SoderbomF (2007) The small RNA repertoire ofDictyostelium discoideum and its regulation by components ofthe RNAi pathway Nucleic Acids Res 35 6714ndash6726

44 PakJ and FireA (2007) Distinct populations of primary andsecondary effectors during RNAi in C elegans Science 315241ndash244

45 KlahreU CreteP LeuenbergerSA IglesiasVA andMeinsF Jr (2002) High molecular weight RNAs and smallinterfering RNAs induce systemic posttranscriptionalgene silencing in plants Proc Natl Acad Sci USA 9911981ndash11986

46 BraunsteinTH MouryB JohannessenM and AlbrechtsenM(2002) Specific degradation of 30 regions of GUS mRNA inposttranscriptionally silenced tobacco lines may be related to 50-30

spreading of silencing RNA 8 1034ndash1044

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47 YangN and KazazianHH Jr (2006) L1 retrotransposition issuppressed by endogenously encoded small interfering RNAs inhuman cultured cells Nat Struct Mol Biol 13 763ndash771

48 WatanabeT TakedaA TsukiyamaT MiseK OkunoTSasakiH MinamiN and ImaiH (2006) Identification andcharacterization of two novel classes of small RNAs in the mousegermline retrotransposon-derived siRNAs in oocytes and germlinesmall RNAs in testes Genes Dev 20 1732ndash1743

49 WatanabeT TotokiY ToyodaA KanedaM Kuramochi-MiyagawaS ObataY ChibaH KoharaY KonoTNakanoT et al (2008) Endogenous siRNAs from naturallyformed dsRNAs regulate transcripts in mouse oocytes Nature453 539ndash543

50 SijenT and PlasterkRH (2003) Transposon silencing in theCaenorhabditis elegans germ line by natural RNAi Nature 426310ndash314

51 ChungWJ OkamuraK MartinR and LaiEC (2008)Endogenous RNA interference provides a somatic defense againstDrosophila transposons Curr Biol 18 795ndash802

52 GhildiyalM SeitzH HorwichMD LiC DuT LeeS XuJKittlerEL ZappML WengZ et al (2008) EndogenoussiRNAs derived from transposons and mRNAs in Drosophilasomatic cells Science 320 1077ndash1081

53 BernsteinE CaudyAA HammondSM and HannonGJ(2001) Role for a bidentate ribonuclease in the initiation step ofRNA interference Nature 409 363ndash366

54 KettingRF FischerSE BernsteinE SijenT HannonGJand PlasterkRH (2001) Dicer functions in RNA interferenceand in synthesis of small RNA involved in developmental timingin C elegans Genes Dev 15 2654ndash2659

55 KnightSW and BassBL (2001) A role for the RNase IIIenzyme DCR-1 in RNA interference and germ line developmentin Caenorhabditis elegans Science 293 2269ndash2271

56 SijenT SteinerFA ThijssenKL and PlasterkRH (2007)Secondary siRNAs result from unprimed RNA synthesis andform a distinct class Science 315 244ndash247

57 MakeyevEV and BamfordDH (2002) Cellular RNA-dependentRNA polymerase involved in posttranscriptional genesilencing has two distinct activity modes Mol Cell 101417ndash1427

58 MoissiardG ParizottoEA HimberC and VoinnetO (2007)Transitivity in Arabidopsis can be primed requires the redundantaction of the antiviral Dicer-like 4 and Dicer-like 2 and iscompromised by viral-encoded suppressor proteins RNA 131268ndash1278

59 CurabaJ and ChenX (2008) Biochemical activities ofArabidopsis RNA-dependent RNA polymerase 6 J Biol Chem283 3059ndash3066

60 AllenE XieZ GustafsonAM and CarringtonJC (2005)microRNA-directed phasing during trans-acting siRNA biogenesisin plants Cell 121 207ndash221

61 SchiebelW HaasB MarinkovicS KlannerA and SangerHL(1993) RNA-directed RNA polymerase from tomato leaves IPurification and physical properties J Biol Chem 26811851ndash11857

62 SchiebelW HaasB MarinkovicS KlannerA and SangerHL(1993) RNA-directed RNA polymerase from tomato leaves IICatalytic in vitro properties J Biol Chem 268 11858ndash11867

Nucleic Acids Research 2014 Vol 42 No 5 3345

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