The internet is a large source of health information and has the capacity to influence its users. However, the information found on the internet often lacks scientific rigor, as anyone may upload content. This factor is a cause of great concern to scientific societies, governments, and users.The objective of our study was to investigate the information about the prevention of coronavirus disease 2019 (COVID-19) on the internet.On February 29, 2020, we performed a Google search with the terms “Prevention coronavirus,” “Prevention COVID-19,” “Prevención coronavirus,” and “Prevención COVID-19”. A univariate analysis was performed to study the association between the type of authorship, country of publication, and recommendations to avoid COVID-19 according to the World Health Organization (WHO).In total, 80 weblinks were reviewed. Most of them were produced in the United States and Spain  by digital media sources and official public health organizations . The most mentioned WHO preventive measure was “wash your hands frequently” . A less frequent recommendation was to “stay home if you feel unwell” . The analysis by type of author  revealed significant differences regarding the recommendation to wear a mask when you are healthy only if caring for a person with suspected COVID-19 (odds ratio [OR] 4.39). According to the country of publication (Spain versus the United States), significant differences were detected regarding some recommendations such as “wash your hands frequently” (OR 9.82), “cover your mouth and nose with your bent elbow or tissue when you cough or sneeze” (OR 4.59), or “stay home if you feel unwell” (OR 0.31).It is necessary to urge and promote the use of the websites of official public health organizations when seeking information on COVID-19 preventive measures on the internet. In this way, users will be able to obtain high-quality information more frequently, and such websites may improve their accessibility and positioning, given that search engines justify the positioning of links obtained in a search based on the frequency of access to them. Internet access has increased worldwide during the past decade, reaching 79.6% of the European population and 48% of the world population in 2017 .As with previous epidemics such as Ebola or Zika infections, the internet has become a favored mechanism for the spread of misinformation ,5. This At present, an outbreak of coronavirus disease 2019 (COVID-19) has occurred and has spread throughout China and to dozens of countries . As in oIn this context, we aimed to conduct an infodemiological study ,9 to invOn February 29, 2020, we performed a Google search and selected the first 20 links  of the GP<.05), this was quantified with the odds ratio (OR) and its 95% CI obtained from univariate logistic regression analysis. The agreement between the two reviewers regarding the adherence to the WHO basic protective measures was analyzed using the Kappa index. All analyses were performed using SPSS v20.0 (IBM Corp) and EpiInfo (Centers for Disease Control and Prevention).We performed a descriptive analysis of all the variables and evaluated the association of the independent variables (type of authorship and country of publication) with the degree of adherence to the WHO basic protective measures by means of a chi-square test or Fisher exact test. When a significant association was found . In particular, 3 Spanish links indicated “maintain a distance of approximately one meter between people.” One Spanish link mentioned that “for people without respiratory symptoms a surgical mask is not required, although masks can be worn in some countries according to local cultural customs.” One link in Spain and another in the United States specified that “someone should only wear a mask if a healthcare professional recommends it.” One link in the United States mentioned, “If you're going to around a lot of sick people, like if you're visiting a friend in the hospital, a mask might be a good idea,” and one link in the United States recommended, “Stay three feet away from people when you talk to them.”Search term: Prevention coronavirushttps://www.cdc.gov/coronavirus/2019-ncov/about/prevention-treatment-sp.htmlhttps://www.cdc.gov/coronavirus/2019-ncov/about/prevention-treatment.htmlhttps://www.ecdc.europa.eu/en/current-risk-assessment-novel-coronavirus-situationhttps://www.who.int/emergencies/diseases/novel-coronavirus-2019/advice-for-publichttps://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/infection-prevention-and-controlhttps://choice.npr.org/index.html?origin=https://www.npr.org/2020/02/27/810016611/coronavirus-101-what-you-need-to-know-to-prepare-and-preventhttps://www.nytimes.com/2020/02/26/health/coronavirus-cdc-usa.htmlhttps://cuidateplus.marca.com/enfermedades/infecciosas/Coronavirus.htmlhttps://www.osha.gov/SLTC/covid-19/https://www.conehealth.com/services/primary-care/coronavirus-get-the-facts-on-symptoms-and-prevention-with-cynthi/https://edition.cnn.com/2020/02/28/health/how-to-wash-hands-coronavirus-trnd/index.htmlhttps://www.nbcnews.com/health/health-news/main-focus-preventing-coronavirus-spread-should-be-hand-hygiene-not-n1144346https://www.businessinsider.com/wuhan-coronavirus-face-masks-not-entirely-effective-2020-1?IR=Thttps://abc7news.com/5971803/https://www.mobihealthnews.com/news/coronavirus-prevention-may-be-your-pockethttps://www.washingtonpost.com/gdpr-consent/?next_url=https%3a%2f%2fwww.washingtonpost.com%2fhealth%2f2020%2f02%2f26%2f how-to-prepare-for-coronavirus%2fhttps://www.cnbc.com/2020/02/26/cdc-confirms-first-possible-community-spread-coronavirus-case-in-us.htmlhttps://foreignpolicy.com/2020/02/28/taiwan-who-coronavirus-china-international-organizations/https://parade.com/987803/lisamulcahy/coronavirus/https://www.canada.ca/en/public-health/services/diseases/2019-novel-coronavirus-infection/prevention-risks.htmlSearch term: Prevention COVID-19http://www.euro.who.int/en/health-topics/health-emergencies/coronavirus-covid-19/news/news/2020/2/joint-who-and-ecdc-mission-in-italy-to-support-covid-19- control-and-prevention-effortshttps://openwho.org/courses/COVID-19-IPC-ENhttps://www.ecdc.europa.eu/en/novel-coronavirus-chinahttps://www.ecdc.europa.eu/en/publications-data/infographic-covid-19http://bvsalud.isciii.es/covid-19/https://abc7news.com/5971803/https://www.japantimes.co.jp/opinion/2020/02/27/editorials/covid-19-preventing-medical-system-breakdown/#.XlrmyahKg2whttps://jamanetwork.com/journals/jama/fullarticle/2762130https://www.iata.org/contentassets/7e8b4f8a2ff24bd5a6edcf380c641201/airport-preventing-spread-of-coronavirus-disease-2019.pdfhttps://www.cdc.gov/coronavirus/2019-ncov/about/prevention-treatment-sp.htmlhttps://www.cdc.gov/coronavirus/2019-ncov/index-sp.htmlhttps://www.osha.gov/SLTC/covid-19/controlprevention.htmlhttps://www.cnbc.com/2020/02/27/coronavirus-latest-updates-outbreak.htmlhttps://www.mica.edu/campus-operating-status-updates/coronavirus/best-practices-and-preventive-measures/https://www.kuow.org/stories/new-coronavirus-cases-found-in-king-and-snohomish-countieshttps://www.euronews.com/2020/02/26/coronavirus-prevention-how-effective-are-masks-closed-borders-screenings-and-quarantineshttps://www.health.govt.nz/our-work/diseases-and-conditions/covid-19-novel-coronavirus/covid-19-novel-coronavirus-health-advice-general-publichttps://www.canada.ca/en/public-health/services/diseases/2019-novel-coronavirus-infection/prevention-risks.htmlhttps://www.bmj.com/content/368/bmj.m810https://vietnamnews.vn/society/652839/pm-pushes-for-covid-19-preventive-measures.htmlSearch term: Prevención COVID-19https://www.cdc.gov/coronavirus/2019-ncov/index-sp.htmlhttps://www.saludcastillayleon.es/profesionales/es/enfermedades-infecciosas/nuevo-coronavirus-covid-19/plan-especifico-prevencion-riesgos-laborales- nuevo-coronavihttps://www.alimente.elconfidencial.com/bienestar/2020-02-29/coronavirus-covid19-que-es-sintomas-contagio_2431343/https://www.saludcastillayleon.es/profesionales/es/enfermedades-infecciosas/nuevo-coronavirus-covid-19https://www.who.int/es/emergencies/diseases/novel-coronavirus-2019/advice-for-public/q-a-coronaviruseshttps://www.ibsalut.es/es/info-ciudadania/cuidar-la-salud/3710-preguntas-y-respuestas-sobre-el-nuevo-coronavirus-2019-n-covhttps://www.campusvirtualsp.org/es/curso/virus-respiratorios-emergentes-incluido-el-2019-ncov-metodos-de-deteccion-prevencion-respuestahttps://www.alimente.elconfidencial.com/bienestar/2020-02-29/coronavirus-covid19-que-es-sintomas-contagio_2431343/https://www.semfyc.es/como-prevenir-infecciones-por-virus-respiratorios-como-el-coronavirus-que-causa-la-enfermedad-covid-19/http://bvsalud.isciii.es/covid-19/https://www.mscbs.gob.es/profesionales/saludPublica/ccayes/alertasActual/nCov-China/documentos/20200224.Preguntas_respuestas_COVID-19.pdfhttps://www.mscbs.gob.es/profesionales/saludPublica/ccayes/alertasActual/nCov-China/documentos/Documento_Control_Infeccion.pdfhttps://www.lasexta.com/noticias/internacional/coronavirus-covid19-que-puedes-hacer-protegerte-como-actuar_202002245e53fcca0cf2547d2a31e546.htmlhttps://www.unicef.org/es/historias/coronavirus-lo-que-los-padres-deben-saberhttps://www.lavanguardia.com/vida/20200229/473828128008/coronavirus-espana-madrid-barcelona-wuhan-china-italia-covid-19-contagios- sintomas-fallecidos-ultima-hora-hoy-en-directo.htmlhttps://www.univision.com/local/philadelphia-wuvp/prevencion-del-coronavirus-que-funciona-para-evitar-la-propagacion-de-covid-19http://bvsalud.isciii.es/covid-19/https://medlineplus.gov/spanish/ency/article/007768.htmhttps://sano-y-salvo.blogspot.com/2020/02/infografias-para-prevenir-la-infeccion.htmlhttps://www.bbc.com/mundo/noticias-51683330Search term: Prevención coronavirushttps://cuidateplus.marca.com/enfermedades/infecciosas/Coronavirus.htmlhttps://www.quironprevencion.com/es/campanas-prevencion-riesgos-laborales/coronavirus-covid-2019https://medlineplus.gov/spanish/coronavirusinfections.htmlhttps://www.cdc.gov/coronavirus/2019-ncov/about/prevention-treatment-sp.htmlhttps://www.saludcastillayleon.es/profesionales/es/enfermedades-infecciosas/nuevo-coronavirus-covid-19/plan-especifico-prevencion-riesgos-laborales- nuevo-coronavihttps://elpais.com/elpais/2020/02/25/ciencia/1582645440_172885.htmlhttps://www.elperiodico.com/es/sanidad/20200225/coronavirus-que-es-sintomas-contagio-prevencion-7814261https://vacunasaep.org/profesionales/noticias/coronavirus-desarrollo-de-vacunashttps://www.hola.com/estar-bien/20200123158838/coronavirus-sintomas-prevenir-contagio/https://www.alimente.elconfidencial.com/bienestar/2020-02-29/coronavirus-covid19-que-es-sintomas-contagio_2431343/https://www.diariocordoba.com/noticias/sociedad/que-es-coronavirus-sintomas-contagio-prevencion-virus_1351515.htmlhttps://www.who.int/csr/disease/coronavirus_infections/ipc-mers-cov/es/https://www.intramed.net/contenidover.asp?contenidoid=95410https://www.bbc.com/mundo/noticias-51683330https://www.semes.org/semes-divulgacion/medidas-de-prevencion-ante-la-neumonia-por-coronavirus/https://www.lavanguardia.com/seguros/empresa/20200217/473630100957/mwc-alerta-sanitaria-contagio-corona-virus-riesgos-laborales-seguros.htmlhttps://chile.gob.cl/chile/medidas-de-prevencion-ante-el-nuevo-coronavirushttps://www2.cruzroja.es/-/-como-puedes-reducir-el-riesgo-de-infeccion-del-coronavirus-https://temas.sld.cu/coronavirus/coronavirus/medidas-preventivas/https://www.lavanguardia.com/ciencia/20200225/473756254816/coronavirus-covid-19-mascarilla-prevencion.htmlUnivariate analysis by type of author  revealed statistically significant differences regarding the recommendation to wear a mask if you are healthy only if caring for a person with suspected COVID-19 OR 4.39; . The anaThis study is the first to evaluate the adherence of the information available on the internet to the WHO basic protective measures against COVID-19. It shows a level of adherence that can be improved and a difficulty in obtaining such information, since it was only available in 32.5%-81.3% of the links.The difficulty of finding WHO-promoted measures to prevent other infectious diseases on the internet has also been described previously by other authors, such as Covolo et al . The autLess than half of the weblinks provided information on the correct use of masks and, together with the fact that some of the links provided information that was ambiguous or did not adhere to the WHO guidance, may have contributed to the misuse of masks by the population and with the subsequent shortage of these devices that is occurring worldwide ,15.As with other studies that evaluated information on the internet on preventive measures for other infections , our worAccording to the analysis by country, the Spanish links provided more information on measures to prevent COVID-19 that adhered to the WHO than did the links produced in the United States. The measures to prevent COVID-19 by the Centers for Disease Control and Prevention  are the One of the limitations of our study is intrinsic to the nature of internet, namely that information changes continuously; like others ,11,13,19In conclusion, it is necessary to urge and promote the use of the websites of official public health organizations  when seeking information on COVID-19 preventive measures on the internet. In this way, they will be able to obtain high-quality information more frequently, and such websites’ accessibility and positioning may improve, given that search engines justify the positioning of links obtained in a search based on the frequency of access to them.


Xist lncRNA mediates X chromosome inactivation (XCI). Here we show that Spen, an Xist-binding repressor protein essential for XCI , binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen loss activates a subset of endogenous retroviral (ERV) elements in mouse embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing. ERV RNA and Xist A-repeat bind the RRM domains of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in strictly local gene silencing in cis. These results suggest that Xist may coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.The  The genetic material inside cells is often packaged into thread-like structures called chromosomes. In humans, mice and other mammals, a pair of sex chromosomes determines the genetic or chromosomal sex of each individual. Those who inherit two “X” chromosomes are said to be chromosomally female, while chromosomal males have one “X” and one “Y” chromosome. This means females have twice as many copies of genes on the X chromosome as a male does, which turns out to be double the number that the body needs.Xist is responsible for switching off the extra X genes in female cells. It does this by coating the entirety of the second X chromosome.To solve this problem, mammals have developed a strategy known as dosage compensation. The second X chromosome in females becomes “silent”: its DNA remains unchanged, but none of the genes are active. A long noncoding RNA molecule called Xist, however, are RNAs that have taken on different jobs inside the cell. Researchers believe that the ancestral Xist gene may have once encoded a protein but changed over time to produce only a noncoding RNA. Carter, Xu et al. therefore set out to find out how exactly this might have happened, and also how Xist might have acquired its ability to switch genes off.Normally, RNA molecules transmit the coded instructions in genes to the cellular machinery that manufactures proteins. “Noncoding” RNAs like Xist silence the X chromosome. In female cells lacking Spen, the second X chromosome remained active. Other chromosomes in male and female cells also had stretches of DNA that became active upon Spen’s removal. These DNA sequences, termed endogenous retroviruses, were remnants of ancestral viral infections. In other words, Spen normally acted as an antiviral defense.Initial experiments used mouse cells grown in the laboratory, in which a protein called Spen was deleted. Spen is known to help Xist, a region which was needed for Xist to work properly. Inserting fragments of endogenous retroviruses into a defective version of Xist lacking this region also partially restored its ability to inactivate genes, suggesting that X chromosome silencing might work by hijacking cellular defenses against viruses. That is, female cells essentially ‘pretend’ there is a viral infection on the second X chromosome by coating it with Xist (which mimics endogenous retroviruses), thus directing Spen to shut it down.Analysis of genetic sequences showed that Spen recognized endogenous retrovirus sequences resembling a key region in This research is an important step towards understanding how female cells carry out dosage compensation in mammals. More broadly, it sheds new light on how ancient viruses may have shaped the evolution of noncoding RNAs in the human genome. Xist is a 17 kb long noncoding RNA that acts through specific interactions between its distinct RNA domains and nuclear effector proteins. The Xist RNA-associated protein complex was identified in 2015 using both genetic and affinity-based methods, and consists of multiple pleiotropic proteins, many of which are highly conserved throughout evolution and act on chromatin structure and gene regulation in myriad systems  that inserted into this locus. Xist contains six tandem repeat regions (A-F), all of which show sequence similarity to TEs, suggesting they arose from eutheria-specific TE insertions  that contains four canonical RNA binding domains, as well as a SPOC domain to facilitate protein-protein interactions. Spen is a co-repressor that binds to several chromatin remodeling complexes, including histone deacetylases (HDACs), and the NuRD complex  is a ~ 400 kDa  complex . Though d humans . Spen bip in XCI .Xist transgene either in the wild-type (WT) context or with a full deletion of Spen (Spen KO)  . Followiromosome . In two sibility . This cosibility , confirmsibility .Xist evolved the ability to recruit Spen is of great interest. We hypothesized that understanding Spen’s role in autosomal gene regulation and its target specificity might lend clues into how Spen was exapted to participate in the Eutherian-specific process of XCI , in contrast to unchanging peaks and those that are less accessible, which encompassed both promoters and distal sites  elements . When members of the TE silencing machinery, such as Kap1 and Setdb1, are inactivated, H3K9me3 is lost, and these TE insertions can be expressed and function as ectopic promoters and enhancers . We hypoSpen KO showed a dramatic loss of H3K9me3 and a gain of both histone H3 lysine 27 acetylation (H3K27Ac) and histone H3 lysine four trimethylation (H3K4me3) marks  marks . Converselements . This deKO mESCs . At the KO mESCs . Genome-KO mESCs . This isKO mESCs .pen KO to those not upregulated in Spen KO, and found that those repressed by Spen have diverged less than those that are not repressed by Spen  from the media and allowed cells to grow for 6 days. After 6 days, WT mESCs downregulated pluripotency factors such as Oct4 and expressed early differentiation markers such as Nestin. In contrast, Spen KO mESCs maintained Oct4 expression and mESC morphology despite LIF removal , which allows for identification of direct RBP binding sites on RNA . Becausein vitro . We exprXist was the top bound gene, with much higher binding strength than other RNAs, consistent with its known interaction with the A-repeat region of Xist and role in XCI , on a total of 4732 transcripts . Comparee in XCI . Spen RRe in XCI . This coA itself . This isERV RNAs are bound by RRM2-4 as well as full-length Spen , covering 2267 individual TEs . Of thesgth Spen .ERV RNAs in the same manner, we first investigated the sequence features in Spen-bound mRNAs, lncRNAs, and ERVs. We found that RRM2-4 binding sites on mRNA and lncRNAs, as well as ERVs, are enriched for AU content, but did not find any longer, more complex motifs shared amongst the classes. Furthermore, we did not find sequences that showed strong homology to the highly conserved A-Repeat regions , and higher than the affinity of Spen RRM2-4 for the SRA hairpin (KD = 450 nM)  . The binERV RNAs . Finallyr manner . These dXist RNA, with a significant contribution from the RRM3 domain. The observation that the A-repeat may be derived from an ancient ERV insertion  recruit and bind to Spen and 2) silence X-linked genes in cis (Xist gene on the X as well as a deletion of the A-repeat region (Xist-ΔA)  leads to silencing of the single X chromosome, Xist expression in Xist-ΔA mESCs has no effect on X chromosome gene expression or accessibility  . While isibility . We usedsibility . We chosng sites . Furtherng sites .Xist-ERV is able to bind directly to and recruit Spen. To do this, we expressed the FLAG-tagged Spen RRM2-4 in our cell lines containing Xist-WT, Xist-ΔA, or Xist-ERV transgenes (two independent clones), and measured Xist-RRM binding under optimized expression conditions (more below), by RIP-qPCR. We found that Xist-ERV is bound by Spen RRM2-4, similar to Xist-WT and in contrast to Xist-ΔA which could not bind Spen  or an HDAC3 inhibitor (HDAC3i) for 24 hr before Xist induction. We found that both HDAC1/2i and HDAC3i de-repressed the transgene and Xist-ERV was expressed . Induction of Xist was confirmed using qPCR and Xist RNA FISH. qPCR primers used for Xist have the following sequences: Forward 5’GCTGGTTCGTCTATCTTGTGGG3’ and Reverse 5’CAGAGTAGCGAGGACTTGAAGAG3’.For Xist with doxycycline and continued for 24 hr during Xist induction . –HDACi samples were treated with DMSO as a control.For HDAC inhibition experiments, cells were treated with either HDAC1/2i  or HDAC3i  at 10 uM final concentration. HDAC inhibition was initiated 24 hr prior to induction of Xist induction, adherent cells on coverslips were fixed using 4% paraformaldehyde for 10 min. Cells were permeablized in 0.5% triton for 10 min on ice and then stored in 70% EtOH. After dehydration in EtOH, cells were rehydrated in wash buffer (2xSSC + 10% formamide) for 5 min. Probe  was then added at a concentration of 125 nM (in wash buffer + 10% Dextran Sulfate) and incubated overnight at 37’C. Following washes in wash buffer + formamide, coverslips were mounted on slides with Vectashield + DAPI.Following TTGGGCTAGAGAAGGATGTGGTT3’, Oct4-R: 5’ GGAAAAGGGACTGAGTAGAGTGTGG3’ ActB-F: 5’TCCTAGCACCATGAAGATCAAGATC3’, ActB-R: 5’CTGCTTGCTGATCCACATCTG3’.NPC differentiation from mESCs was performed as previously described . BrieflyImmunofluorescence for Oct4 and Nestin was performed on adherent cells fixed with 4% paraformaldehyde. After fixation, cells were permeablized with 0.5% PBS/Triton for 10 min on ice. Following permeablization, cells were blocked in 10% FBS in 0.1% PBS/Tween for 1 hr at room temperature. After blocking, cells were incubated for 2 hr in primary antibody , washed in 0.1% PBS/Tween and then incubated for 2 hr in secondary antibody . After washing, cells were stained with DAPI and imaged at 40x magnification.Cells were fixed with 1% formaldehyde for 10 min at room temperature and subsequently quenched with 0.125M glycine. Cells were then snap frozen and stored at −80’C. Cells were then lysed  for 10 min at 4’C. Nuclei were lysed  for 10 min at room temperature. Chromatin was resuspended in sonication buffer  and sonicated using a Covaris Ultrasonicator to an average length of 220 bp. For all ChIPs, 5 million cells per replicate were incubated with 5 ug (Histone) or 7.5 ug (Oct4) antibody overnight at 4’C . Antibody-bound chromatin was incubated with Protein G Dynabeads  for 4 hr at 4’C and eluted in Tris buffer . Crosslinks were reversed by incubation overnight at 65’C followed by treatment with 0.2 mg/mL proteinase K  and 0.2 mg/mL RNAse A (Qiagen). DNA was purified using Qiagen Minelute Columns . For library preparation, 4 ng ChIP DNA was incubated with transposase  for 10’ at 55’C. DNA was then amplified using Nextera barcoding adapters and sequenced on an Illumina Nextseq (2 × 75 bp reads).ChIP-seq libraries for histone modifications were sequenced on an Illumina NextSeq with paired-end 75 bp reads. Reads were mapped to the mm9 genome build using Bowtie2 . Unique Peaks were called using MACS2 with a q-value cutoff of 0.01 with input as background . Peaks cATAC-seq library preparation was performed exactly as described in P values in DESEq2, for each gene the counts were modelled using a generalized linear model (GLM) of negative binomial distribution among samples. The two-sided Wald statistics test was processed for significance of the GLM coefficients. The Benjamini-Hochberg correction was applied to all p-values to account for multiple tests performed. This gives the final adjusted p-values used for assessing significance.Reads were trimmed using CutAdapt  and mappATAC-seq reads were trimmed and mapped as described above. Peaks were called in all samples (+ and – doxycycline) and bedtools multicov was used to count the number of reads falling within each peak in each sample. In order to avoid bias in normalization in the case of a global loss of accessibility on the X chromosome, we first normalized count data using all peaks genome-wide. We then calculated the ratio of average normalized reads in +dox / -dox samples at each peak on the X chromosome.RNA-seq library preparation was performed using the TruSeq Stranded mRNA Library Prep Kit . In place of poly-A selection, we performed Ribosomal RNA depletion using the RiboMinus Eukaryote System v2 (Life Tech. A15026). Each library was prepared from 200 ng starting RNA, and two replicates were made for each cell line. RNA-seq libraries were sequenced on an Illumina HiSeq 4000 with paired 2 × 75 bp reads.https://www.gencodegenes.org/mouse_releases/1.html). Differential genes were called using DESeq2  (RNA-seq reads were mapped to the mm9 genome using STAR . Followi <0.001) . For GO  <0.001) .HOMER was used for enrichment of genomic locations in ATAC-seq and ChIP-seq data . SpecifiSpen KO Clone 2 mESCs were transduced with a lentivirus carrying Spen RRMs 2–4 with the SV40 nuclear localization signal, 2x HA tags, and 3x FLAG tag . Following transduction, GFP+ cells were FACS sorted and single colonies were picked. Western blotting was used to confirm the expression of SpenRRM-FLAG at 37 kDa. irCLIP was performed exactly as described  and sonicated using a Bioruptor (Diagenode) (six cycles 30’ on 45’ off). Clarified lysates were incubated overnight with Protein A dynabeads conjugated to mouse anti-FLAG (Sigma Aldrich F3165) or anti-IgG antibody overnight at 4C. Following IP, beads were washed in high stringency buffer , high salt buffer , and low salt buffer . After washing, RNA was digested using RNase I at 1:2000. RNA ends were then dephosphorylated and IR-conjugated linker (/5Phos/AGATCGGAAGAGCGGTTCAGAAAAAAAAAAAA/iAzideN/AAAAAAAAAAAA/3Bio/) was ligated overnight. Following ligation, samples were run on a 4–12% Bis-Tris gel, transferred to nitrocellulose and imaged using an Odyssey LiCOR scanner. RNA-protein complexes were eluted from nitrocellulose and treated with proteinase K to eliminate protein. Following RNA elution, Oligo(dT) Dynabeads were added to capture the polyadenylated irCLIP adapter. Next, the TruAmp sequencing adapter was added and RNA was reverse transcribed using SuperScript IV. Following reverse transcription, DNA was captured with streptavidin beads, circularized on the bead, and then PCR amplified. irCLIP libraries were then purified using PAGE gel purification and sequenced on an Illumina NextSeq using single-end 75 bp reads.For in vivo irCLIP-seq experiments, V6.5 male mESCs, or escribed . BrieflyE. coli expression.The following constructs for SPEN (Split Ends protein), SHARP , were designed and cloned into pET28a for SPEN-1=6 His-GST-TEV- RRM 2,3,4-SG-FlagSPEN-4=6 His-GST-TEV- RRM 2,3,4-RRM3mutant-SG-FlagDNA for each was transformed into BL21 (DE3) Star competent cells, the transformation mix plated on LB-Kan agar and single colonies obtained the following day. 4 × 50 ml overnight pre-cultures were setup with single colonies for each construct in Terrific Broth (Teknova) + Kanamycin (50 ug/ml) (TB-Kan), at 37C, 250 rpm. Pre-cultures were used to inoculate 1li volumes of TB-Kan expression media at 10 ml/li (gives a starting OD600 = 0.15). Expression cultures were incubated at 37C, shaken at 250 rpm, for 4 hr then chilled to 18C for 1 hr and induced overnight with 0.25 mM IPTG at 18C. Cultures were harvested and resulting cell pellets were stored frozen at −80C. Frozen cell pellets were resuspended in 5 ml/g of Lysis Buffer, Lysozyme (1 ug/ml), 5 mM 2-ME, 2 x Roche protease Inhibitor tablet/50 ml and 1 mM PMSF). Resuspended pellets (in suspensions) were homogenized by Polytron and lysed by two rounds through a Microfluidizer (18000 psi). Lysates were clarified by centrifugation at 33000 x g for 60mins, the supernatant extracted and subjected to batch binding with 5 ml of NiNTA (Qiagen) for 2 hr, 4C with end-to-end rotation. Resin was washed with 20Cv’s of Lysis Buffer containing 20 mM Imidazole, followed by elution of bound protein with 3 × 1 CV of Elution Buffer (Lysis Buffer + 250 mM Imidazole). Eluted fractions were pooled and dialysed overnight against 4li of 50 mM Hepes;pH 7.5, 300 mM NaCl, 1 mM TCEP with the addition of TEV protease to effect in-situ cleavage of the N-terminal 6His-GST tag. TEV cleaved solution was passed through a 0.5 ml NiNTA and the flow-through collected and concentrated 3–5 fold to give 4–10 mg/ml samples which were aliquotted and stored frozen at −80C.2, 0.3% NP-40 and 10% glycerol) with 100 Units SUPERase-In. Cells were incubated on ice for 10 min, vortexes, and centrifuged for 3 min at 1000xg at 4C. The pelleted nuclei were then washed 3x in hypotonic lysis buffer and centrifuged for 2 min at 200xg at 4C. Pellet was then resuspended in TRIzol and RNA extraction was carried out per the manufacturer’s instructions. 20 μg nuclear RNA was then fragmented , and incubated with 5 μg recombinant Spen RRMproteins (RRM2-4 WT or RRM2-4:RRM3 Mt) at 4C for 2 hr. RNA-protein complexes were then transferred to a 3 cm cell culture dish on ice and crosslinked (254 nM UV-C) at 0.3 J/cm2. Following crosslinking, RNA-protein complexes were incubated with antibody-bound Protein A Dynabeads (anti-FLAG [Sigma Aldrich F3165] or anti-IgG) at 4C for 2 hr. Following IP, beads were washed with IPP buffer , low salt buffer , and high salt buffer . After washes, the protocol was performed exactly as in in vivo irCLIP from dephosphorylation to membrane imaging(above).In vitro irCLIP-seq was performed similarly to in vivo irCLIP with the following modifications. First, nuclear RNA was isolated as previously described . BrieflyirCLIP was performed as previously described . In brieTo identify expressed transposable elements in RNA-seq data, we counted all unique reads mapping to the database of repeat element loci from RepeatMasker for the mm9 genome. Bedtools multicov was used to count the number of reads per sample in each element. Read counts were then converted to fpkm. All elements with an FPKM >= 1 in all samples were considered expressed and used for downstream analysis.GCGGGATTCGCCTTGATTTG) and 20 ug HDR donor vector. Cells were nucleofected with RNP complex and donor vector using the Amaxa Nucleofector (Lonza VPH-1001). Clonal colonies were picked and genotyped by PCR followed by Sanger sequencing. PCR of the Tet operator array was done using the following primers: TetO-F: 5’CCTACCTCGACCCGGGTACC3’, TetO-R: 5’GGCCACTCCTCTTCTGGTCT3’.For Xist rescue experiments with ETn elements, we utilized the TXY ΔA cells which harbor a doxycycline inducible Xist transgene with the A Repeat region deleted. We designed donor plasmids for homology directed repair containing homology arms to the transgenic Xist present in TXY ΔA cells that contain a 154 bp portion of an ETn that binds to Spen, repeated 9x. Homology arms flanking the ETn insertions were cloned into the PCR4-Blunt TOPO vector (Thermo Fisher K287520). Cells were then nucleofected with a Cas9 RNP complex directed to the A Repeat region. 10 ug Cas9 protein (PNA Bio CP01-20) was mixed with 10 ug modified guideRNA . Following transduction, GFP+ cells were FACS sorted and expression of the RRM-FLAG construct was confirmed by Western Blot using an anti-FLAG antibody (Sigma F3165). SpenRRM-FLAG expressing cells were treated with 10 mM HDAC1/2 inhibitor  for 24 hr, followed by treatment with HDAC1/2i + 2 mg/mL doxycycline for 24 hr. 5 million cells were then washed in PBS and lysed in RIP lysis buffer  in the presence of RNAse inhibitor . Cells were sonicated on a Covaris , and debris was pelleted for 15 min at 21,000xg at 4’C. Cleared supernatant was then added to 100 uL FLAG magnetic beads . After 2 hr incubation at room temperature, beads were washed three times in cell lysis buffer and resuspended in Trizol. RNA was extracted using the RNEasy mini kit . qRT-PCR was run on a Roche Lightcycler 480 using the Brilliant II SYBR Green qPCR Master Mix  using the following primers: Xist-F: 5’After finding that SPEN regulates repeat regions, we re-analyzed the ChIP-seq and ATAC-seq data to include repetitive regions, which are usually removed in the standard analysis. We allowed each multimapping read to randomly assign to one place in the genome, without filter reads by mapping quality. The differential peaks is consistent to the results from unique mapped reads. We used the alignment files with multiple mapped reads for all the meta-analysis to avoid losing information.Multiple alignments of the SPEN CDS sequence were download from UCSC, including human, chimpanzee, rhesus, mouse, dog, opossum, platypus, chicken, zebrafish. We separated the CDS of SPEN into three domain regions .dN), the number of synonymous substitutions per synony- mous site (dS), and the dN/dS ratio, for each region using maximum likelihood as implemented in the codeml program of the PAML software package  were compared. icSHAPE data analysis.The icSHAPE data from Sun et al. were downloaded from GEO under accession GSE117840. The sequence data reads were collapsed to remove PCR duplicates and trimmed to remove 3’ adapters following the icSHAPE computational pipeline . The proSequencing output reads were processed by bbmap to remove the duplication on fastq level . RemaineThe RBP binding loci as suggested by the irCLIP method, was defined as one nucleotide shift to the 5’ end of each mapped read. Each loci was extended five nucleotides both up and downstream into a 11-nt-long local interval. Only intervals overlapped between two biological replicates were considered reliably detected and were included in the downstream analysis. Overlapping intervals were merged into one cluster. Five nucleotides were trimmed from each side of the cluster to shape the final cluster boundary. Cluster length is required to be longer than one nucleotide for downstream analysis.SPEN binding clusters on mouse genome and on repeat-regions were identified respectively. Cluster annotations were processed against the Genocode NCBI37/mm9 GTF file and RepeatMasker.For each experiment condition of each cell type, only clusters with quantification tenfold higher than the observation in its corresponding IgG samples, would be included for the downstream analysis.Quantification of SPEN binding on each expressed gene or expressed repeat element was calculated as the total amount of read in all binding clusters covering the gene/repeat element. The quantifications were then normalized by gene/repeat expression level.RNA motif enrichment analysis were process on SPEN binding clusters on mouse genome and on repeat-regions respectively (Homer version 4.10). Strand-specific sequences defined by SPEN irCLIP clusters were searched for short sequences elements 5-mer, 6-mer, 7-mer and 8-mer in each analysis of hypergeometric enrichment calculations. The searching background was set as expressed transcriptome and expressed repeat regions.2D-structures were predicted by the RNAstructure tool (version 6.0.1) with the maximum free energy model and according the icSHAPE results. 2D-structures of MMETn repeat1125276, ETnERV repeat355428 and RLTR13G_2D were process by algorithm AllSub. 2D-structure of Xist repeat A region #4 and #5 were processed with algorithm DuplexFold.Wild type mouse Spen cDNA was kindly provided in a D-TOPO vector from the Guttman lab . Mutant Spen cDNA corresponding to RRMs 2–4  was ordered as a gBlock from IDT . Primers containing SalI and XhoI restriction enzyme cut sites on the 5’-end and 3’-end respectively of the cDNA corresponding to RRMs 2–4 (aa 336–644) were used to PCR amplify both wild type and RRM3 Mt cDNA inserts for ligation into a pET28b vector containing an N-terminal 10xHis-SUMO tag using the Quick Ligation kit . Plasmids containing the cDNA of interest were verified by Sanger sequencing prior to expression .600 between 0.6–1.2. Cells were flash-cooled on ice for 10 min, then 1 mM IPTG was added to induce protein expression overnight at 20°C. Cells were harvested, and pellets were frozen and stored at −20°C until purification. All proteins were subject to a 3-column purification protocol; the wild-type buffers were pH 7.5, while the mutant buffers were pH 8.3 due to differences in predicted pI and anticipated solubility requirements. Specifically, one cOmplete EDTA-free EASYpack protease inhibitor tablet  was added to each cell pellet corresponding to 1 L of growth. The tablet and frozen cell pellet were resuspended in 80 mL of lysis buffer . The cell slurry was sonicated on ice with a Misonix Sonicator 3000  for 4–6 total minutes in pulses of 15 s followed by 35 s of rest. Membranes and other cellular debris were sedimented away from soluble protein on a Beckman J2-21 large floor centrifuge . Supernatant containing the recombinant protein was incubated with 10 mL of precleared Ni-NTA beads for 10 min at 4°C. Unbound cellular proteins were cleared by gravity flow. Beads were washed twice; once with 150 mL of lysis buffer, and once with 100 mL of modified lysis buffer containing 20 mM imidazole. Protein was then eluted with 50 mL of similar buffer containing 300 mM imidazole. For the washes and elution, beads were resuspended and incubated with the buffer for 10 min prior to gravity flow through. The elution was dialyzed in 4 L of size exclusion column buffer  for about 2 hr at 4°C. 1–2 mL of 1 mg/mL in-house His-ULP1  was then added, and SUMO tag cleavage of recombinant Spen protein and further dialysis continued overnight at 4°C. Protein precipitate, when present, was pelleted or filtered prior to flowing over the second Ni-NTA column (11 mL beads precleared in column buffer). The Spen fragment flowed through the second Ni column while the His tagged protease and SUMO expression tag were retained on the resin. Flowthrough containing the cleaved Spen construct was concentrated to 2 mL for injection onto the Superdex 200 size exclusion column. Size exclusion fractions are checked for the presence of purified protein by SDS-PAGE. Fractions containing purified protein are combined, concentrated to between 100 µM - 1 mM, aliquoted, flash frozen in liquid nitrogen, and stored at −70°C until binding assays are performed.BL21(DE3) expression cells were transformed with pET28b vector containing the cDNA of the protein of interest. 1 L cultures of 2XYT media were grown at 37°C for 3 hr to an OD2+, 40 mM Tris pH 8.0, 10 mM DTT, 2 mM spermidine, 0.01% Triton X-100, IPPase (>0.01 U/µL), and in-house T7 polymerase . Transcription reactions were gel purified on a 5–12% acrylamide 8M urea 1X TBE slab gel. Reaction products were UV shadowed, appropriate sized bands were excised, and RNA was extracted through a 0.5X TE crush and soak . Gel bits were filtered and RNA was buffer exchanged in 0.5X TE until the calculated residual urea was less than 10 µM. RNA was concentrated prior to use; purity and concentration were assessed by the A260 and the A260/A280 ratio. Concentrations were calculated using the extinction coefficient provided for each RNA sequence by the Scripps extinction coefficient calculator .RNA generation cDNAs of RNAs of interest with a 5’ T7 promoter were ordered as gBlocks from IDT . Primers flanking the cDNA containing the EcoRI and BamHI cut sites on the 5’ and 3’ ends respectively were used to PCR amplify the cDNA for restriction enzyme cloning into a pUC19 vector for cDNA amplification. Ligated plasmids were verified using Sanger sequencing  and utilized for transcription template generation. Standard PCR using a 3’ transcription primer was used to generate in vitro transcription template for run-off transcription. For in vitro transcription, 300 nM PCR template was incubated at 37°C for 2–4 hr with 4 mM each rNTP in 24 mM Mg4 for 20 min in the dark at room temperature. Excess NaIO4 was precipitated using 26 mM KCl (10 min on ice followed by a hard spin). Supernatant containing the RNA was then ethanol precipitated with the aid of 1 µg glycogen. The RNA pellet was washed 1x with 70% ethanol, and then resuspended in 100 µL of freshly made labeling solution  and reacted at 37°C in the dark for 1–2 hr. 280 µL of cold 100% ethanol was then added to precipitate the RNA (ice for ≥30 min). The labeled RNA pellet was then washed thoroughly at least 4X with 400 µL of 70% ethanol, resuspended in 30 µL of water, and run through a Sephadex MicroSpin G-25 column  to remove excess FTSC. Labeled products were qualitatively assessed for purity and labeling efficiency via denaturing gel analysis; the Cy2 filter on the Typhoon imager  was used to visualize the attached fluorophore. A260 measurements post-labeling were used to determine RNA concentration. Labeling efficiencies typically allowed for the use of RNA in binding assays between 1–3 nM. FTSC-labeled RNA was stored in dark amber tubes at −20°C until use.350 pmol of purified RNA were treated with 20 mM NaIO⊥,I‖) were measured using a ClarioStar Plus FP plate reader (BMG Labtech). Anisotropy values were calculated for each protein titration point where anisotropy = (I‖ - I⊥)/(I‖ + 2*I⊥). Protein concentration was plotted versus associated anisotropy, and data were fit to the simplified binding isotherm (anisotropy ~fraction bound=S*(P/(KD+P))+O with KaleidaGraph where S and O were saturation and offset respectively, and P was the protein concentration.Wild-type and mutant Spen binding were performed at pH 7.5 and 8.3 respectively. RNA concentrations were held as low as possible while still obtaining enough fluorescent signal (1–3 nM). Briefly, purified labeled RNA was snap cooled in 1X binding buffer  at 2X the final concentration, and likewise protein titrations were performed separately in 1X binding buffer at 2X final concentration. Protein and RNA were then mixed in a 1:1 vol ratio in a 20 µL reaction and were allowed to come to equilibrium at room temperature in the dark for 40–60 min. Flat bottom low flange 384-well black polystyrene plates  were used. Perpendicular and parallel fluorescence intensities  for 1 hr at 37°C. RNA was purified via phenol chloroform extraction followed by ethanol precipitation. RNA was resuspended and γ-ATP was added to the 5’ end of the RNA with T4 polynucleotide kinase (NEB) for 30 min at 37°C. Water was added to dilute the RNA to 1 µM, and the reaction was then run through a Sephadex MicroSpin G-25 column  to remove excess γ-ATP. 32P-labeled A repeat concentrations were kept constant at 10 µM and 1 µM respectively, and the competition was done at 50 mM Tris (pH 7.5), 45 mM KCl, 5 mM NaCl, and 2.5% glycerol. Different amounts of unlabeled competitor RNA were added, and the amounts were relative to the amount of labeled RNA. Briefly, RNAs were snap cooled in RNA buffer (50 mM Tris (pH 7.5), 45 mM KCl, 5 mM NaCl). Labeled RNA was snap cooled at 4X the final concentration, while competitor RNA was snap cooled at 2X the final concentration. Protein was diluted in protein buffer (50 mM Tris (pH 7.5), 45 mM KCl, 5 mM NaCl, 10% glycerol) to 4X the final concentration. 2.5 µL of 4X protein, 2.5 µL of 4X labeled RNA, and 5 µL of 2X competitor RNA were allowed to come to equilibrium at room temperature for 40–60 min. The reactions were then loaded onto a pre-run 5% acrylamide 0.25X TBE mini native gel at 200V. The gel was run at room temperature at 200V for 10–15 min, dried, then exposed overnight to a phosphoscreen. Phosphoscreen images were taken on the Typhoon  at 50 µm resolution.Wild-type Spen and GGAACTCGTTCCTTCTCCTTAGCCCATCGGGGCCATGGATACCTGCTTTTAACCTTTCTCGGTCCATCGGGACCTCGGATACCTGA-repeat from Xist (85 nt): GGAGCAGGTATGTGATGACATCAGCCGACGCCTGGCACTGCTGCAGGAACAGTGGGCTGGAGGAAAGTTGTCAATACCTGTASRA RNA (82 nt): ERV 205 (86 nt):GAUUCCAGGCAGAACUGUUGAGCAUAGAUAAUUUUCCCCCCUCAGGCCAGCUUUUUCUUUUUUUAAAUUUUGUUAAUAAAAGGGAGERV 509 (114 nt):GGUUAAAACUGCUAUUGUUCCAUUGACUGCAGCUUGCAGUUUGAUUUCAAAUUUAAGAUCUUUAAUUCACCUGUAUACUGUAAUUAAGAUAAUUACAAGAGUAAUCAUCUUAUGERV 495 (113 nt):GAAAUUUCUCUCUGGGCCUUAUAGCAGGAGUACUCUGUUCCCUUUUGUGUCUUGUCUAAUGUCCGGUGCACCAAUCUGUUCUCGUGUUCAAUUCAUGUAUGUUCGUGUCCAGUERV 411 (109 nt):GGUACCUUAAAUCCUUGCUCUCACCCAAAAGAUUCAGAGACAAUAUCCUUUUAUUACUUAGGGUUUUAGUUUACUACAAAAGUUUCUACAAAAAAUAAAGCUUUUAUAAERV 302 (111 nt):GAUCAGAGUAACUGUCUUGGCUACAUUCUUUUCUCUCGCCACCUAGCCCCUCUUCUCUUCCAGGUUUCCAAAAUGCCUUUCCAGGCUAGAACCCAGGUUGUGGUCUGCUGGAnalysis methods used for next generation sequencing data in this paper are described in the appropriate sections of the methods. No new software packages were generated for the analysis of data in this paper. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.eLife. Your article has been reviewed by three peer reviewers, including Gene W Yeo as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by James Manley as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Molly Hammell (Reviewer #3).Thank you for submitting your article "Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Xist interaction, also binds to ERV elements embedded in transcripts. Structurally, ERV elements appear to be similar to the SPEN binding site in Xist. The authors provide data that indicate SPEN represses ERV elements in mouse ES cells. Inactivation of SPEN leads to increase in chromatin accessibility, active histone marks and RNA transcription of ERV element-encoded transcripts. The authors identify that RRM3 domain of SPEN is responsible for binding to ERV and insertion of ERV into an A-repeat deficient Xist can rescue binding of Xist RNA to SPEN, which result in local gene silencing. Overall the paper is an interesting observation that has for the most part rigorous assays that contribute to our understanding of how Xist evolved new domains to recruit protein interactors for gene expression control. There are, however, major concerns that need to be addressed.Carter et al. demonstrates that SPEN, an RNA binding protein involved in Major concerns:1) Can the authors clarify how the chromatin changes observed at ERV genomic loci change upon SPEN depletion in mESCs as there are some inconsistencies. The high expression of the ERV elements in WT ESCs would be expected to reflect open chromatin at these loci. But are these ERVs then repressed normally? The model of RNA recruitment of SPEN to the ERVs can be better explained to account for these inconsistencies. Also, what fraction of the ERVs with altered chromatin marks have associated expressed transcripts and which ones are differentially expressed in SPEN KO cells? Can the authors present the data as standard DEseq-like scatter/volcano plots?2) The preference for ERVs, and especially ERV-K's is still unclear. The only thing that links ERVK type TEs to each other is the presence of a lysine-tRNA in the primer binding site (PBS) of those ERV internal sequences. So, given that SPEN is not looking for a specific sequence, but a structure. And, given that SPEN doesn't seem to care about the ERV PBS region, it is difficult to understand why it prefers ERVK elements. Can the authors show that these are structurally different than the other TEs that are not bound? Eg., do they lack the single stranded bubbles associated with SPEN preferred shapes? Or, are these really just following what's expressed at this stage?Xist with the ERV sequence needs more clarification. The RIP-PCR data does not appear to demonstrate significant binding of SPEN to ERV-XIST for clone 1 and the CLIP-PCR data in the supplement does not show significance testing and have differing data between the two Xist primer pairs. The results regarding the repression of ERV-XIST needs to be clarified. It seems that HDAC1/2 inhibition has the largest effect on derepression, even though SPEN is thought to function through HDAC3 in XCI. Which HDACs might be involved in SPEN recruitment to ERV-XIST? In that same line of inquiry, the authors should explain some inconsistencies. They report SPEN induces silencing of the Xist RNA instead of silencing the Xist-bound chromosome. This suggests that there is some information that could be useful in the comparisons between which TEs were directly bound in the Clip-seq data, which of them had chromatin-based alterations, and which induced changes at the RNA level. There should have been one summary figure of the genomics data that shows how consistent these are with each other. Perhaps what happens at a locus depends on where SPEN binds ? Perhaps the number of SPEN binding sites on the locus influences how those transcripts are regulated ? Even if this can't explain why SPEN seems to regulate TEs differently from how it contributes to XCI, these comparisons should be explored.3) The results from the experiments replacing the A-repeat in  Major concerns:1) Can the authors clarify how the chromatin changes observed at ERV genomic loci change upon SPEN depletion in mESCs as there are some inconsistencies. The high expression of the ERV elements in WT ESCs would be expected to reflect open chromatin at these loci. But are these ERVs then repressed normally? The model of RNA recruitment of SPEN to the ERVs can be better explained to account for these inconsistencies. Also, what fraction of the ERVs with altered chromatin marks have associated expressed transcripts and which ones are differentially expressed in SPEN KO cells? Can the authors present the data as standard DEseq-like scatter/volcano plots?Spen KO cells, some ERV loci gain chromatin access, lose heterochromatic marks, and have elevated RNA expression. These results are consistent with a Spen-mediated surveillance of ERV loci via RNA. We now present this data in Figure 2—figure supplement 1A-F. In panels a-c, we show that there are family-specific changes in chromatin accessibility, H2K9me3 modification, and expression of specific subfamilies of transposable element as determined by DESeq2 in Spen KO mESCs. In panels d-f, we show the changes in RNA expression and chromatin modification at the sites that gain accessibility in Spen KO mESCs. Consistent with the data presented in Figure 2, we demonstrate that sites gaining accessibility in Spen KO mESCs show increased RNA expression, increased enhancer-associated marks (H3K27Ac), and decreased repressive marks (H3K9me3).The reviewers make a great point, that the relationship between changes in chromatin accessibility or modifications and RNA expression can be better presented. In general, there is low but detectable level of ERV RNA expression in wild type cells. In 2) The preference for ERVs, and especially ERV-K's is still unclear. The only thing that links ERVK type TEs to each other is the presence of a lysine-tRNA in the primer binding site (PBS) of those ERV internal sequences. So, given that SPEN is not looking for a specific sequence, but a structure. And, given that SPEN doesn't seem to care about the ERV PBS region, it is difficult to understand why it prefers ERVK elements. Can the authors show that these are structurally different than the other TEs that are not bound? Eg., do they lack the single stranded bubbles associated with SPEN preferred shapes? Or, are these really just following what's expressed at this stage?The reviewers raise a good point, that Spen’s apparent preference for ERVKs cannot be explained entirely by their belonging to the ERVK family of TEs. We believe that Spen’s specificity for this subset of ERVK subfamilies has at least two components: (i) the developmental stage during ES cell differentiation, and (ii) the structure of the recognition site on the RNA. First, we observed that Spen does not bind to nor repress all ERVKs. Rather, only specific subsets of ERVK families are targeted, notably the ETn family and RLTR9 and 13 . These subsets of ERVKs have the potential to be expressed in the mESC state, and thus are not as heavily repressed by DNA methylation and other chromatin modifications as other TE families. Thus, they may display some aberrant transcription in mESCs, leading Spen to recognize their RNA products and recruit silencing machinery to silence the locus. Consistent with this idea, we see ectopic occupancy of Oct4, the pluripotency transcription factor on these subsets of ERVKs .Spen KO mESCs show the highest expression level  The results from the experiments replacing the A-repeat in Xist with the ERV sequence needs more clarification. The RIP-PCR data does not appear to demonstrate significant binding of SPEN to ERV-XIST for clone 1 and the CLIP-PCR data in the supplement does not show significance testing and have differing data between the two Xist primer pairs. The results regarding the repression of ERV-XIST needs to be clarified. It seems that HDAC1/2 inhibition has the largest effect on derepression, even though SPEN is thought to function through HDAC3 in XCI. Which HDACs might be involved in SPEN recruitment to ERV-XIST? In that same line of inquiry, the authors should explain some inconsistencies. They report SPEN induces silencing of the Xist RNA instead of silencing the Xist-bound chromosome. This suggests that there is some information that could be useful in the comparisons between which TEs were directly bound in the Clip-seq data, which of them had chromatin-based alterations, and which induced changes at the RNA level. There should have been one summary figure of the genomics data that shows how consistent these are with each other. Perhaps what happens at a locus depends on where SPEN binds ? Perhaps the number of SPEN binding sites on the locus influences how those transcripts are regulated ? Even if this can't explain why SPEN seems to regulate TEs differently from how it contributes to XCI, these comparisons should be explored.Xist-ERV was significantly reduced compared to Xist-WT or Xist-△A upon doxycycline treatment and we found that this failure to induce the chimeric Xist RNA was due to epigenetic silencing and not genetic changes in the Tet operator in our cells . Recent work from Edith Heard’s lab  showed that HDAC3 inhibition partially blocks X chromosome inactivation. Interestingly, they also found that HDAC1/2 inhibition leads to upregulation of transgenic Xist through epigenetic derepression. Thus, we tested whether HDAC1/2i or HDAC3i could relieve Spen-mediated silencing of Xist-ERV. We found that both HDAC1/2 and HDAC3 relieved Spen-mediated Xist repression, and chose to perform functional experiments in the presence of HDAC1/2i, since we knew this would allow expression of our chimeric Xist presumably without affecting the ability of this lncRNA to silence the X chromosome which we wished to test. Our data also indicate that Spen and HDAC3 are responsible for Xist-ERV mediated gene repression, as the repression is relieved by knock down of Spen and inhibition of HDAC3 .We apologize for the confusion regarding the Xist-ERV experiments and hope to clarify here and in the main manuscript text. First, we observed that expression of Xist-ERV expression was significantly lower than the expression of Xist-WT, making the RIP-qPCR results for this cell line significantly noisier than those obtained with Xist-WT. The results are plotted as the percent of input RNA, and in the case where the input RNA is lowly expressed, this value is inherently noisier. While the result is not statistically significant, this is driven in large part by two outliers . Thus the results for Xist-ERV clone 1 suggest that Spen RRM binds to Xist-ERV and the results from clone 2 show that Spen RRM binding to Xist-ERV is significant.We also wish to clarify the results of the Spen RRM RIP experiments with Xist-ERV clone 1. Even in the presence of HDACi, Xist expression, the noise in the assay warrants the removal of these outliers, as the experiment is operating at the lower bound of dynamic range. From this analysis, we conclude that the results in Xist-ERV clones 1 and 2 show that the Spen RRMs bind to Xist-WT and Xist-ERV, but not to Xist-ΔA.In We also understand the confusion regarding the CLIP-qPCR presented in the supplemental figure. The reason that these panels did not have significance indicated is that they were performed with technical but not biological replicates. We opted to perform more experiments  for the RIP-qPCR instead of the CLIP-qPCR, so we have chosen to remove the CLIP-qPCR results from the manuscript. We don’t believe this affects the results presented in Figure 6.Xist-ERV chimeric RNA.Regarding the mechanism by which Spen silences target loci, we believe that our results are consistent with a model in which Spen recognizes and binds certain transcripts, including ERV-derived transcripts, recruits silencing machinery, including HDACs, and silences the promoter. We provide the list of altered loci with each assay in Figure 2—figure supplement 1. This model is consistent with our results at ERVs as well as our results in the context of the The matter of the overlap between ERVKs bound by Spen RRMs (irCLIP-seq) and the ERVKs that become de-repressed at the level of chromatin modifications and RNA expression is complex. We find consistently that the sites that change at the chromatin and RNA level belong to specific subsets of ERVKs including ETns, and we found that these ERVK subfamilies are also bound by Spen RRMs in multiple cell lines and conditions (Spen FL and RRM-FLAG). However, we find very little overlap between the specific genomic loci that change in accessibility and produce RRM-bound transcripts (1/288 sites that gain accessibility). We believe that this is likely due to differences in when these experiments were performed and the passage of the cells used. For the irCLIP-seq experiments, cells had to be expanded, infected with RRM-FLAG viruses and subcloned. Thus, if Spen’s recognition of ETns and other ERVKs is consistent, but the source of these aberrant expressed ETns in the genome is dynamic, the results are consistent with what we observe. Unfortunately, for technical reasons, these experiments were performed multiple passages apart.Xist itself can be expressed from the inactive X chromosome when this lncRNA is recruiting multiple powerful gene silencing complexes. This duality of potent expression on the inactive X while silencing the remainder of the chromosome is an evolved function of the modern lncRNA. In the context of wild type Xist, Spen is able to bind to Xist and spread across the chromosome in order to silence distal gene promoters. We found that Xist-ERV, our model of a proto-Xist lncRNA, mainly executes cis silencing of its own locus. This implies that additional features are missing in Xist-ERV. We agree with the reviewer that the number or quality of RNASpen interaction may determine the difference between local vs. distant gene silencing. It is also worth recalling that Xist A-repeat interacts with m6A modification machinery  as well as RNF20 . Some of these interactions are likely missing in Xist-ERV. Very recently it was suggested that Xist A-repeat has transcriptional anti-terminator activity , that allows Xist RNA to be transcribed in the face of regulatory factors that normally terminate transcription. We believe that solving this longstanding mystery in the field is beyond the scope of the present work, and respectfully suggest our work has clarified the distinct functions that may be gained by a lncRNA when it exapts an endogenous retroviral element.A fascinating mystery of the X inactivation field is how


To compare three-dimensional (3D) balanced steady-state free precession (SSFP), prospective self-gating technique  without Data were acquired in 15 subjects  using a 1.5 T system with a five element cardiac array coil. In each subject a standard multi-slice, multi-breathhold 2D cine SSFP sequence was performed with complete ventricular coverage. Additionally, a three-dimensional cine sequence with prospective self-gating [Good agreement between LVEDV, LVESV, LV mass, LVEF, RVEDV, RVESV, and RVEF calculated for the standard 2D and the 3D prospective self-gating method . The mean bias  for each parameter was; LVEDV: -0.3% (-5.2 to 4.6), LVESV: 0.3% (-5.4 to 6.0), LV mass: -0.8% (-8.3 to 6.8), LVEF: -0.2% (-2.7 to 2.4), RVEDV: 4.5% (-9.6 to 18.6), RVESV: 3.8% (-11.2 to 18.7), RVEF: 1.0% (-4.7 to 6.7). The overall image quality score for prospective self-gating (2.7 ± 0.8) was lower when compared to standard SSFP . Figure Three-dimensional, free-breathing, prospective self-gating MRI enabled accurate assessment of LV and RV quantitative parameters when compared to standard multi-slice, multi-breathhold SSFP cine imaging. Image quality with prospective self-gating was rated lower relative to the reference ECG triggered, multiple breathhold scans due to lower image contrast between blood and myocardium and residual motion artefacts.


N6-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am), 8-oxo-7,8-dihydroguanosine (8-oxoG), pseudouridine (Ψ), 5-methylcytidine (m5C), and N4-acetylcytidine (ac4C), have been shown to regulate mRNA stability, consequently affecting diverse cellular and biological processes. In this review, we discuss our current understanding of the molecular mechanisms underlying the regulation of mammalian mRNA stability by various RNA modifications.Many studies have highlighted the importance of the tight regulation of mRNA stability in the control of gene expression. mRNA stability largely depends on the mRNA nucleotide sequence, which affects the secondary and tertiary structures of the mRNAs, and the accessibility of various RNA-binding proteins to the mRNAs. Recent advances in high-throughput RNA-sequencing techniques have resulted in the elucidation of the important roles played by mRNA modifications and mRNA nucleotide sequences in regulating mRNA stability. To date, hundreds of different RNA modifications have been characterized. Among them, several RNA modifications, including  Messenger RNA molecules play their key role in directing the manufacture of proteins via translating the genetic information in DNA. Sung Ho Boo and Yoon Ki Kim at Korea University in Seoul review current understanding of the significance of modifications to messenger RNA with respect to RNA stability in mammalian cells. Recent advances in RNA sequencing technology have revealed hundreds of different modifications which, by influencing RNA stability, can control gene expression. The modifications largely involve small chemical groups added to any of the four nucleotide groups that make up an RNA molecule. This can affect all aspects of mature RNA biogenesis, including transcription, pre-mRNA splicing, RNA export form the nucleus to the cytoplasm, translation, and stability. According to the MODOMICS database, ~170 different RNA modifications have been identified in coding and noncoding RNAs9. In certain types of modifications, the specific nucleotide sequences and positions targeted for RNA modification have been well characterized due to recent advances in specialized high-throughput RNA-sequencing technologies10.Many recent studies have demonstrated that RNA undergoes various modifications in a manner similar to DNA. These RNA modifications play a role in many cellular and biological processes, thereby opening up an emerging research field known as epitranscriptomics7: (i) writer proteins (RNA-modifying enzymes), which transfer a specific chemical group to a target position on an RNA molecule; (ii) RNA-binding proteins (RBPs), which specifically recognize the modified nucleotides (reader proteins); and (iii) eraser proteins, which remove specific chemical groups from the modified nucleotides, converting them back into unmodified nucleotides. In certain cases, endogenous or exogenous chemical damage can also generate RNA modifications without the involvement of writer proteins12. In addition, some modifications are reversible, while others are irreversible.Generally, the fate of a modified transcript is determined by the coordinated actions of the following three effector proteins Fig. 1,3–7: , N6,2′-O-dimethyladenosine (m6Am), 8-oxo-7,8-dihydroguanosine (8-oxoG), pseudouridine (Ψ), 5-methylcytidine (m5C), and N4-acetylcytidine (ac4C).These RNA modifications can affect a variety of molecular processes, such as transcription, pre-mRNA splicing, RNA export, mRNA translation, and RNA degradation6A is the most abundant internal mRNA modification, and it affects various cellular and physiological processes, such as maternal-to-zygotic transition (MZT), cortical neurogenesis, and the regulation of cancer stem cells in acute myeloid leukemia19. A transcriptome-wide analysis for identifying the consensus sequence motifs for the m6A modification in the human transcriptome revealed that m6A sites (Gm6AC or Am6AC) are found in noncoding RNAs and mRNAs, with a greater number within long exons and adjacent to stop codons21.m6A modification is cotranscriptionally generated in nascent transcripts by a methyltransferase complex comprising methyltransferase like 3 (METTL3), METTL14, WTAP, and KIAA1429 , which remove the methyl group from m6A. The α-ketoglutarate-dependent dioxygenase alkB homolog 5 protein (ALKBH5) is known to be the primary and specific m6A demethylase30. In addition, fat mass and obesity-associated protein (FTO) demethylase is known to have a weak preference for m6A32.The m6A modification plays a regulatory role in diverse molecular processes, such as transcription, pre-mRNA splicing, mRNA export, mRNA stability, and translation19. The molecular events that occur through the m6A modification are guided by various m6A-recognizing reader proteins, such as YT521-B homology (YTH) domain-containing proteins. The YTHDF2 protein is the most representative m6A reader protein involved in the decay of m6A-containing RNA 38 to the m6A-containing mRNA via a direct interaction with the N-terminus of YTHDF2 and the SH domain of CNOT1, a component of the CCR4–NOT complex33. The resulting deadenylated m6A-containing mRNA would be more vulnerable to 3′–5′ exoribonucleolytic cleavage by the exosome complex or DIS3-like enzymes43.The m6A-containing mRNAs can be initiated by the interplay among YTHDF2, heat-responsive protein 12 , and an endoribonuclease RNase P/MRP complex , binds to the UGGAC motif54, which overlaps with the m6A motif, and increases the half-life of m6A-containing mRNA54. In addition to IGF2BP, other m6A reader proteins or RBPs, such as fragile X mental retardation protein (FMRP), Ras-GTPase-activating protein SH3 domain-binding protein (G3BP1), proline-rich coiled-coil 2 A (PRRC2A), and human antigen R , have been shown to stabilize m6A-containing mRNA at the transcriptome or gene-specific level60.mBPs Fig. . For ins7G cap structure in mRNA is generally methylated on the ribose ring at the 2′-OH position7. In particular, when the first nucleotide is adenosine, the methylated adenosine at the 2′-OH position, known as 2′-O-methyladenosine (Am), is further methylated at the N-6 position of Am, generating m6Am 65, resulting in the increased mRNA stability  and are also generated by various environmental stresses, such as ultraviolet irradiation and heat shock67. It should be noted that ROS oxidize RNA bases and generate numerous forms of oxidized RNAs67. These oxidized bases include 8-oxoG, 8-oxo-7,8-dihydroadenosine, 5-hydroxyuridine, 5-hydroxycytidine, and cytosine glycol , ultraviolet light, and alkylating agents73. For instance, the oxidation of mRNA  inhibits the efficiency of peptide bond formation by >1000-fold, regardless of the codon position71. This inhibition at the elongation step of translation causes the accumulation of stalled ribosomes71, which triggers the rapid mRNA degradation via the no-go decay (NGD) pathway, one of the mRNA surveillance pathways in eukaryotes  from the ribosomes stalled because of the mRNA oxidation.The oxidation of mRNA affects multiple steps of mRNA fate determination, including mRNA stability and translationtes Fig. 41,74–76.80. A recent study also identified poly(rC)-binding protein 1 (PCBP1) as an 8-oxoG reader protein. Notably, unlike AUF1, which recognizes a single 8-oxoG, the binding of PCBP1 to RNA requires two 8-oxoGs located in close proximity. In addition, the binding of PCBP1 to oxidized RNA is associated with apoptosis—under conditions of oxidative stress81—rather than with the rapid degradation of 8-oxoG–containing mRNAs, as has been observed in the case of AUF180. Several important questions regarding the molecular mechanisms of mRNA decay remain unanswered. First, how do 8-oxoG reader proteins trigger the rapid mRNA degradation? Second, how do the 8-oxoG reader proteins recruit general RNA-degrading enzymes? Third, is 8-oxoG reversibly converted into a normal guanosine base by a specific enzyme, as observed in the case of many other RNA modifications?In addition to NGD, 8-oxoG–containing mRNAs are degraded by 8-oxoG reader proteins via unknown mechanisms Fig. . Y-box b83. More recently, another transcriptome-wide profiling study identified thousands of Ψ sites in human mRNAs84. The conversion of uridine into Ψ is catalyzed by either RNA-independent or RNA-dependent mechanisms86. In the RNA-independent mechanism, Ψ is deposited by various Ψ synthases (PUSs) with different substrate specificities, different chemical reactions, and different subcellular localizations. In contrast, the RNA-dependent mechanism is guided and catalyzed by Box H/ACA small nucleolar RNAs (snoRNAs).Ψ is generated by the C–C glycosidic isomerization of a uridine base Fig. . Althoug86. For instance, Ψ makes the phosphodiester backbone more rigid, and the base pairing between Ψ and adenine is stronger than that between uridine and adenine. Because of these properties, the presence of Ψ in mRNAs can affect the local secondary structures and the protein-coding potential of the mRNA. Therefore, despite the lack of sufficient experimental evidence, it is plausible that Ψ may directly or indirectly influence pre-mRNA splicing, mRNA translation, mRNA localization, and/or mRNA stability. Indeed, the artificially targeted conversion of U-to-Ψ in translation termination codons  turns them into missense codons87. In particular, the U-to-Ψ change at premature termination codons (PTCs) can inhibit the rapid mRNA degradation triggered by nonsense-mediated mRNA decay (NMD)87, an mRNA surveillance mechanism by which faulty  mRNAs are specifically recognized and removed before the production of truncated  polypeptides , which catalyzes the deposition of a methyl group at the 5 position of cytosine  in the cold-shock domain of YBX195. This interaction contributes to the stabilization of m5C-containing RNA, consequently affecting physiological events, such as the MZT  and oncogene activation in human urothelial carcinoma of the bladder (UCB)95. During early MZT in zebrafish, the interaction between m5C and YBX1 stabilizes a subset of maternal mRNAs by recruiting poly(A)-binding protein cytoplasmic 1 (PABPC1)95. Failure of this stabilization leads to early gastrulation defects in zebrafish embryos95. Another recent report also showed that, in human UCB, a subset of oncogenic mRNAs have hypermethylated m5C sites and that the levels of these mRNAs are upregulated in an NSUN2-dependent manner94. In addition, the levels of NSUN2 and YBX1 proteins are higher in UCB than those in normal cells94. Mechanistically, YBX1 binds to and stabilizes oncogenic mRNAs with hypermethylated m5C sites  by recruiting HuR, thus indicating an essential oncogenic role of m5C in UCB94.A possible role of mied Fig. 98,100. D4C, in human cells showed that ac4C is widely distributed within noncoding RNAs and coding RNAs, with greater abundance near the translation initiation codon in mRNA  reduces the level of ac4C modification on RNA, indicating that NAT10 is a primary ac4C writer protein (RNA cytosine acetyltransferase)101. In yeast, orphan box C/D snoRNAs specifically guide Kre33 (a yeast homolog of human NAT10) to ac4C target sites in rRNA102, similar to the way that Ψ is guided by the H/ACA snoRNAs86. However, it remains unknown whether human NAT10 also uses box C/D snoRNAs to generate ac4C on its target RNAs. To date, neither ac4C reader protein nor an active deacetylation process has been reported. It is also unknown whether ac4C modification is a reversible process. Therefore, future studies should address the molecular mechanism underlying the stabilization of ac4C-containing mRNAs.Recent transcriptome-wide profiling of another cytidine modification, acRNA Fig. 101. mRNAion Fig. 101. Knoccis-acting elements  and trans-acting factors . Furthermore, recent advances have been made toward understanding the roles of RNA modifications in regulating mRNA stability. Although only certain types of modifications are discussed in this review, recent studies imply that several other RNA modifications might have the ability to influence mRNA stability. For instance, 2′-O-methylation (Nm)—in which a methyl group is added to the 2′-OH of the ribose ring—is highly enriched in the first and second transcribed nucleotides next to the cap structure2. The presence of Nm is known to increase the levels of peroxidasin mRNA103. Furthermore, in vitro decapping experiments have shown that a capped RNA with an Nm modification is resistant to hydrolysis by the decapping exoribonuclease DXO, which specifically recognizes and removes pre-mRNAs harboring a defective cap structure104. Therefore, these two recent reports suggest that Nm functions as an mRNA stabilizer. As they are oxidized, RNAs can also be alkylated upon exposure to alkylating agents that are either endogenously produced during normal metabolic processes or exogenously provided in the environment11. Bases, riboses, and the phosphate backbone of RNA are all vulnerable to alkylation because they contain oxygen and nitrogen atoms. As a result, numerous alkylated nucleosides can be generated in RNA, possibly affecting the RNA structure and/or the protein-coding potential of mRNA. A recent study even showed that alkylated mRNA is subject to rapid degradation via NGD12. Finally, the presence of internal N7-methylguanosine (m7G) in mRNA is known to promote translation. The positive charge of internal m7G may affect the RNA secondary structure and thereby affect the mRNA stability106.Cellular mRNA levels are determined by various quantity control pathways  and quality control pathways (such as NMD and NGD). All of these molecular events are mediated by diverse 74 are associated with the mRNA degradation caused by other types of RNA modifications.Future investigations should aim to determine the transcriptome profiles of all RNA modifications, extend a list of RNA modifications that affect mRNA stability, and elucidate the underlying molecular mechanisms. In addition, considering that several mRNA modifications, such as Ψ, oxidation, and alkylation, are associated with mRNA surveillance pathways, it will be interesting to investigate whether mRNA surveillance pathways


Caenorhabditis elegans, the piRNA (21U RNA) pathway is required to establish proper gene regulation and an immortal germline. To achieve this, PRG‐1‐bound 21U RNAs trigger silencing mechanisms mediated by RNA‐dependent RNA polymerase (RdRP)‐synthetized 22G RNAs. This silencing can become PRG‐1‐independent and heritable over many generations, a state termed RNA‐induced epigenetic gene silencing (RNAe). How and when RNAe is established, and how it is maintained, is not known. We show that maternally provided 21U RNAs can be sufficient for triggering RNAe in embryos. Additionally, we identify PID‐2, a protein containing intrinsically disordered regions (IDRs), as a factor required for establishing and maintaining RNAe. PID‐2 interacts with two newly identified and partially redundant eTudor domain‐containing proteins, PID‐4 and PID‐5. PID‐5 has an additional domain related to the X‐prolyl aminopeptidase APP‐1, and binds APP‐1, implicating potential N‐terminal proteolysis in RNAe. All three proteins are required for germline immortality, localize to perinuclear foci, affect size and appearance of RNA inheritance‐linked Z granules, and are required for balancing of 22G RNA populations. Overall, our study identifies three new proteins with crucial functions in C. elegans small RNA silencing.In  PID‐2 and its newly‐identified interactors PID‐4 and PID‐5 modulate size and appearance of Z granules to establish and maintain stable heritable silencing (RNAe) by maternal piRNAs. Therefore, genome stability should be tightly controlled in these cells. The integrity of the genome is constantly threatened not only by external factors, such as irradiation and mutagenic agents, but also by intrinsic factors resident in the genome, such as transposable elements (TEs). Consequently, organisms have evolved a variety of mechanisms to counteract these threats. Among these, small RNA pathways often play important roles in controlling TE activity. In many animals, TEs are recognized and silenced in the germline by a specific small RNA pathway: the Piwi pathway. Piwi proteins represent a specific subclade of Argonaute proteins that exert their silencing function upon loading with their cognate small RNA, named piRNA (Piwi‐interacting RNA), that specifies the target transcript. The Piwi/piRNA pathway is essential in most organisms for TE silencing, but also TE‐unrelated effects have been well‐described —while in other studied model systems this does not happen. Upon target recognition by PRG‐1, an RNA‐dependent RNA polymerase (RdRP) is recruited to the targeted transcript, which is used as a template for the synthesis of complementary small RNAs, named 22G RNAs. For this step, the RdRP RRF‐1 is required, as well as so‐called Mutator proteins    screen   that causes a premature stop codon (W122X) in the gene Y48G1C.1. This gene encodes a protein with disordered N‐ and C‐terminal regions , even though the defect is less severe compared with Mutator mutants   mutants  mutants, particularly at elevated temperature  animals  mutants  is a weaker allele than pid‐2(xf23), and as such only has a very weak phenotype in the more stringent sensor (RNAe) assay, while it has an easily scored phenotype in the sensor(+) assay. RT–qPCR showed that pid‐2‐mediated reactivation of the 21U sensor (RNAe) transgene resulted in RNA expression levels that were very similar to that of 21U sensor(+) in a pid‐2 mutant background . In this state, its silencing no longer depends on PRG‐1, but does still rely on 22G RNAs (Ashe NAe) Fig . Nonetheants Fig . Continuture Fig . Such lomals Fig . Given tants Fig , we assuound Fig . We concpid‐2(xf23) impaired silencing of the DNA transposon Tc1. We found that pid‐2(xf23) animals displayed activation of Tc1, but at a frequency that is significantly below that observed in, for instance, a wago‐1; wago‐2; wago‐3 triple mutant  with pid‐1 heterozygous hermaphrodites that did not carry a 21U sensor transgene. The pid‐1 homozygous mutant offspring from this cross cannot produce 21U RNAs themselves, but do receive maternal PRG‐1 and 21U RNAs. In other words, any 21U RNA‐driven activity in these embryos stems from the maternal pool alone. We observed that a large fraction (~ 40%) of such animals was able to silence the 21U sensor(+), indicating that the maternal 21U RNAs were sufficient for silencing  mutants . In addition, a pseudo‐male animal  was detected , and hrde‐1 and mut‐7 mutants in which the RNAe status was disrupted  and pid‐2(tm1614) on these 22G RNA populations. For this, we crossed the 21U sensor into pid‐2 mutants, either from a mut‐7 mutant background, in which it was expressed (+), or from a prg‐1 mutant background, in which it was under control of RNAe  state   mutants did not show this effect on either 26G RNAs  Fig . ConsistRNAs Fig  or assocRNAs Fig . The effRNAs Fig . Also whpid‐2 mutants. This revealed that many genes displayed consistently either up‐ or down‐regulated 22G levels in pid‐2(xf23) mutants , very similar effects were found , and a polyclonal antibody that we raised against the endogenous protein  followed by quantitative label‐free mass spectrometry (MS) on gravid adults, using both the transgenic line expressing the rescuing, C‐terminally tagged PID‐2::eGFP fusion protein ) in pid‐4 and pid‐5 mutants. Independent of the initial status of the 21U sensor, both pid‐4 and pid‐5 mutants were silencing‐proficient   dropped significantly, comparable to what we observed in pid‐2 mutants  Fig , with exants Fig . The 21Uants Fig , but likring Fig . We alsonts Figs  and EV4Apid‐4, pid‐5 and pid‐4;pid‐5 mutants, although 26G RNAs tended to be reduced  is not only a characteristic of prg‐1 mutants initiation of silencing within the PGCs of the next generation. In case of de novo silencing, maternal 21U RNAs initiate the 22G RNA response, and PID‐2 is required to do so effectively. In case of inheritance, inherited 22G RNAs re‐trigger 22G RNA production, and again, PID‐2 is required for effectivity. The fact that we see a stronger requirement for PID‐2 in establishment of silencing compared with maintenance could be due, for instance, to differences in 21U and 22G RNA inheritance. Seen from this perspective, PID‐2 may play an important role in interpreting inherited small RNA populations from the parents and in using these to establish silencing within the PGCs in the embryo.We also show that PID‐2 is required for stable inheritance of RNAe, and Wan et al  show thapid‐2 mutants. Hence, it seems more likely that PID‐2 regulates factors that in turn execute the silencing response. We also note that PID‐2 is not specific to the PRG‐1 pathway, as pid‐2 mutants do show effects on overall 22G RNAs, including those of the CSR‐1 pathway, as well as relatively strong effects on 26G RNAs. Possibly, PID‐2 may have a general function in the regulation of RdRP activity and thus affect both 22G and 26G RNAs. The fact that, in pid‐2 mutants, Mutator target genes lose 22G RNAs preferentially from their 5ʹ regions also supports this idea: PID‐2 could be involved in the processivity of the RdRP enzyme RRF‐1. We note that this effect is opposite to what has been observed for znfx‐1 mutants that lose 22G RNAs preferentially within the 3ʹ end of target genes   Fig , but notEscherichia coli OP50 and grown at 20°C, unless otherwise stated  on a slide, washed and then fixed with acetone (2 × 80 µl). After acetone has evaporated, worms have been washed 2 × 10 min with 80 µl of PBS‐Triton X‐100 0.1%. After removing the excess of PBS‐Triton X‐100 0.1%, the worms have been mounted on a coverslip with Fluoroshield™ with DAPI (5 µl) .3 have been added to paralyse the worms. After removing the excess of M9, a slide prepared with 2% agarose (in water) has been placed on top of the coverslip and worms have been imaged directly.Alternatively, for live imaging, 20–25 worms have been picked to a drop of M9 (80 µl) on a slide, washed and then 2 µl of 1 M NaNImages have been acquired either at a Leica DM6000B microscope  or at a Leica TCS SP5 STED CW confocal microscope . Images have then been processed with Leica LAS software and ImageJ. Images representing the expression of PID‐2, PID‐4, PID‐5, and P and Z granules markers have been processed with the Huygens Remote Manager v3.6 and deconvoluted .For scoring the 21U sensor as active or silenced, we have used a Leica M165FC widefield microscope. The 21U sensor has been scored as active, if the fluorescence was easily visible with a lower magnification ; faint, if the fluorescence was only visible with a higher magnification ; and silenced, if no fluorescence was visible. The worms have been later used also for live imaging with a Leica DM6000B microscope as described above.et al, et al, z‐stack, and analysed the two fluorescence channels of interest at the time. First, we cropped an area containing 1–8 nuclei within the pachytene zone of the gonad arm, adding the area to the ROI manager, to ensure cropping of the same area in both channels being analysed. We then used DiAna_Segment to apply a mask to the images, considering all objects with size from 1 to 2,000 pixels and then adjusted the threshold to ensure a faithful segmentation of the images. After segmentation, we performed the analysis using DiAna_Analyse and measured the surface area (µm2), the distance between the two signals (µm) and the number of objects present in the cropped area. We then represented the distance between centres as a measure of colocalization. For each couple of fluorescent proteins, we analysed 4–10 images of gonads from individual animals.In order to perform colocalization analysis of the fluorescently tagged proteins PID‐2, PID‐4, PID‐5 and of the P and Z granules markers, we used the DiAna plugin of (Fiji Is Just) ImageJ  were added to the RNA and incubated at 37 °C for 30 min with gentle shaking. The reaction has been stopped by adding 5 µl of 10× TURBO™ DNase Inactivation Reagent. Samples have been centrifuged at 10,000 × g for 90 s and RNA transferred to a fresh tube. RNA quality has been assessed at NanoDrop and on agarose gel, and then, samples have been further processed for enrichment of small RNA populations.Synchronized gravid adults have been collected with M9 and fast‐frozen on dry ice in 250 µl of Worm Lysis Buffer . 30 µl of Proteinase K  has been added to dissolve the worms for 90 min at 65°C with gentle shaking. Lysate has been centrifuged at maximum speed for 5 min at room temperature (RT), and the supernatant was transferred on a Phase Lock Gel tube . 750 µl TRIzol LS  has been added per 250 µl of sample, and after homogenization, the samples have been incubated for 5 min at RT to allow complete dissociation of the nucleoprotein complex. Then, 300 µl of chloroform  was added per 750 µl of TRIzol LS, and the samples were incubated for 15 min at room temperature after mixing. Samples have been centrifuged at 12,000 × et al, For each strain, three biological replicates have been used for RNA extraction and library preparation. RNA was treated with RppH  to dephosphorylate small RNAs and specifically enrich for 22G RNAs, as previously described , using the NEXTFlex 3ʹ SR adaptor and 5ʹ SR adaptor , quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life Technologies), and sequenced on a NextSeq 500/550 (Illumina).http://www.bioinformatics.babraham.ac.uk/projects/fastqc).Before mapping to the reference sequences, reads were processed in the following manner: (i) trimming of sequencing adapters with cutadapt v1.9 (‐a TGGAATTCTCGGGTGCCAAGG ‐a AGATCGGAAGAGCACACGTCT ‐O 5 ‐m 26 ‐M 38) Martin, ; (ii) reC. elegans genome (WBcel235) to which the 21U sensor sequence  insertion were singled into a 10 cm NGM plate seeded with 100 µl of OP50 and grown at 20°C. Plates were regularly checked for reversion events until they were starved. When starved, the population was estimated at 10,000 animals. Average animal populations on non‐starved plates were estimated by counting sectors of a number of plates. Transposition frequencies at each time point were calculated using the following formula: f = −ln [(T − R)/T]/N, where T = total number of plates scored, R = number of plates with revertants and N = number of worms on the plate. A table with the numbers used to make the graph in Fig For each analysed genotype, mutant worms carrying the 2) seeded with 300 µl OP50, grown at 25°C and followed over time. Worms have been picked every 4–6 days, before starvation, and we assumed 2–3 generations, respectively, have passed. Worms have been passed to fresh plates every 4–6 days until all the mutants died.Before starting the experiment, mutants have been outcrossed four times. For the assay, N2 has been used as wild‐type strain and the desired mutant strains have been tested. For each strain, 6 L3 worms have been picked onto 15 NGM plates  and two deletion alleles of W03G9.2/pid‐4 (xf184 and xf185). Each allele has been sequenced to pinpoint the exact deletion at nucleotide resolution. The mutant strains have been outcrossed two times against wild‐type N2 strain to remove any potential off‐target effect of Cas9 and used for further experiments.Wild‐type worms have been injected with an injection mix containing 50 ng/µl pJW1259 , co‐injet al, rol‐6) were singled out. The tagged strains have been outcrossed two times against wild‐type N2 strain to remove any potential off‐target effect of Cas9 and used for further experiments.In order to introduce an epitope tag at endogenous loci, we used the co‐conversion approach as previously described  for quantitation and the Ensemble E. coli REL606 database (Version Oct 2018) to filter potential contaminations.Label‐free quantitative mass spectrometry has been performed as described in Ref. (Almeida Information available in the pid‐2 and performed initial analyses. SD and FB performed quantitative mass spectrometry. All authors were involved in discussion of the data. MP and RFK wrote the manuscript with input from all authors.MP, AMJD and RFK planned experiments. MP generated strains, and performed genetics, microscopy and molecular biology. RFK and MP performed Tc1 reversion assays. AMJD analysed sequencing data. JS generated strains and performed microscopy. SH generated strains. BFMA identified The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Table EV1Click here for additional data file.Table EV2Click here for additional data file.Source Data for Expanded View and AppendixClick here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 5Click here for additional data file.Source Data for Figure 6Click here for additional data file.Source Data for Figure 7Click here for additional data file.