Patent Application: US-201314400131-A

Abstract:
the present invention relates to small rnas , inhibitors thereof , inhibitors of enzymes producing thereof , and their use to modulate the response of a cell to a dna damaging event . the invention concerns also a method to detect the presence or quantify dna damage .

Description:
early passage bj cells , wi38 and mrc - 5 ( the american type culture collection , atcc ) were grown under standard tissue culture conditions ( 37 ° c ., 5 % co 2 ) in mem supplemented with 10 % fetal bovine serum , 1 % l - glutamine , 1 % non - essential aminoacids , 1 % na pyruvate . hela , phoenix ecotrophic and hek293t cell lines were grown under standard tissue culture conditions ( 37 ° c ., 5 % co 2 ) in dmem , supplemented with 10 % fetal bovine serum , 1 % glutamine , 1 % penicillin / streptomycin . rko , hct 116 and dld1 colon cancer cell lines 25 were cultured in mc &# 39 ; coy 5a medium + 10 % fetal calf serum , 1 % penicillin / streptomycin . nih2 / 4 35 where grown in dmem , supplemented with 10 % fetal bovine serum , 1 % glutamine , gentamicine ( 40 μg / ml ), and hygromycin ( 400 μg / ml ). h - rasv12 overexpressing senescent bj cells were generated as in 20 . brdu incorporation assays were carried at least a week after cultures had fully entered the senescent state , as determined by ceased proliferation , ddr activation , sahf formation , and senescence - associated β - galactosidase expression . ionizing radiation ( ir ) was induced by a high - voltage x - rays generator tube ( faxitron x - ray corporation ). in general , cultured cells were exposed to 2 grays for the foci formation assay . the authors used 5 grays for the g2 / m checkpoint assays and 10 grays for the g1 / s checkpoint assays . cherry - lac and i - sce i - restriction endonuclease expressing vector were transfected by lipofectamine 2000 ( invitrogen ) in a ratio of 3 : 1 . 16 h post transfection around 70 % of the cells were scored positive for ddr markers at the lac array . for generation of dicer and drosha knocked - down nih2 / 4 cells were infected with lentiviral particles carrying plko . 1 , shdicer or shdrosha vectors . after 48 hours cells were superinfected with adeno empty vector or adeno i - sce i [ anglana et al . nucl ac res 1999 ]. nuclei were isolated the day after the adenoviral infection . transient expression of er - i - ppoi endonucleases in hela cells was carried out by lipofectamine 2000 transfection and 16 hours later tamoxifen ( 0 . 1 μm ) was added to culture medium to induce the activation of the endonuclease . 4 hours later cells were fixed for immunostaining or used for rna extraction . cherry - lac transfected ( mock ) cells were used as control in these experiments . cultured cells and lna transfection ( for the experiments in fig3 ). nih2 / 4 cells where grown in dmem , supplemented with 10 % fetal bovine serum , 1 % glutamine , gentamicine ( 40 mg / ml ), and hygromycin ( 400 mg / ml ). cherry - lac and i - sce i - restriction endonuclease expressing vectors were transfected with lipofectamine 2000 ( invitrogen ) with a 3 : 1 ratio . lna were first boiled at 90 ° c . for 5 minutes and quickly chilled at 4 ° c . for 5 minutes and then added in different combinations to the cherry - lac and i - sce i transfection mix , at the final concentration of 200 pm . 24 h post transfection cells were scored for ddr markers at the lac array . t19 fibrosarcoma cells ( van steensel , cell 1998 ) were grown in dmem supplemented with 10 % fetal bovine serum , 1 % glutamine and doxycycline ( 100 ng / ml ). for induction , cells were grown without doxycycline for at least 7 - 8 days . cre - er trf2 flox / flox mefs ( lazzerini denchi and de lange , nature 2007 ) were grown in dmem supplemented with 10 % fetal bovine serum and 1 % glutamine . for induction , cells were grown in presence of 4 - hydroxytamoxifen ( 600 nm ) for 48 hours . for brdu incorporation , cells were labeled with 10 μg / ml bromodeoxyuridine ( brdu , sigma ) for 16 hours and incorporation was evaluated by immunofluorescence after dna denaturation . mouse anti - γh2ax , anti - h3k9 me3 , rabbit polyclonal anti - ph3 ( upstate biotechnology ); anti - ps / tq ( cell signaling technology ); anti - h2ax , anti - h3 and anti dicer ( 13d6 ) ( abcam ); rabbit polyclonal anti - 53bp1 ( novus biological ) and mouse monoclonal anti - 53bp1 ( a gift from thanos halazonetis ); anti - mre11 ( a gift from s . jackson ); anti ph3 , anti - brdu ( becton dickinson ); rabbit polyclonal anti - mcm2 ( a gift of marine melixetian ); anti mre11 rabbit polyclonal raised against recombinant mre11 ; anti - patm ( rockland ); mouse monoclonal anti - atm and anti - mdc1 ( sigma ); anti - vinculin ( clone hvin - 1 ), anti - β - tubulin ( clone aa2 ) and anti - flag m2 monoclonal antibodies ( sigma ). cells were grown on poly - d - lysinated coverslips ( poly - d - lysine was used at 50 μg / ml final concentration ) and plated ( 15 - 20 × 10 3 cells / cover ) one day before staining ddr and brdu staining was performed as in 20 . cells were fixed in 4 % paraformaldehyde or methanol : acetone 1 : 1 . nih2 / 4 mouse cells were fixed by 4 % paraformaldehyde as in 35 . images were acquired using a wide field olympus biosystems microscope bx71 and the analysis or the metamorph software ( soft imaging system gmbh ). comparative immunofluorescence analyses were performed in parallel with identical acquisition parameters ; at least 100 cells were screened for each antigen . cells with more than 2 ddr foci were scored positive . foci intensity quantifications were performed using cell profiler software 2 . 0 . confocal sections were obtained with a leica tcs sp2 aobs confocal laser microscope by sequential scanning . cells were fixed with 1 : 1 methanol / acetone solution for 2 minutes at room temperature , or 4 % paraformaldehyde for 10 minutes at room temperature . after blocking , cells were stained with primary antibodies for 1 h at room temperature , washed and incubated with conjugated secondary antibodies for 40 minutes at rt . nuclei were stained with dapi ( 1 μg / ml ). flag - dicer , flag - dicer44ab and flag - dicer110ab were a kind gift of r . shiekhattar . plko . 1 shdicer expressing vector was a kind gift of wc . hahn . short hairpin sequence for dicer is : ccg gcc aca cat ctt caa gac tta act cga gtt aag tct tga aga tgt gtg gtt ttt g ( seq id no : 1 ). pretrosuper shp53 as in 20 . short hairpin sequence for p53 was : agt aga tta cca ctg gag tct t ( seq id no : 2 ). cherry - lac - repressor and i - sce i - restriction endonuclease expressing vectors were kind gifts of e . soutoglou 35 . er - i - ppo i - restriction endonuclease expressing vector was a kind gift of michael kastan 33 . shrna against mouse dicer and drosha expressing vectors were a kind gift of w . c . hahn . shrna for mouse dicer : ccg ggc ctc act tga cct gaa gta tct cga gat act tca ggtcaa gtg agg ctt ttt ( seq id no : 3 ). shrna for mouse drosha : ccg gcc tgg aat atg tcc aca ctt tct cga gaa agt gtg gac ata ttc cag gtt ttt g ( seq id no : 4 ). the dharmacon sigenome smartpool sirna oligonucleotide sequences for human 53bp1 , atm , dicer , drosha were : the dharmacon sigenome si rna sequences for human tnrc6a , b and c were : sirnas were transfected by oligofectamine ( invitrogen ) at a final concentration of 200 nm in ois cells and 100 nm in hnf . in the sirna titration experiment we transfected ois cells in parallel with 20 nm and 200 nm sirna oligos . for sirna transfection with deconvolved sirna oligos the authors used 50 nm for smart pools and 12 . 5 nm for deconvolved sirnas . total rna was isolated from cells using trizol ( invitrogen ) or rnaeasy kit ( qiagen ) according to the manufacturer &# 39 ; s instructions , and treated with dnase before reverse transcription . for microrna isolation the authors used mirvana ™ mirna isolation kit ( ambion ). cdna was generated using the superscript ii reverse transcriptase ( invitrogen ). cdna was used as template in taqman ® gene expression assays ( applied biosystems ) for the evaluation of dicer ( assay id : hs00998580_m1 ) and drosha ( assay id : hs01095030_m1 ) mrna levels . taqman ® microrna assays ( applied biosystems ) were used for the evaluation of mature mir - 21 and rnu44 and rnu19 expression levels ( assay id : 000397 , 001094 and 001003 ). 18s or β - actin was used as a control gene for normalization . mir21 and rnu44 enrichment in the small rna - enriched fraction was evaluated as the ratio between pcr cycles ( ct ) for mir - 21 or rnu44 and for β - actin mrna after normalization to the same ratio in total rna fraction . real - time quantitative pcr reactions were performed on an applied biosystems abi prism 7900ht sequence detection system or on a roche lightcycler 480 sequence detection system . the reactions were prepared using sybr green reaction mix from roche . ribosomal protein p0 ( rpp0 ) was used as a human and mouse control gene for normalization . cells were plated on poly - d - lysinated coverslips and irradiated with 2 gy of ir . 1 h after hela cells were permeabilized with 2 % tween 20 in pbs for 10 minutes at rt while i - sce i - transfected nih2 / 4 cells were permeabilized in 0 . 5 % tween 20 in pbs for 10 minutes at rt . rnase a treatment was carried out in 1 ml of 1 mg / ml ribonuclease a from bovine pancreas ( sigma - aldrich cat n : r5503 ) in pbs for 25 minutes at room temperature . after rnase a digestion , the samples were washed with pbs , treated with 80 units of rnase inhibitor ( rnaseout invitrogen 40 units / μl ) and 2 μg / ml of α - amanitin ( sigma ) for 15 minutes in a total volume of 70 μl . for experiments with mirin , nih2 / 4 cells were incubated at this point also with 100 μm mirin ( sigma ) or dmso for 15 minutes . then , rnase a - treated cells were incubated with total , small or gel extracted rna , or the same amount of trna , for additional 15 minutes at room temperature . if using mirin , nih2 / 4 cells were incubated with total rna in the presence of 100 μm mirin or dmso for 25 minutes at room temperature . cell were then fixed with 4 % paraformaldehyde or methanol : acetone 1 : 1 . in complementation experiments with synthetic rna oligonucleotides , eight rna oligonucleotides with the potential to form four pairs were chosen among the sequences obtained by deep sequencing that map at the integrated locus in nih2 / 4 cells . synthetic rna oligonucleotides were generated by sigma with a monophosphate modification at the 5 ′ end . sequences map to different regions of the integrated locus : two pairs map to a unique sequence flanking the i - sce i restriction site ( oligo 1 + oligo 2 and oligos 3 + oligo 4 ), one to the lac - operator ( oligo 5 + oligo 6 ) and one to the tet - repressor repetitive sequences ( oligo 7 + oligo 8 ). two paired rna oligonucleotides with the sequences of gfp were used as negative control ( oligo gfp 1 + oligo gfp 2 ). sequences are reported below . rnas were resuspended in 60 mm kcl , 6 mm hepes - ph 7 . 5 , 0 . 2 mm mgcl2 , at the stock concentration of 12 . 5 μm , denatured at 95 ° c . for 5 minutes and annealed for 10 minutes at room temperature . dicer rna products were generated as follows . a 550 bp dna fragment carrying the central portion of the genomic locus studied ( three lac repeats , the i - sce i site and two tet repeats ) was flanked by t7 promoters at both ends and was used as a template for in vitro transcription with the turboscript t7 transcription kit ( amsbio ). the 500 nt long rna obtained was purified and incubated with human recombinant dicer enzyme ( amsbio ) to generate 22 - 23 nt rnas . rna products were purified , quantified and checked on a polyacrylamide or an agarose gel . as a control , the same procedure was followed with a 700 bp construct containing the rfp dna sequence . equal amounts of dicer products generated in this way were used in complementation experiment in nih2 / 4 cells following rnase treatment . cre - er trf2 flox / flox mefs ( lazzerini denchi and de lange , nature 2007 ) were induced to generate trf2 knockout and telomere uncapping . 48 hours later cells were permeabilized with 0 . 6 % tween 20 in pbs for 15 min at room temperature . rnase a treatment was carried out in 1 ml of 1 mg / ml ribonuclease a from bovine pancreas ( sigma - aldrich catalogue no . r5503 ) in pbs for 30 minutes at room temperature . lna were first boiled at 90 ° c . for 5 minutes , chilled at 4 ° c . for 5 minutes and transfected with lipofectamine rnaimax ( invitrogen ) at the final concentration of 200 nm . total rna was isolated from cells using trizol ( invitrogen ) according to the manufacturer &# 39 ; s instructions . to generate small rna - enriched fraction and small rna - devoid fraction the authors used mirvana ™ microrna isolation kit ( ambion ) according to the manufacturer &# 39 ; s instructions . the mirvana microrna isolation kit employs an organic extraction followed by immobilization of rna on glass - fiber ( silica - fibers ) filters to purify either total rna , or rna enriched for small species . for total rna extraction ethanol is added to samples , and they are passed through a filter cartridge containing a glass - fiber filter , which immobilizes the rna . the filter is then washed a few times , and finally the rna is eluted with a low ionic - strength solution . to isolate rna that is highly enriched for small rna species , ethanol is added to bring the samples to 25 % ethanol . when this lysate / ethanol mixture is passed through a glass - fiber filter , large rnas are immobilized , and the small rna species are collected in the filtrate . the ethanol concentration of the filtrate is then increased to 55 %, and it is passed through a second glass - fiber filter where the small rnas become immobilized . this rna is washed a few times , and eluted in a low ionic strength solution . using this approach consisting of two sequential filtrations with different ethanol concentrations , an rna fraction highly enriched in rna species ≦ 200 nt can be obtained 25 , 66 . total rna samples were heat - denatured , loaded and resolved on a 15 % denaturing acrylamide gel [ 1 × tbe , 7 m urea , 15 % acrylamide ( 29 : 1 acryl : bis - acryl )]. gel was run for 1 hour at 180 v and stained in gelred solution . gel slices were excised according to the molecular weight marker , moved to a 2 ml clean tube , smashed and rna was eluted in 2 ml of ammonium acetate 0 . 5 m , edta 0 . 1 m in rnase - free water , rocking overnight at 4 ° c . tubes were then centrifuged 5 minutes at top speed , the acqueous phase was recovered and rna was precipitated and resuspended in rnase free water . wi38 , bj and mrc - 5 cells were irradiated with 10 gy and 1 hour afterwards incubated with brdu for 7 h ; hct 116 and rko cells were irradiated 2 gy and incubated with brdu for 2 h . cells were fixed with 4 % paraformaldehyde and probed for brdu immunostaining at least 100 cells per condition were analyzed . hek 293 calcium phosphate transfected cells were irradiated with 5 gy and allowed to respond to ir - induced dna damage in a cell culture incubator for 12 , 24 or 36 hours . then , at these three time points post irradiation , together with not irradiated cells , 1 × 10 6 cells were collected for fluorescence activated cell sorting ( facs ) analysis , fixed in 75 % ethanol in pbs , 30 minutes on ice . afterwards , cells were treated 12 hours with 40 μg / ml of rnase a and incubated at least 1 h with propidium iodide ( pi ). facs profiles were obtained by the analysis of at least 5 × 10 5 cells . in the complementation experiments hek 293 were transfected using lipofectamine rnai max ( invitrogen ) and 48 hours later irradiated with 5 gy . cells were then treated as explained above . cells were lysed in sample buffer and 50 - 100 μg of whole cell lysate were resolved by sds - page , transferred to nitrocellulose and probed as in 29 . for zebrafish immunoblotting protein analysis , 72 hours post fertilization ( hpf ) larvae were deyolked in krebs ringer &# 39 ; s solution containing 1 mm edta , 3 mm pmsf and proteases inhibitor ( roche complete protease inhibitor cocktail ). embryos were then homogenized in sds sample buffer containing 1 mm edta with a pestle , boiled 5 min and centrifuged 13000 rpm for 1 min . protein concentration was measured with the bca method ( pierce ) and proteins ( 50 μg - 900 μg ) were loaded in an sds - 12 % ( for γh2ax and h3 ) and sds - 6 % polyacrylamide gel ( for patm and atm ), transferred to a nitrocellulose membrane , and incubated with anti - γh2ax ( 1 : 2000 , a gift from j . amatruda 67 ), h3 ( 1 : 10000 , abcam ), patm ( 1 : 1000 , rockland ), atm ( 1 : 1000 , sigma ). immunoreactive bands were detected with horseradish peroxidase - conjugated anti - rabbit or anti - mouse igg and an ecl detection kit ( pierce , springfield , ill ., usa ). protein loading was normalized to equal amounts of total atm and h3 . zebrafish embryos at the stage of 1 - 2 cells were injected with a morpholino against dicer1 29 diluted in danieau buffer . the morpholino oligonucleotide was injected at a concentration of 5 ng / nl , and a volume of 2 nl / embryo . to assess the efficiency of the morpholino to block microrna maturation , the authors co - injected the morpholino with in vitro synthesized mrna , encoding for red fluorescent protein ( rfp ) and carrying 3 binding site for mirl26 in the 3 ′ utr 28 . the oligonucleotides carrying the binding sites for mirl26 used for construction of pcs2 : rfpmir126 sensor are : the construct was verified by sequencing and used to synthesize mrna in vitro using the mmessage kit ( ambion ). messenger rna encoding for rfpmir126 sensor was injected alone or in combination with dicer1 morpholino at a concentration of 10 pg / nl . dicer morpholino was injected at a concentration of 5 ng / nl , and a volume of 2 nl / embryo . for cell transplantation experiments , the authors injected donor embryos with a mixture of dicer1 morpholino and mrna encoding for gfp ( 5 pg / nl ). approximately 20 cells were transplanted from donor embryos at dome ( 5 hpf ) stage to uninjected host at the same stage . successfully transplanted larvae ( displaying gfp + cells ) were irradiated as described below . mature mirna were reverse transcribed to produce 6 different cdna for taqman ® microrna assay ( 30 ng of total mrna for each reaction ; applied biosystems ). real - time pcr reactions based on taqman reagent chemistry were performed in duplicate on abi prism ® 7900ht fast real - time pcr system ( applied biosystems ). the level of mirna expression was measured using c t ( threshold cycle ). fold change was generated using the equation 2 − ct . for immunofluorescence in zebrafish larvae : 72 hpf larvae were irradiated with 12 gy , fixed in 2 % paraformaldehyde for 2 hours at room temperature . after equilibration in 10 and 15 % sucrose in pbs , larvae were frozen in oct compound on coverslips on dry ice . sections were cut with a cryostat at a nominal thickness of 14 □ m and collected on superfrost slides ( bdh ). antisera used were zebrafish γh2ax — a kind gift of j . amatruda 67 — and patm ( rockland ). gfp fluorescence in transplanted embryos was still easily visible in fixed embryos . images were acquired with a confocal ( leica sp2 ) microscope and 63 × oil immersion lens . nuclear rna shorter than 200 nt was purified using mirvana ™ microrna isolation kit . rna quality was checked on a small rna chip ( agilent ) before library preparation ( supplementary fig2 a ). for illumina hi seq version3 sequencing , spike rna was added to each rna sample in the rna : spike ratio of 10 , 000 : 1 before library preparation and libraries for illumina ga iix were prepared without spike . an improved short rna library preparation protocol was used to prepare libraries 68 . in brief , adenylated 3 ′ adapters were ligated to 3 ′ ends of 3 ′- oh short rnas using a truncated rna ligase enzyme followed by 5 ′ adapter ligation to 5 ′- monophosphate ends using rna ligase enzyme , ensuring specific ligation of undegraded short rnas . cdna was prepared using a primer specific to the 3 ′ adapter in the presence of dimer eliminator and amplified for 12 - 15 pcr cycles using a special forward primer targeting the 5 ′ adapter containing additional sequence for sequencing and a reverse primer targeting the 3 ′ adapter . the amplified cdna library was run on a 6 % polyacrylamide gel and the 100 bp band containing cdnas up to 33 nt was extracted using standard extraction protocols . libraries were sequenced after quality check on a dna high sensitivity chip ( agilent ). multiplexed barcode sequencing was performed on illumina ga - iix ( 35 bp single end reads ) and illumina hi seq version3 ( 51 bp single end reads ). sequences of all the ddrnas identified in this study will be available for free downloading by the time of publication at short read archive . results are shown as means plus / minus standard error ( s . e . m .). p - value was calculated by chi - squared test . qrt - pcr results are shown as means of a triplicate plus / minus standard deviation ( s . d .) and p - value was calculated by student &# 39 ; s t - test as indicated . * indicates p - value & lt ; 0 . 05 . results in fig3 b and c are shown as means plus standard error ( s . e . m .). p - value was calculated by chi - squared test . * indicates p - value & lt ; 0 . 05 , ** indicates p - value & lt ; 0 . 01 , *** indicates p - value & lt ; 0 . 005 . results in fig3 - 36 are shown as means plus standard error of the mean ( s . e . m .). p - value was calculated by chi - squared test . * indicates p - value & lt ; 0 . 05 , *** indicates p - value & lt ; 0 . 001 . statistical significance of downregulation of normalized mirnas in dicer and drosha knockdown samples were calculated using the wilcoxon signed - rank test . the differences in the fraction of 22 - 23 nt vs total short rnas at the locus between the wildtype , dicer knockdown , and drosha knockdown before and after cut was calculated by fitting a negative binomial model to the srna count data and performing a likelihood ratio test , keeping the fraction of 22 - 23 nt vs total short rnas at the locus fixed across conditions under the null hypothesis and allowing it to vary between conditions under the alternative hypothesis . inactivation of dicer and drosha inhibits ddr and allows senescent cells to re - enter into cell cycle oncogene - induced senescence ( ois ) is a non - proliferative state characterized by a sustained ddr 20 , 21 ( caused by high level of endogenous dna damage ) and senescence - associated heterochromatic foci ( sahf ) 22 . since the rnai - machinery has been involved in heterochromatin formation 23 , the authors investigated whether the inactivation of components of the rnai machinery could have an impact on escape from senescence induced in human fibroblasts by transduction of h - rasv12 ( referred here as ois cells ). the authors therefore suppressed the expression of dicer or drosha in ois cells using a pool of small interfering rnas ( sirna ) and monitored cell - cycle progression into s - phase with brdu labeling . the authors observed that dicer or drosha knockdown , as well as atm knockdown used as positive control for escape from senescence 20 ( fig1 a ), result in an increased fraction of brdu - positive cells ( fig7 b ), re - expression of markers of chromosomal dna replication ( fig7 c - e ) and entry into mitosis ( fig7 f - g ). these results could be reproduced over a range of sirna concentrations ( fig8 a - c ) and with four individual sirna oligonucleotides ( fig8 d , e ). perhaps unexpectedly however , in dicer - or drosha - inactivated cells the authors failed to detect any overt impairment in heterochromatin formation and sahf components accumulation as detected by dapi staining and by immunostaining and immunoblotting for the trimethylated form of the histone h3 ( h3k9me3 ) ( fig9 a , b ). since ddr plays a crucial role in the maintenance of the proliferative arrest in ois cells 20 , 21 , the authors monitored whether dicer or drosha inactivation had an impact on ddr foci maintenance . the authors therefore stained cells for markers of active ddr such as the autophosphorylated form of atm ( patm ), phosphorylated substrates of atm and atr ( ps / tq ), 53bp1 and γh2ax . the authors observed that dicer or drosha inactivation significantly reduces the number of 53bp1 , patm and ps / tq foci positive cells ( fig1 a , b and 9 a ) even though 53bp1 , atm or h2ax protein levels are not reduced ( fig9 c ). the authors also observed that the percentage of γh2ax - positive cells did not show a significant variation , although the intensity of γh2ax foci was generally reduced ( fig1 a and b ). these effects could be observed over a range of sirna concentrations ( fig9 d - f ). importantly , inactivation of gw182 / tnrc6a , the main component of the rnai machinery involved in mrna translational control , did not impact on ddr foci detection ( fig1 ), nor the simultaneous inactivation of all three gw182 - like proteins tnrc6a , b and c with two independent pools of sirnas ( fig1 ). therefore , dicer or drosha inactivation impairs ddr signaling and overcomes the ddr - induced proliferative arrest of ois cells . the authors next asked whether the involvement of dicer and drosha in ddr activation is specific for the senescence condition or whether dicer or drosha inactivation has also an impact on ionizing radiation ( ir )- induced ddr activation in proliferating non - senescent cells . therefore , the authors transiently inactivated dicer or drosha by a pool of sirna in human normal fibroblasts ( hnf — wi38 ; fig1 a and b ), exposed them to ir and a few hours later the authors stained them for markers of activated ddr . the authors observed that , despite not reduced levels of protein expression ( fig1 c ), formation of patm , ps / tq , mdc1 , but not γh2ax , foci are impaired in dicer - or drosha - inactivated hnf ( fig1 c and d ). these observations can be reproduced by using four individual sirnas ( fig1 d - g ) and in a different hnf cell line ( bj , data not shown ). under these conditions ( dicer - or drosha - knocked down hnf analyzed 7 hours post ir ), the authors did not observe the dramatic impairment of 53bp1 foci previously observed in ois cells . however , an analysis performed at earlier time points ( 10 ′ after ir ) showed a significant reduction of 53bp1 foci formation in dicer - or drosha - inactivated hnf ( fig1 a ), suggesting that dicer or drosha inactivation delays 53bp1 foci formation . in order to exclude off target effects , the authors expressed an rnai - resistant form of dicer in dicer - knocked down hela cells . the authors observed that re - expression of wild - type dicer , but of not a mutant allele ( dicer44ab ) previously shown to abolish its rna endonuclease activity 24 , allows ddr foci formation to an extent similar to wild type cells , thus confirming dicer - dependency of the effects observed ( fig7 b - d ). finally , the effects observed are independent of mrna translational control , as gw182 knockdown has no significant impact on ddr foci formation ( fig1 a - c ), consistent with the results in ois cells ( fig1 ). as an additional control , the simultaneous inactivation of tnrc6a , b and c or dicer in hela cells expressing a reporter mrna encoding for red fluorescent protein ( rfp ) carrying three binding sites for mir - 126 , used as a sensor for microrna - dependent translational repression , showed that both gw - like proteins and dicer inactivation result in comparable rfp up - regulation , due to the abolished mir - 126 - dependent rfp translational repression ( fig1 a , b and d ); nevertheless , only dicer inactivation affects ddr foci stability ( fig1 c ). to further confirm the involvement of dicer in ddr activation , the authors used a colon cancer cell line ( rko ) carrying a homozygotic genetic deletion of exon 5 in dicer gene and therefore expressing a hypomorphic allele of dicer ( dicer exon5 ); this cell line is defective in micrornas maturation 25 . in dicer exon5 - hypomorphic cells , the level of expression of atm , mdc1 , 53bp1 or h2ax proteins is not reduced ( fig1 a ). however , while in wild - type ( wt ) cells ir induces patm , ps / tq , mdc1 and γh2ax foci , in dicer exon5 - hypomorphic cells ddr foci formation is impaired ( fig1 e and f ). in a time - course experiment , the authors observed that 53bp1 foci formation is delayed in dicer exon5 - hypomorphic cells ( fig1 b and c ). also in this system , the authors observed that γh2ax foci are only mildly affected ( fig1 e and f ). the defects observed in dicer exon5 - hypomorphic cells could be reversed by the re - introduction of wild - type but not of an endonuclease mutant allele of dicer ( dicer44ab ) ( fig1 ). similar conclusions were reached in an additional cell line ( hct 116 ) carrying the same dicer deletion ( data not shown ). next , the authors tested if the absence of ddr foci observed in dicer - or drosha - inactivated cells was due to a defect in actual ddr activation or ddr foci assembly . therefore , the authors performed a set of immunoblot analyses both in dicer - or drosha - interfered hnf and in dicer exon5 - hypomorphyc cell lines . the authors &# 39 ; analyses revealed that ir - induced atm autophosphorylation is impaired in dicer - or drosha - inactivated fibroblasts ( fig1 a and b ) and in irradiated rko dicer exon5 - hypomorphic cells , compared to wild - type cells ( fig1 c ). this indicates that dicer and drosha control atm activation and not just its accumulation in foci . combined , these results reveal that dicer or drosha inactivation impairs ddr activation induced by exogenous sources of dna damage in manner independent from canonical rnai translational repressors ( gw - like proteins ). dicer or drosha inactivation impairs g1 / s and g2 / m dna damage checkpoints . dna damage elicits ddr signal transduction leading to checkpoint - dependent cell - cycle arrest at two critical transition steps : the g1 / s checkpoint and the g2 / m checkpoint 1 . the authors tested whether impaired ddr activation in dicer - or drosha - inactivated cells has an impact on g1 / s and g2 / m checkpoints . to test the g1 / s checkpoint - dependent arrest , cells were irradiated and pulse - labeled with brdu . the authors observed that dicer - and drosha - inactivated hnf ( wi38 ; fig1 a and b ) have an impaired irradiation - induced g1 / s - phase arrest compared to control cells ( fig2 a ) and that the extent of checkpoint deficiency is comparable to that of 53bp1 - interfered cells , used as a positive control 26 ( fig2 a and 19 c ). g1 / s checkpoint impairment was confirmed with four individual sirna oligonucleotides ( fig1 d , e ) and in two additional hnf cell lines ( mrc - 5 and bj ) in separate sets of experiments ( fig1 f , g and data not shown ). consistent with the results in hnf , dicer exon5 - hypomorphic cells , from two distinct cell lines ( rko and hct116 ; fig2 b and 20 a , respectively ), also show an impaired g1 / s transition arrest . to confirm the dependency of the checkpoint on dicer in these cell lines , the authors re - expressed wild - type dicer - cdna in dicer exon5 - hypomorphic cells . indeed , dicer cdna expression restored the g1 / s checkpoint in both dicer exon5 - hypomorphic cell lines ( fig2 b and 20 a ). next , the authors asked if the rna - endonuclease activity of dicer is required for the dna damage - induced checkpoint activation . with this aim , the authors complemented dicer exon5 - hypomorphic cells with two distinct dicer mutants carrying the amino acid substitution asp44 to ala44 ( dicer44ab ) or glu110 to ala110 ( dicer110ab ) known to abolish dicer rna endonuclease activity 24 . while wild - type dicer expression rescued the g1 / s checkpoint defect of dicer exon5 - hypomorphic cells , both dicer mutants failed to do so ( fig2 b ), despite similar levels of dicer expression ( fig2 b ). the authors conclude that the rna processing activity of dicer is necessary to enforce the g1 / s checkpoint . the authors also tested whether dicer is required to arrest cell - cycle progression at the g2 / m boundary following dna - damage . thus , the authors suppressed dicer , or p53 as positive control , in hek293 cells and the authors tested g2 / m checkpoint activation by monitoring the cell - cycle progression profile over time through fluorescence - activated cell sorting ( facs ). as expected , irradiated empty - vector ( ev ) transfected cells progressively accumulate in the g2 phase of the cell cycle , as a consequence of the checkpoint enforcement . differently , dicer , as well as p53 knocked - down cells , did not arrest upon dna damage and passed through the g2 / m transition ( fig2 c and d ). this result was further confirmed by monitoring the percentage of mitotic cells in control and dicer - inactivated cells following ir , based on histone h3 phosphorylation ( ph3 ) ( fig2 c and d ) 27 . furthermore , the defect in g2 / m checkpoint activation in dicer - knocked down cells could be rescued by the expression of a sirna - resistant dicer expressing construct ( fig2 e - g ). these results indicate that dicer - inactivated cells are deficient in the activation of both g1 / s and g2 / m checkpoints and that dicer &# 39 ; s rna processing activity is necessary to enforce the checkpoint after dna damage . to study if dicer is required for ddr activation upon irradiation in a living organism , the authors tested the impact of dicer inactivation in danio rerio ( zebrafish ) larvae , as a model system . zebrafish embryos were injected with morpholino oligonucleotides against dicer1 . efficiency of dicer1 inactivation was assessed by the ability of the morpholino oligonucleotide to block microrna maturation and therefore impede the suppression of the co - injected reporter rfp - mir - 126 28 . in addition , the authors investigated the levels of six different mature micrornas using qpcr to confirm inactivation of dicer1 . larvae originated from embryos injected with morpholino oligonucleotides against dicer1 displayed upregulated rfp expression and the developmental defects previously reported for dicer1 - inactivated larvae 29 ( fig2 a , b ) together with reduced levels of micrornas ( fig2 c , d ). irradiated larvae were stained with antibodies against patm and γh2ax . not irradiated larvae showed no or weak staining ( fig3 ). irradiation induced a strong patm and γh2ax activation in all the cells throughout the sections of wild - type larvae head . differently , irradiated dicer1 morpholino - injected larvae showed a dramatic impairment both in patm and γh2ax signal ( fig3 a ). the observed impact on γh2ax suggests a stronger dependency of γh2ax on dicer1 in zebrafish in vivo , compared with cultured mammalian cells . an immunoblot analysis of protein extracts from wild - type and dicer1 morpholino - injected larvae treated in parallel showed that the impairment in patm and γh2ax accumulation is present in the whole animal ( fig3 b ). the differential response to irradiation of cells with reduced dicer1 activity due to morpholino injection versus dicer1 proficient cells was further confirmed in reciprocal cell transplantation experiments . briefly , cells from embryos injected with dicer1 morpholino and mrna encoding for gfp were transplanted at blastula stage into control uninjected embryos . chimaeric larvae were irradiated at 3 days post fertilization ( dpf ) and stained with antibodies against γh2ax ( fig3 c ). in the reciprocal experiment , control cells from embryos injected with mrna encoding for gfp were transplanted into dicer1 morpholino injected embryos . chimaeric larvae were irradiated as above and stained with antibodies against γh2ax . in both cases , cells with reduced dicer1 activity displayed reduced γh2ax signals compared to their neighboring , dicer1 - proficient cells . ( fig3 c ). the authors conclude that dicer1 is required for ddr activation in vivo in living zebrafish larvae . in an in vitro cell system ddr foci are sensitive to rnase a and dicer and drosha rna products allow ddr foci reformation . the authors then sought an experimental system amenable for the study of the potential direct contribution of dicer and drosha rna products in ddr activation . it has been previously shown that mammalian cells can withstand a transient membrane permeabilization and rnase treatment . this approach allowed the study of the contribution of rna to heterochromatin structure and protein association with chromatin 30 , 31 . the authors therefore utilised this technique to address the contribution of rna in ddr activation . ir - exposed human cells ( hela ) were permeabilized by a mild detergent and treated with the broad - specificity rna nuclease rnase a . this treatment leads to the degradation of both messenger rnas and mirnas including the mrnas of ddr genes ( fig2 a , b ), without significantly affecting ddr protein levels ( fig2 c ). untreated and rnase a - treated irradiated cells were stained for markers of ddr activation . the authors observed that rna degradation strongly impairs 53bp1 , patm , ps / tq and mdc1 foci formation ( fig4 a and b ). this result is consistent with the reported sensitivity of 53bp1 - gfp to ribonuclease treatment 31 . similar to the authors &# 39 ; observations in dicer - and drosha - inactivated cells , γh2ax accumulation is only slightly affected by rnase a ( fig4 a and b ). noteworthy , unperturbed γh2ax signals indicate that rnase a treatment does not dramatically alter chromatin structure or nuclear integrity . intriguingly , 53bp1 , mdc1 and γh2ax triple staining shows that rnase a reduces 53bp1 and mdc1 accumulation at individual γh2ax - foci , which are instead maintained ( fig2 d ), thus suggesting that rna molecules act to favor mdc1 and 53bp1 focus formation once h2ax has been phosphorylated . the authors also noticed that when rnase a is inactivated by an rnase a inhibitor ( rnaseout , a small protein inhibitor ), ddr foci progressively reappear within minutes . in addition , the authors also observed that foci reformation can be prevented by the rna polymerase ii - specific inhibitor a - amanitin ( fig2 a , b ). this suggests that that ddr foci formation is dependent on rna polymerase ii rna products . next , the authors tested if ddr foci that are lost after rnase a treatment can reform following the addition of purified rna to rnase a - treated cells . therefore , irradiated rnase a - treated cells were washed , incubated with rnaseout and a - amanitin and incubated with hela - purified total rna . strikingly , the authors observed that the addition of total rna , but not trna used as control , robustly restores focal accumulation of all ddr factors tested ( fig4 d , e ) within a relatively short time ( 15 minutes ) at room temperature . as ir may induce different kinds of dna lesions , the authors expressed the site - specific endonuclease ppoi 32 , 33 which generates several genomic dsbs . also in this system , the authors could demonstrate that 53bp1 , patm and ps / tq signals assemble in ddr foci that are sensitive to rnase a treatment and that their reformation can be induced by the addition of rna extracted from the same cells ( fig2 ). similar conclusions were reached when using an inducible form of the restriction enzyme asisi 34 ( data not shown ). next , the authors attempted to characterize the rna species involved in ddr - foci reformation by incubating rnase a - treated cells with different rna populations . to gauge the length of the rna molecules involved in ddr focus reformation , the authors enriched total hela rna for short rnas by chromatography (& lt ; 200 nt ; fig2 a ) and the authors used proportional volumes of total and short rna to restore ddr foci in rnase a treated cells . the authors observed that the short rnas - enriched fraction was sufficient to restore patm , ps / tq and 53bp1 foci indicating that this fraction contains the active rna molecules ( fig2 b , c ). to attain better rna size separation , the authors resolved total rna on a polyacrylamide gel and recovered rnas of different lengths : longer than 100 nt , between 100 nt and 35 nt and between 35 nt and 20 nt ( fig4 c and 26 ). using equal amounts of rna from each fraction , the authors observed that only rnas in the 20 - 35 nt size range are active in restoring ddr foci formation ( fig4 c ). thus , two different separation approaches indicate that rna components required for ddr foci assembly are short and in the size range of the rna products generated by dicer and drosha . since the authors observed that inactivation of dicer and drosha affects ddr foci formation in living cells and organisms , the authors reasoned that its small rna products could indeed be responsible for ddr foci restoration in this in vitro cell system . thus , the authors investigated if dicer rna products directly contribute to ddr foci formation . to do so , the authors extracted total rna from wild - type and dicer exon5 - hypomorphic cells and the authors used these two rna preparations to restore ddr foci in rnase a - treated irradiated cells . total rna preparations from the two cell lines are expected to have the same composition apart from the population of dicer rna products 25 . strikingly , while rna extracted from wild - type cells does restore patm , ps / tq or 53bp1 foci , rna extracted from dicer exon5 hypomorphic cells does not ( fig4 d , e ). importantly , rna from dicer exon5 hypomorphic cells transfected with a vector expressing wild - type but not mutant dicer allows ddr foci reformation ( fig2 ). these results can be quantitatively reproduced using rna preparations from two additional cell lines ( hct116 and dld1 ) carrying the same hypomorphic dicer mutation 25 ( fig2 a ) and by the use of rna extracted from cells transiently knocked - down for dicer ( fig2 b - d ). to test if also rna purified from drosha - inactivated cells is unable to restore ddr foci , the authors knocked - down drosha , and gfp as control , by sirna in hela cells , purified rna and used these rna preparations to attempt to restore ddr foci . the authors &# 39 ; experiments revealed that total rna from sigfp control cells restores ddr foci , while rna purified from drosha - inactivated cells does not ( fig2 e ). overall , these observations are consistent with a model in which small rna molecules generated by dicer and drosha are necessary to form ir - induced ddr foci . one conceivable mechanism is that small rna products from dicer and drosha activity suppress the translation of a hypothetical ddr inhibitor . however , the observation that after rnase a treatment ( which degrades both mrnas and micrornas ) ( fig2 a , b ) and a - amanitin treatment ( which inhibits transcription ), gel - purified 20 - 35 nt short rna promote ddr foci reformation , strongly indicates that dicer and drosha rna products control ddr directly and independently from potential mrna targets and translational modulation . moreover , the translation inhibitor cyclohexamide has no impact on ddr foci formation in this system ( data not shown ). these conclusions are consistent with the observation that inactivation of dicer or drosha , but not gw - like proteins involved in translational inhibition , impacts on ddr activation in living cells . ddr focus formation at a defined damaged genomic site requires damage site - specific rnas . ionizing radiations induce the formation of dna lesions that are heterogeneous in nature and random in their location . to reduce this diversity , the authors studied a single dsb at a unique , defined and traceable genomic locus . the authors therefore took advantage of nih2 / 4 mouse cells carrying an integrated copy of the i - sce i restriction site flanked by an array of lac - repressor ( lac ) binding sites and tet repeats 35 . in this system , the expression of the i - sce i restriction enzyme together with the fluorescent protein cherry - lac - repressor ( cherry - lac ) allows the visualization in the nucleus of the site - specific dsb generated by the nuclease . indeed , co - expression of i - sce i and cherry - lac - repressor in nih2 / 4 cells induces a 53bp1 and γh2ax focus that overlaps with a focal cherry - lac signal ( fig5 a ). the authors observed that , also in this system , rnase - a treatment causes the disappearance of the 53bp1 focus from i - sce i - induced dsb ( fig5 a and b ) and a - amanitin prevents 53bp1 focus reformation at the same site ( data not shown ). next , the authors tested if total rna re - addition , following rnase a treatment , restores ddr focus at the i - sce i - induced dna lesion . therefore , rnase a - treated nih2 / 4 cells were incubated with increasing amounts of total rna extracted from cells treated in parallel . when rna was added to the rnase a - treated samples , nih2 / 4 cells re - acquired a bright 53bp1 focus co - localizing with the cherry - lac - repressor in a manner dependent on the rna amount used ( fig5 a and b ). therefore , the very same ddr focus generated on a defined dsb can disassemble and reassemble in a manner dependent on rna . collectively , the results described so far demonstrate that dicer and drosha short rna products control ddr foci formation . however , as such , they do not allow to discriminate whether rnas are generated in cis using the damaged genomic locus as a template or in trans from a distinct locus . to discriminate between these two possibilities , the authors took advantage of the fact that the i - sce i - induced dsb is generated within an exogenous sequence , which is not present in the parental cell line . the authors therefore transfected i - sce i and the cherry - lac - repressor in nih2 / 4 cells and in the nih3t3 parental cell line and the authors used rna extracted from either cell lines to attempt to restore 53bp1 focus formation in rnase a - treated nih2 / 4 cells that had experienced the i - sce i - induced dsb : the two rna preparations are expected to differ only in the potential presence of rna transcripts generated from the exogenous integrated construct carrying the lac and tet repeats and the i - sce i site . excitingly , the 53bp1 focus assembly on the i - sce i - induced dsb , as labeled by cherry - lac - repressor , was efficiently recovered only by the addition of rna purified from nih2 / 4 cells and not by rna extracted from the nih3t3 parental cell line ( fig5 c ). this result indicates that ddr - focus formation requires an rna component , which originates from the damaged genomic locus . the mre11 / rad50 / nbs1 ( mrn ) complex is a key dna damage sensor and a necessary cofactor of the apical ddr regulator atm 1 . also mre11 focus formation upon i - sce i induction is sensitive to rnase a - treatment ( fig2 a ). to probe the molecular mechanisms of action of rnas at sites of dna damage , the authors used mirin , a specific small molecule inhibitor of mrn 36 which , as expected , prevents atm activation also following i - sce i induction ( fig2 b ). the authors therefore tested whether rnas involved in ddr modulation engage mrn . the authors observed that in the presence of mirin , nih2 / 4 rna is unable to induce 53bp1 or patm focus reformation ( fig5 d and e ). this result demonstrates that rnas at sites of dna damage modulate ddr in a mrn - dependent manner . to detect potential short rnas originating from the integrated locus , the authors isolated nuclear rna from parental nih 3t3 cells transfected with the i - sce i ( mock ), nih 2 / 4 cells transfected with cherry - lac - repressor ( uncut ) and from nih 2 / 4 cells transfected with the i - sce i ( cut ) and further selected them for length (& lt ; 200 nt )— this procedure enriches for rnas active in ddr foci reformation 40 folds ( data not shown ). libraries prepared from these samples were sequenced by illumina gaii - x to obtain 15 - 32 bp cdna reads ( fig3 a - d ). their analyses revealed transcripts arising from the exogenous locus which were absent in the parental nih 3t3 cells which had a length distribution of mapped tags peaking around 22 nt , the length of canonical mirnas ( fig2 e ). thus , even an exogenous integrated locus lacking mammalian transcriptional regulatory elements is transcribed and generates short rna transcripts . in order to test whether the short rnas identified at the damaged locus are biologically active in ddr activation , the authors chemically synthesized four potential pairs of short rnas as identified at the cut genomic locus by short rna sequencing as described above and the authors used them to attempt ddr focus reformation in rnase a - treated cells carrying a dsb at the locus . by testing them over a large range of concentrations , the authors could demonstrate that the addition of these rnas , but not equal amounts of control ones , promote site - specific ddr activation at the damaged site ( fig6 a ). these chemically - synthesized short rnas are biologically active both when added to the cells together with total rna from parental cells ( cells not carrying the integrated endogenous locus ; fig6 a ) or with yeast trna ( fig3 , thus in the absence of any additional mammalian rna . in addition , to prove the biological activity of locally generated dicer rna products , the authors cloned the locus , and an unrelated control dna , in a plasmid to allow its transcription in vitro by t7 polymerase and the authors processed the resulting rnas with recombinant dicer protein in vitro . the resulting short rnas ( fig3 g ) were purified and tested in rnase a - treated cells . the authors observed that also these locus - specific dicer - generated rnas , but not equal amounts of control rnas , allow ddr focus reformation both when mixed with rna from parental cells ( fig6 b ) and when mixed with yeast trna ( fig3 h ). thus , in vitro generated dicer rna products promote ddr focus reformation at the dna damaged site in a sequence - specific manner . overall , these results indicate that short rnas with the sequence of the damaged locus play a direct role in ddr activation at the damaged site . in order to further investigate the nature of the rnas generated at the locus , the authors performed deeper sequencing experiments of nuclear rnas & lt ; 200 nt from wildtype nih 2 / 4 samples before and after cut using the illumina hi seq version3 ( fig3 ). to study the biogenesis of these rnas , the authors also sequenced & lt ; 200 nt nuclear rnas from nih 2 / 4 cells following dicer or drosha knockdown ( wt uncut , dicer kd uncut , drosha kd uncut , wt cut , dicer kd cut , drosha kd cut ) ( fig3 a ). to ease normalization , each rna preparation was spiked with a short rna “ spike ” before library preparation . reads were mapped to mirbase 18 and , after spike normalization , were demonstrated to be significantly reduced after dicer or drosha knockdown in uncut and cut samples when compared with wildtype uncut and cut samples respectively ( fig3 b ). this validates the functional efficacy of the knockdowns performed . by analyzing the locus , the authors found that in wildtype samples the bulk of rnas from the locus were in the 22 - 23 nt size range ( 45 . 2 % in wt uncut and 67 . 6 % in the wildtype cut , fig6 c and 33 a ). fitting a negative binomial model to the sequence count data and application of the likelihood ratio test showed that this increase in the fraction of 22 - 23 nt vs total short rnas in wildtype cut sample is statistically significant respect to the uncut sample ( p = 0 . 020 ) ( fig6 c ). further , the authors found that the fraction of 22 - 23 nt vs total short rnas at the locus decreases upon dicer knockdown , both in the uncut ( p = 4 . 8e - 7 ) and cut ( p = 0 . 029 ) conditions , suggesting that the 22 - 23 nt rnas at the locus are indeed dicer dependent ( fig6 c ). the fraction of 22 - 23 nt vs total short rnas at the locus also decreases upon drosha knockdown . to further exclude that the majority of tags arising from the locus were products of random degradation , the authors compared the fraction of 22 - 23 nt vs total rnas at the locus to the same fraction at non - mirna genomic loci — at such loci , any 22 - 23 nt rnas are most likely products of random degradation or dicer / drosha independent enzymatic processing . the authors found that the fraction of 22 - 23 nt vs total short rnas is significantly larger than the fraction found in non - mirna genomic loci before cut ( p = 0 . 049 ) and after cut ( p = 0 . 022 , fig3 b ). finally , the authors observed that the distribution of nucleotides at the 5 ′ and the 3 ′ end of rna sequences from the locus is significantly different from both the genomic background nucleotide distribution ( p = 0 . 012 at the 5 ′ end and 0 . 008 at the 3 ′ end ) as well as the background nucleotide distribution at the locus ( p = 0 . 014 at the 5 ′ end and 1 . 2e - 6 at the 3 ′ end ). specifically , 82 . 9 % sequences start with an a / u and 48 . 6 % sequences end with a g ( fig6 d ). by these analyses the authors therefore conclude that 22 - 23 nt rnas are the bulk of the rna species detected at the locus , they depend on dicer and , to an extent , on drosha , and their proportion increases upon dna damage . their unlikelihood to be random degradation products is further indicated by their differential abundance compared to the rest of non - mirna loci and the observed 5 ′ and 3 ′ base bias . sequence - specific inhibitory oligonuncleotides ( lnas ) reduce ddr activation at a specific genetic locus . the authors previously showed that ddrnas identified by deep sequencing are biologically active and have a causative role in sequence - specific ddr focus reformation at the damaged site , following removal of all cellular rnas by rnasea treatment ( fig6 a ; fig3 f ). the authors next aimed to test whether ddrna functions could be inactivated in living cells by sequence - specific locked nucleic acids ( lna , modified dna oligonucleotides avidly binding and inactivating complementary rna species ) ( jepsen et al ., oligonucleotides , 2004 ; machlin et al ., curr gene ther ., 2012 ). the authors thus got 4 lna molecules synthesized ( exiqon ) with their sequence fully complementary to the individual ddrnas they previously showed to be biologically active and able to restore ddr signaling and focus formation in rnasea - treated cells ( fig6 a ; fig3 . given the repetitive dna sequence structure of the locus , these are likely to anneal also to other ddrnas generated at the locus ( fig3 a ). cells carrying the integrated locus were co - transfected with cherry - lac and i - sce i - restriction endonuclease - expressing vectors and with either no lna ( sample 1 ), control lna carrying an unrelated sequence which is not part of the locus ( sample 2 ) or different sets of lna ( samples 3 - 7 ) ( fig3 b ). 24 hours post transfection , cells were fixed and stained for ddr markers . the authors analyzed the samples at the confocal microscope and scored as positive those cells that showed a ddr signal at the locus . as shown in fig3 b , the portion of cells showing a specific γh2ax focus co - localizing with the cherry - lac signal was not significantly affected by the transfection of any lna . this is consistent with the authors &# 39 ; data showing that any impairment of the biogenesis of ddrnas or removal of rnas by rnasea treatment makes no significant impact on γh2ax ( fig1 ; fig4 a , b , d , e ; fig5 a ). differently , 53bp1 accumulation at the locus , a marker of activated ddr , is significantly reduced upon transfection of lna with the sequence matching the ddrnas ( samples 3 - 6 ), compared to control lna ( sample 2 ) or to the same lna inactivated ( sample 7 ; fig3 c ). lna were inactivated by annealing them to each other in vitro before transfection , thus becoming unable to bind and interfere with the action of other complementary nucleic acids . the decrease in 53bp1 accumulation was observed , to different extents , for all lna sets tested ( samples 3 - 6 ). in summary , these results demonstrate that sequence - specific lnas can specifically inactivate the known biological functions of ddrnas . sequence - specific inhibitory oligonucleotides ( i . e . lnas ) with a telomeric sequence reduce ddr activation at dysfunctional telomeres . the authors previously showed that short rnas with the sequence of a damaged locus ( named ddrnas ) are necessary for ddr activation and maintenance specifically at that locus , upon ionizing radiations or endonuclease cleavage ( fig4 , 5 ). however , nothing is known about the role of ddrnas at telomeres . telomeres are the end of linear chromosomes , and they are protected by a protein complex named shelterin ( de lange , genes dev . 2005 ). removal of this protection causes telomere uncapping , so telomeres are recognized as dna dsbs . this may lead to ddr activation , cellular senescence , chromosomal fusions and genome instability ( sfeir and de lange , science 2012 ). in order to investigate the role of ddrna at dysfunctional telomeres , the authors used cre - er trf2 flox / flox mouse embryonic fibroblasts ( mefs ) ( lazzerini denchi and de lange , nature 2007 ). cells were grown in presence of 4 - hydroxytamoxifen to induce cre recombinase localization into the nucleus , thus generating a trf2 - knockout ( trf2 −/− ) cell line . trf2 is one of the shelterin component and its removal induces a strong ddr activation at telomeres ( lazzerini denchi and de lange , nature 2007 ). to test the role of ddrna at the telomeres , we treated mefs trf2 −/− with rnase a or bsa as a control . consistent with the authors &# 39 ; previous results , they observed that γh2ax foci resist , while 53bp1 foci are sensitive to rnase a treatment ( fig3 a - b ). this suggests that , like all other dsb lesions , also at uncapped telomeres , ddrnas with telomeric sequence are generated and they are necessary for ddr cascade activation . cells can accumulate damaged telomeres during ageing , due to telomeric shortening ( harley , nature 1990 ; herbig , mol cell 2004 ; d &# 39 ; adda di fagagna , nature 2003 ) or to endogenous or exogenous dna damage occurred at telomeres . dna damage accumulate and ddr signaling persists at telomeres as they are not repairable ( fumagalli , nat cell biol 2012 ). in both cases this persistent ddr activation at telomeres leads to cellular senescence . if ddrnas are necessary for ddr at damaged telomeres , inhibiting their action could suppress ddr activation and potentially prevent or revert the senescence phenotype . to test this hypothesis , the authors used a human cell line , t19 fibrosarcoma cells , that express an inducible dominant negative ( dn ) allele of flag - tagged trf2 ( van steensel , cell 1998 ). the expression of this allele is induced culturing cells in the absence of doxycycline . after 7 - 8 days of dn trf2 expression , telomeres are dysfunctional and cells show a strong ddr activation at telomeres ( data not shown and van steensel , cell 1998 ). induced cells , in which flag - dn trf2 was expressed , are positive for flag immunostaining ( flag +). at day 13 from induction , the authors transfected t19 cells with lna molecules with sequences complementary to either strands of telomeric dna repeats ( lna 5 and 6 ) or an unrelated sequence as control ( lna 3 , cntrl ). the authors observed that , with time , in flag + cells , both lnas transfected individually with telomeric sequence decrease the percentage of 53bp1 - positive cells , to a different extent , while control lna had no effect ( fig3 a ). importantly , in uninduced undamaged cells ( flag −), lna molecules are not inducing any dna damage , excluding that they can be genotoxic per se . furthermore , the authors monitored the passage through s phase of cell cycle , looking at brdu incorporation in induced t19 cells , three days after lna transfection . the authors observed that flag + cells , transfected with telomeric lnas , proliferate significantly more than control cells ( fig3 b ), suggesting that lna , by inactivating ddr at telomeres , can promote cell cycle reentry of cells otherwise activating a dna - damage checkpoint and reducing brdu incorporation . in contrast there is no significant difference in flag − cells ( fig3 c ). here the authors show that different sources of dna damage , including oncogenic stress , ionizing irradiation and ppoi or i - sce i endonucleases engage the ddr in a manner dependent on dicer and drosha rna products . these ddr - regulating rnas ( ddrnas ) control ddr - foci formation and maintenance , checkpoint enforcement and cellular senescence . this occurs both in cultured human and mouse cells and in different cell types in living zebrafish embryos . oncogene activation can trigger ddr and ddr - induced cellular senescence acts as tumor suppressive mechanism 2 , 37 . dicer inactivation enhances tumor development in a k - ras - induced mouse model of lung cancer 38 , 39 and inactivation of various components of dicer and drosha complexes stimulate cell transformation and tumorigenesis 38 . more recently , mutations of dicer and tarbp2 , a dicer cofactor affecting its stability , have been described in human carcinomas 4 , 41 . however , individual micrornas have been reported both to promote and to reduce cell proliferation by regulating stability and translation of mrnas encoding proteins with different roles in cell proliferation 18 : it is therefore presently unclear how rnai apparatus inactivation favors tumorigenesis . in the light of the authors &# 39 ; novel findings pointing to a role of ddrnas in ddr control , a known tumor suppressive mechanism 37 , it is tempting to suggest that , in addition to their well - characterized functions in the modulation of gene expression , dicer and drosha rna products may curb cancerous cell proliferation by sustaining ddr activation and this generates the selective pressure for the inactivation of factors involved in their biogenesis . the authors also report that in an in vitro cellular system , ddr foci are lost in irradiated cells following rnase a treatment and that site - specific ddrnas , even if generated by chemical synthesis or upon in vitro cleavage by recombinant dicer , are required to restore them . this suggests that ddrnas are locally generated and favor the assembly of ddr factors in the shape of detectable ddr foci at the dna damaged site . indeed rna sequencing confirmed the presence of short rnas arising from the integrated exogenous locus which are induced upon cut . comparison with short rnas generated at other non mirna genomic loci indicates that they are distinct from products of rna degradation and their nucleotide bias at 5 ′ end and 3 ′ end indicates that these rnas are processed at preferential rna precursors sites . although at present how ddrnas act to control ddr activation has not been elucidated in full , the observation that they act in a manner dependent on the mrn complex place them upstream of the canonical ddr signaling cascade . although novel and unanticipated , the authors &# 39 ; results are consistent with the emerging evidence supporting a role for rna molecules in ddr . indeed , an epistasis map generated in fission yeast has recently shown that ddr components display genetic interactions with the rnai machinery 56 and components of the large drosha complex have been identified in a atm - dependent phosphoproteome screen 57 . in drosophila , repeated dna integrity is dependent on rnai pathway 58 . in saccharomyces cerevisiae and in oxytricha trifallax rna orchestrates recombination and rna can function as a template for dna repair events in s . cerevisiae 59 , 60 , 61 . it is also intriguing to observe that like several ddr factors , that are inactivated early in apoptosis in order to prevent ddr activation 62 , also dicer is specifically cleaved by caspases during apoptosis 63 . recently , atm has been shown to directly modulate the biogenesis of dicer and drosha rna products by phosphorylating ksrp 64 . finally , it is worth noticing that the here - described novel functions of components of the rnai machinery in the modulation of the response to dna damage are consistent with its well - established and evolutionary - conserved role of preserving genome integrity from viral invaders , transposons and retroelements 65 . 1 jackson , s . p . & amp ; bartek , j . nature 461 , 1071 - 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