Sexual and reproductive health (SRH) communication is most likely to promote healthy sexual practices and to reduce risky sexual behavior among adolescents. Communication is the principal means for parents to transmit sexual values and knowledge to their children. This study aimed to assess adolescent-parent communication on SRH issues among high school students in Woldia town.A cross-sectional study was conducted from December 15-31, 2016. Systematic random sampling technique was used to select 693 students from Grades 9 and 10. Data were entered into Epi-info version 3.5.1 and analyzed by using SPSS version 20. Logistic regression with Odds Ratios (OR) and 95% Confidence intervals (CI) was used to identify the independent predictors of adolescent-parent communication.674 students accepted to participate in this study giving a response rate of 97.3%. Only 205(30.4%) had discussed on two or more SRH topics with their parents. In logistic regression analyses, mothers who could read and write  and had diploma certificate  were more likely to discuss on SRH issues with their children. Adolescents who agreed on the importance of discussion on SRH issues , whoever got SRH information  and those whoever had sexual intercourse  were more likely to discuss on different SRH issues with their parents.This study revealed that parent-adolescent communication on SRH issues was inadequate. Therefore, there is a need to equip and educate parents on different adolescents' SRH issues. An increased incidence of sexually transmitted disease (STD) including human immunodeficiency virus (HIV) infections in adolescents in developing and developed countries has led researchers to examine factors that influence young people's sexual behaviors . ImproviDifferent Studies revealed that adolescents in Ethiopia have very low health-seeking behavior mainly to their SRH matters and even the current reproductive health services are not adolescent-centered , 8. FurtStudy area and period: The school-based cross-sectional study was conducted among high school students in Woldia town, North Wollo Zone, Amhara Regional State from December 15-31, 2016. Woldia town is located in the Northeastern part of Ethiopia at 521km from the capital city, Addis Ababa. According to 2007 Ethiopian population census report, the total population of the town was 46,139 with sex distribution of 49.8% “males” and 50.2% “females”. Moreover, from the total population, 13,027(28%) found in the age group of 15-24 for both sexes [th sexes . The towStudy population: The study population was all students of grade 9 and 10 of both high schools in Woldia town during the study period. Unmarried adolescents in the age group 15-24 years were included in the study. The sick, visually impaired and those unwilling to participate were excluded from the study.Sample size determination: The sample size was calculated using single population proportion formula considering the following assumptions: P=28.9% [α/2=1.96, margin of error 5% (d=0.05). Assuming 10% non-response rate and design effect 2, the final sample size was 693. P=28.9% , signifiSampling procedures: A multi-stage sampling technique was used. Simple random sampling was used to select the sections from each grade in the two schools. To select the study unit, students" roster was used as a sampling frame. To determine the number of students from each grade, proportional allocation to their size was used and in every step, a simple random sampling technique was used to select the grade and students from each section. Finally, the study subjects were selected using simple random sampling technique.Dependent variable: Levels of communication on SRH issues.Independent (explanatory) variables: Determinant factors for parent-adolescent communication like socio-demographic characteristics ; behavioral factors (perceived openness of parents to discuss on SRH issues); sexual behavior were assessed.Communication on SRH issues: Students who discussed at least two SRH issues  with their parents in the last 12 months.Parents: Biological parents, step-parents or foster parents including elder siblings.Adolescents: Segment of the population who are unmarried and fall to the age range of 15-24years.Data collection instruments: Data were collected using pretested structured self-administered questionnaire. The questionnaire consisted of socio-demographic characteristics of parents and adolescents, behavioral factors of parents, and sexual behaviors of the adolescents. The questionnaire was prepared in English, and it was translated into Amharic language  for appropriateness and easiness. The Amharic version was again translated back to English language to check the content validity of the original version. Translation of questionnaire was done by language experts in both cases.Data collectors: Three individuals who had completed grade 12, preferably who has experience of data collection were selected. Confidentiality and privacy were given attention during training and the trainees participated during pre-testing of the questionnaire. The aim of the study was explained to the selected adolescents. Data collectors were supervised by two B.Sc degree holder nurses and problems faced during data collection were solved on time. The principal investigator had checked filled questionnaires and solution was given by discussing with the supervisors and data collectors. Finally, filled questionnaires were signed by supervisors after checking for its completeness.Data quality control: Data collectors were trained for one day on the objectives of the study, sampling procedure, about the questionnaire and how to check the completeness of questionnaires. To ensure the quality of the data, all the filled questionnaires were checked for incompleteness and inconsistency. The investigator also discussed with the supervisors on how to supervise the data collectors and how to solve problems encountered during the data collection process. Data collectors were supervised by B.Sc degree holder nurses. Moreover, pre-test was conducted on 10% of all the total sample size from other high school to ensure its completeness and consistency in providing the information needed for the study.Data processing and analysis: In this study, the term “parent-adolescent communication on SRH issues refers to parents opening a discussion on SRH issues or parental involvement in SRH discussion initiated by the adolescents or both”. It was measured with a “Yes” for those who had a parental discussion and a “No” option for those who had no parental discussion. Data were coded, edited, and entered using EPI Info version 3.5.1 and then transported to SPSS version 20 for statistical analysis. Descriptive statistical analysis was used to compute frequency and a percentage of independent and dependent variables. Binary logistic regression analysis was used to ascertain the association between explanatory variables and outcome. Variables with significant associations in the bivariate analysis were entered into a multivariate analysis to determine the independent associated factors of adolescent-parent communication on SRH issues. Variables with a P value<0.05 was considered as significant.Ethical consideration: Ethical clearance was obtained from Woldia University; Faculty of Health Science institutional research ethics review committee. Permission was obtained from school administration and schools' parent-teachers union. Informed verbal consent/assent was obtained from every participant after explaining the purpose of the study in detail. Confidentiality of information was kept anonymously.Socio-demographic variables of respondents: The response rate for this study was 674/693(97.3%). Of these, 345 (51.2%) were females. The median age of the respondents was 18.96. Four hundred eighty-nine (72.6%) were Orthodox Christians. Adolescents who were living with their fathers and mothers account 480(71.2%) followed by those who were living with their mothers 102(15.1%). Three hundred sixty-nine (54.7%) were grade nine students followed by grade ten students who account 305 (45.3%) ( (45.3%) . Only, 1 (45.3%) .Adolescent-parent communications on SRH issues: Majority of the adolescents, 528(78.3%) believed that it is important to discuss on SRH matters with their parents. Parents are recommended to have regular communication on SRH issues with their adolescents. However, only 205(30.4%) of adolescents had a discussion with either of the parents on at least two topics of SRH issues  of adolescents mentioned that they got SRH information. Among mentioned source of information for SRH issues, Television, and school account 332 (56.3%) and 293 (49.2%), respectively  in this study mentioned that they got SRH information. Among mentioned sources of information for SRH issues, Television and schools account 56.3% and 49.2%, respectively. This may be due to the fact that currently adolescents are more exposed to Television and information communicated through school mini-media, teachers and the incorporation of SRH matters in their grade level science subjects. In this study, from the assessed factors associated with communication on SRH issues, only mothers who could read and write, adolescents' positive perception on the importance of discussion on SRH issues, adolescents whoever had sexual intercourse and whoever got SRH information showed significant positive association with communication on SRH issues. This result is inconsistent with the study conducted in Debre Markos, Ethiopia . Those aIn this study parent-adolescent communication on SRH issues was inadequate. Adolescents whose mothers could read and write and adolescents whoever got SRH information had communication on SRH issues with their parents than others. Moreover, adolescents who had positive perception on the importance of discussion on SRH issues with parents and adolescents whoever had sexual intercourse were more likely to communicate SRH issues with their parents as compared to others. Majority of adolescents preferred their friends/peers other than parents to discuss on different SRH issues. School-based education is important to improve adolescent-parent communication about SRH issues. Equip and educate parents on different SRH issues, initiate comprehensive family life education for the adolescents and parents are needed. Moreover, a qualitative study could be done on adolescents and parents communication.Adolescent sexual and reproductive health has been overlooked historically despite the high risks that countries face for its neglect; Some of the challenges faced by adolescents across the world include early pregnancy and parenthood, unintended pregnancy, difficulties accessing contraception and safe abortion;Young people are currently the group most severely impacted by HIV/AIDS and other STIs;Most youth-friendly services and health care providers in Ethiopia are not well prepared in addressing adolescents' SRH desires; healthcare workers often act as a barrier to care by failing to provide young people with supportive, nonjudgmental, youth-appropriate services.Majority of adolescents preferred their peer than parents to discuss all SRH issues; so incorporating peer to peer sexuality education program into school curriculum is needed;Adolescents whoever got SRH information were more likely to discuss on some SRH issues with their parents; hence, providing behavioral change communication, conducting sustainable advocacy works targeting parents and communities on young people's SRH is important to improve adolescent-parent communication on SRH issues;Programmes/policies related to youth and adolescents' SRH should address not only individual or behavioral factors but also cultural and social factors that negatively influence parents' communication on SRH issues.The authors declare no competing interests.


The present data are reported in the article “Parenting by Lying in Childhood is Associated with Negative Developmental Outcomes in Adulthood” . Data were collected using online survey. In this dataset, there are 377 responses from young adults from Singapore who reported on their childhood exposure to parenting by lying, their current deceptive behaviors toward parents, and their psychosocial adjustment. Path analysis was performed to better understand parenting by lying - a prevalent, but under-studied parenting practice. We measured exposure to parenting by lying in childhood , lying b2All the data were collected online using Qualtrics. The data consist of demographic information, responses from parenting by lying questionnaire The parenting by lying questionnaire is a 16-item questionnaire developed by Heyman and colleagues The lying to parents questionnaire is a 12-item survey assessed the current frequency of participants’ lying to their parents The Adult Self-Report questionnaire included 126 items assessing adults’ general adaptive functioning, as well as specific psychosocial dysfunctions The Levenson self-report psychopathy scale consisted of 26 items which assessed psychopathic attributes among the noninstitutionalized population


Retinoblastoma is a rare pediatric tumor of the retina, caused by the homozygous loss of the Retinoblastoma 1 (RB1) tumor suppressor gene. Previous microarray studies have identified changes in the expression profiles of coding genes; however, our understanding of how non-coding genes change in this tumor is absent. This is an important area of research, as in many adult malignancies, non-coding genes including LNC-RNAs are used as biomarkers to predict outcome and/or relapse. To establish a complete and in-depth RNA profile, of both coding and non-coding genes, in Retinoblastoma tumors, we conducted RNA-seq from a cohort of tumors and normal retina controls. This analysis identified widespread transcriptional changes in the levels of both coding and non-coding genes. Unexpectedly, we also found rare RNA fusion products resulting from genomic alterations, specific to Retinoblastoma tumor samples. We then determined whether these gene expression changes, of both coding and non-coding genes, were also found in a completely independent Retinoblastoma cohort. Using our dataset, we then profiled the potential effects of deregulated LNC-RNAs on the expression of neighboring genes, the entire genome, and on mRNAs that contain a putative area of homology. This analysis showed that most deregulated LNC-RNAs do not act locally to change the transcriptional environment, but potentially function to modulate genes at distant sites. From this analysis, we selected a strongly down-regulated LNC-RNA in Retinoblastoma, DRAIC, and found that restoring DRAIC RNA levels significantly slowed the growth of the Y79 Retinoblastoma cell line. Collectively, our work has generated the first non-coding RNA profile of Retinoblastoma tumors and has found that these tumors show widespread transcriptional deregulation. The protein produced from the Retinoblastoma 1 (RB1) tumor suppressor gene, pRB . The actTo identify transcriptional changes to both messenger RNA (mRNA) and long non-coding RNAs (LNC-RNAs) dependent on pRB-loss, we used RNA-sequencing (RNA-seq) to profile Retinoblastoma tumors and normal retinal tissue from patients. Retinoblastoma is a rare pediatric tumor of the Retina that affects around 6,000–8,000 children every year , 12. TheAs these tumors are collected from pediatric patients, they contain very few genomic alterations and represent an ideal resource for understanding the coding and non-coding transcriptional effects driven by pRB-loss. Determining how loss of pRB changes cells is important for expanding our treatment options for patients with Retinoblastomas, and may provide insights into how RB1 contributes to additional tumor types . To expaTo determine how the loss of the Retinoblastoma 1 (RB1) tumor-suppressor gene changes the expression of coding and non-coding RNAs in tumors, we conducted RNA-sequencing (RNA-seq) from 7 Retinoblastoma patients. The tumors were removed from patients between 8 months and 6 years of age, with a mean age of 2 years and 8 months. Importantly, the gender demographics of these patients is equally split between males (4) and females (3), and between invasive (3), and non-invasive (4) tumors. A complete list of the patient and control sample demographics is included in We then used Gene Ontology (GO) analysis , 27 to iPrevious microarray studies of Retinoblastoma tumors have provided important insights into the coding RNA changes in these tumors. However, these technologies are limited in their capacity to detect RNA fusion products. To address this, we searched for RNA-fusions within our RNA-seq dataset and identified 22 putative RNA fusion events. From this collection, we selected two putative RNA hybrids for detailed examination, using RT-PCR. For this RT-PCR based evaluation, we used 10 normal retinal tissue samples and a larger cohort (>13) of Retinoblastoma tumors . From thin trans. We first compared the genomic loci of significantly up or downregulated LNC-RNAs, with the expression of deregulated coding genes that displayed reciprocal patterns. From this assay, we found that de-regulated LNC-RNAs (either up or downregulated) showed statistically significant anti-correlates with coding genes, however, these patterns were not restricted by chromosomal location  (p > 0.05)  . In the  > 0.05) . We next > 0.05) . Putativ > 0.05) . CollectWe next investigated how modulating the levels of a down-regulated LNC-RNA in Retinoblastoma affected the growth of Y79 Retinoblastoma cells. For this, we selected the DRAIC LNC-RNA, as DRAIC levels are strongly decreased in tumors  and in oInactivation of the RB/E2F pathway is a common feature of cancer . In thisIn agreement with previous microarray based studies –25, 33, bone fide fusion transcripts that occurred at high frequencies in Retinoblastoma tumors, relative to normal retinal tissue. Although much more work is needed to test the possible function of these RNAs, this work suggests that RNA-fusion products are common in Retinoblastoma tumors.As RNA-seq provides a unique platform for detecting rare fusion or trans-spliced transcripts, we investigated the frequency of these events in Retinoblastoma. There are multiple potential mechanisms for generating the hybrid RNAs, including chromosomal translocation, deletions or inversions, gene fusion, or aberrant splicing events. We independently evaluated each dataset to identify potential fusion transcripts, as these have been shown to have potential oncogenic  or prediThis work represents the first genome-wide profiles of non-coding RNA changes in Retinoblastoma tumors. Our profiles identified significant and widespread differences in the levels of anti-sense, LNC-RNAs and transposable elements in tumor samples, compared to normal retinal tissue. Although there is some inter-tumoral variation between LNC-RNA levels, the majority show consistent changes which could be independently confirmed in an unrelated Retinoblastoma cohort. Determining the potential roles of LNC-RNAs can be challenging, as they can potentially function via many mechanisms to affect local or distant gene expression or directly interact with other RNAs to alter their stability and/or translation capacity. To address this complex question, we conducted an exhaustive analysis of how altered LNC-RNAs may contribute to the relative mRNA levels in Retinoblastoma.in cis. In agreement with this hypothesis, we detected only 16 LNC-RNAs that showed tight expression correlations with their overlapping, adjoining or proximal gene partners. Second, by generating correlation efficiencies for each of the 400 most deregulated LNC-RNAs in our tumors to the mRNA changes, we found that many LNC-RNAs showed a relative positive or negative relationship to mRNA levels. Third, using new LNC-RNA: mRNA interaction platforms followed by gene ontology analysis of these putative interactions, we found that many up and down regulated LNC-RNAs have strong potential interactions with genes involved in cellular processes that sustain Retinoblastoma growth including cell division and cell attachment. These data provide a complete analysis of the transcriptional changes for both coding and non-coding genes in the pediatric Retinoblastoma tumor.First, we investigated how each altered LNC-RNA correlated with deregulated mRNA levels, based on chromosome location. These predictions suggest that although each LNC-RNA did show anti-correlates with several mRNAs, these were spread throughout the genome, suggesting that most of the changed LNC-RNAs in Retinoblastoma do not function To measure how altered LNC-RNAs may contribute to Retinoblastoma growth, we evaluated how re-expressing a down-regulated LNC-RNA in Y79 Retinoblastoma cell lines altered cell number. DRAIC expression slowed Y79 cell growth compared to controls, in support of previous data from lung cancer, showing that reducing the expression of DRAIC back to physiological levels changes the proliferation potential of cells. These experiments show that modulating the levels the DRAIC LNC-RNA does affect Retinoblastoma cells.The 2 normal Retinas were obtained from donor eye balls from the PU Shah eye bank. The age was 12 and 22 years, respectively. The Retinoblastoma tumors were obtained from informed consent provided by the parents of the children undergoing treatment at Sankara Nethralaya, India. The protocol was approved by institutional review board on Ethical practices for research at Sankara Nethralaya. The pathological characterizations of the tumors are presented in The Retinae and tumors were thawed, and RNA was extracted using TRIzol reagent  according to the manufacturer's instructions. The samples were homogenized in TRIzol reagent and incubated at room temperature for 5 min. Next, chloroform was added, and the tubes were mixed vigorously for 15 s followed by incubation at room temperature for 2–3 min. Next, the samples were centrifuged at 4°C for 15 min at 12,000 g. RNA, which is part of the aqueous phase, is collected cautiously and is precipitated using isopropanol, followed by 75% ethanol washes. After the washes, RNA pellet is dried completely (5–10 min) before re-suspending in nuclease free water. Total RNA thus extracted is qualified on 2,200 Tape Station using the RNA specific Tapes and reagents . Quantification of the RNA was done using Qubit 3.0 Fluorimeter  with an RNA HS Assay .Once the RNA passed QC, it was taken up for library preparation using the TruSeq RNA Library Prep Kit V2 . During the library preparation, the Ribosomal RNA (rRNA) were removed using biotinylated, rRNA specific oligo's combined with Ribo-zero rRNA removal beads . Following purification, the Ribo-depleted RNA is subjected to fragmentation in the presence of divalent cations at elevated temperatures. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Then, second strand cDNA synthesis was performed, using DNA polymerase I and RNase H. These cDNA fragments will go through the process of repairing the ends, addition of a single “A” base, followed by ligation of the adapters. The ligation product was purified using Agencourt AMPure XP beads  and enriched using PCR to create the final cDNA library for sequencing. After a final cleanup with Agencourt AMPure XP beads, the library was quantified using Qubit DNA Assay and the fragments were assessed using DNA Tape Station D1000 Screen Tape . The quantified libraries were then clonally amplified on a cBOT and sequenced on the HiSeq2500 with 100 bp paired end chemistry. 5′AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT3′INDEX]ATCTCGTATGCCGTCTTCTGCTTG3′.The TruSeq Universal Adapter. The TruSeq Illumina Index Adapter consisted of following sequence: 5′GATCGGAAGAGCACACGTCTGAACTCCAGTCAC[ftp://ftp.ensembl.org/pub/release75/gtf/homo_sapiens/Homo_sapiens.GRCh37). The processed data were then checked for the presence of unwanted sequences or contamination , and those contaminations were removed using Bowtie2 v-2.2.4  and the predicted targets were extracted. These mRNAs, their associated SUMENERGY score and change in expression, were used to sub-group the putative LNC-RNA targets in the tumor cohort. These groups were then used in Gene Ontology analysis using DAVID , 45.LNC-RNAs are known to regulate gene expression in cis-acting and trans-acting ways. We carried out neighboring gene analysis and LNC RNA-mRNA interaction analysis to identify the probable targets of LNC-RNA. LNC-RNAs identified to be differentially expressed were classified based on genomic location, and this was done with any other gene (irrespective of coding nature). The following distance parameters were used: Overlap is within any other gene, Adjoining is within 5 kb of another gene, Proximal is within 10 kb of another gene, Others >10 kb of another gene. Those neighboring protein-coding genes that were found to be significantly expressed were considered to be influenced by LNC-RNA.Retinoblastoma samples were provided by the Cooperative Human Tissue Network, a National Cancer Institute supported resource. Other investigators may have received samples from these same tissue specimens. Normal retinal tissue was obtained from warm autopsy samples from Mohamed Abdel-Rahman at the Ohio State University. RNA was extracted from each of the three retinoblastoma tissue samples and four retina tissue samples that were received snap frozen. To each sample, 1 mL TRIzol Reagent (Invitrogen) was added to 10 mg of tissue and the samples were homogenized using a homogenizer and incubated at room temperature for 5 min. 0.2 mL of chloroform was added to the samples and incubated at room temperature for 2 min, then the samples were centrifuged for 15 min at 12,000 × g at 4°C. The upper aqueous phase containing the RNA was transferred to a new tube and 0.5 mL of isopropanol was added, then this mixture was incubated at room temperature for 10 min. The samples were centrifuged for 10 min at 12,000 × g at 4°C and a white pellet formed. The supernatant was discarded and the pellet re-suspended in 1 mL of 75% ethanol. The sample was briefly vortexed and then centrifuged for 5 min at 7,500 × g at 4°C. The supernatant was discarded and the RNA pellet air dried for 10 min. The pellet was re-suspended in 20 μL of RNase-free water and incubated in a 60°C water bath for 15 min. The RNA was then quantified using a BioTek Epoch Microplate Spectrophotometer.http://hegemon.ucsd.edu/RB-p1/fusion/fusion.php).The list was then manually filtered using BLAST on the NCBI website.EricScript framework (Version: 0.5.5b) is used for the discovery of fusion transcripts in paired end RNA-seq data (cite PMID: 23093608). Following steps are performed by the EricScript: (1) Mapping of the reads against the transcriptome , (2) Identification of discordant alignments and building of the exon junction reference, (3) Recalibration of the exon junction reference, (4) Scoring and filtering of the candidate gene fusions. The data is visualized using a custom webserver based on The JBrowse Genome Browser (PMID: 19570905). A simple perl script was written to filter through the list of candidate fusion transcripts and explored using the JBrowse Genome Browser (cDNA was prepared from 400 ng of RNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems), according to manufacturer's protocol.The expression of the specified target genes was quantified by q-RT-PCR, using Fast Start Universal SYBR Green Master with ROX (Roche), and was normalized to the expression of β-actin and normal samples. Custom designed primers were used.5 Y79 cells were seeded in a 24-well plate. Six wells each were transfected with pcDNA-DRAIC and pcDNA3-EmptyVector were transfected using Lipofectamine2000, according to manufacturer's protocol. The plasmids were a kind gift of Anindya Dutta .KD extracted RNA from tumor and normal retinal samples. KD and SR generated cDNA and conducted RT-PCR from extracted RNA for second independent cohort analysis. RK tumor collection and data handling. PR and VK are the surgeons who obtained tumors. SK conducted the pathology on the tumor samples. KS, MB, and DS ran the bioinformatics analysis. LS conducted RNA fusion product analysis and MC conducted LNC-RNA correlation analysis. SE contributed to data handling and designed the experiments. WM designed experiments, helped with analysis, and wrote the manuscript.KS was employed by the company MedGenome. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CAG repeats RNA causes various fatal neurodegenerative diseases exemplified by Huntington’s disease (HD) and several spinocerebellar ataxias (SCAs). Although there are differences in the pathogenic mechanisms, these diseases share the common cause, i.e., expansion of CAG repeats. The shared cause of these diseases raises the possibility for the exploiting the common target as a potential therapeutic approach. Oligonucleotide-based therapeutics are designed earlier with the help of the base pairing rule but are not very promiscuous, considering the nonspecific stimulation of the immune system and the poor cellular delivery. Therefore, small molecules-based therapeutics are preferred for targeting the repeats expansion disorders. Here, we have used the chemical similarity search approach to discern the small molecules that selectively target toxic CAG RNA. The lead compounds showed the specificity towards AA mismatch in biophysical studies including CD, ITC, and NMR spectroscopy and thus aided to forestall the polyQ mediated pathogenicity. Furthermore, the lead compounds also explicitly alleviate the polyQ mediated toxicity in HD cell models and patient-derived cells. These findings suggest that the lead compound could act as a chemical probe for AA mismatch containing RNA as well as plays a neuroprotective role in fatal neurodegenerative diseases like HD and SCAs. Trinucleotide repeats (TNRs) are present in human genes, with only a few known functions. However, the expansion in some repeats above the threshold limits results in more than 40 neurological disorders3. These expansions can occur either in the coding region, like Huntington disease or in the non-coding region such as Fragile X-associated Tremor Ataxia Syndrome (FXTAS). CAG repeats RNA alone is involved in most of the trinucleotide repeats disorders, including Huntington’s disease (HD) and different spinocerebellar ataxia (SCAs). CAG repeats expansion can occur in 5′UTR of the gene PPP2R2B that causes SCA type 12 whereas expansion in exon region causes other late-onset progressive neurodegenerative disorders like HD, spinal and bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DPLA) and several other SCAs.Classically, RNA was considered merely a carrier of genetic information. But modern views have stated RNA as a profound tool to modulate the gene expression as well as other biological processes. Although, RNA has been considered undruggable with the small molecules outside of bacterial ribosome, due to lack of their higher affinity and specificity. However, the use of new biochemical screening methods and design based technologies have provided compelling progress in this field4. However, other homo-polymeric proteins produced by sense and antisense repeat-associated non-ATG (RAN) translation were also reported for diseases progression5. The early age onset and the severity of the TNR disorders are associated with a large number of repeats present in the genes. HD is one of the well-known trinucleotide repeats disease that is caused by the expansion of CAG repeats in the first exon of the IT15 gene (Interesting Transcript 15). Generally, a healthy individual contains up to 36 CAG repeats, while the number of CAG repeats is increased by more than 40 repeats in the coding region of the huntingtin gene in the diseased condition6. The global prevalence of HD varies widely, with a very high incidence in Caucasian while lowest in the Asian population7. Unfortunately, there is no cure for these devastating disease till date.Provided all possible reading frames, expanded CAG repeats could freely encode into three different kinds of amino acids . Earlier, reports stated that only poly Q protein aggregates are responsible for the pathogenesis and therefore these diseases are also known as polyglutamine (poly Q) diseases9. The size and stability of the hairpin are dependent on the CAG repeat length10. The formation of duplex hairpins recruits various RNA binding proteins like muscleblind-like (MBNL), DICER, several transcription factors, and nucleolar protein nucleolin. The sequestration of these crucial proteins results in the loss of their normal functions that further leads to aberrant post-transcriptional regulation13. Recently, repeat-associated non-ATG (RAN) translation in r(CAG)exp has also been studied where four different types of homo-polymeric proteins are encoded by mutant CAG RNA that further results into toxic protein aggregates inside the cells15. All these mutual pathogenic mechanisms lead to a toxic condition inside the neuronal cells.CAG repeats expansion translates into polyglutamine which further forms the polyQ aggregates, a crucial pathological hallmark of HD patients. The transcript of expanded CAG repeats can also fold into the non-canonical three-dimensional duplex hairpin loop structure with non-stranded A-A mismatch17 (2)-Epigallocatechin-3-Gallate (EGCG) has been studied as a poly Q protein inhibitor in HD18. However, HD pathology is not limited to poly Q protein aggregation, but the mutant RNA also plays a vital role in disease development. Hence, targeting the actual toxic RNA rather than poly Q aggregation may provide promising results for HD therapeutics development. Oligonucleotide and peptide-based approaches targeting the toxic CAG RNA has the limitation of the blood-brain barrier, allergic reactions, and poor absorption19. Conversely, small molecule-based therapeutic approaches have shown promising results. Several other groups, as well as we have reported small molecules targeting expanded CAG RNA and providing hope for HD drug development23. However, the high affinity and specificity of small molecules are prerequisite for the therapy as HD treatment needs a long-term continuous dose. To explore compounds with similar shape or chemistry with improved biological activity or pharmacodynamics & pharmacokinetic properties, herein, we have used chemical similarity approach to screen the NCI library with a previously reported molecule, Myricetin as a query molecule.Various strategies have been reported as an HD therapeutics, including protein aggregation inhibitorsInterestingly, we have found some small molecules with a better affinity and less toxicity than Myricetin. We have assessed the selectivity of potential lead compounds using CD, UV melting, various gel-based assays, and NMR spectroscopy. Moreover, the compounds were also tested in HD cellular models, and patient-derived cells to assess the applicability of our decrees to the HD pathogenesis.25. Therefore, we have used chemical similarity search to explore new potent small molecules that could be utilized to target the pathogenic CAG repeats, causing HD and SCAs.The rationally designed small molecules for this toxic 5′CAG/3′GAC RNA could provide a valuable avenue as a therapeutic approach for these Trinucleotide repeat expansion diseases27. Recently, shape-based small molecule screening has been considered as a promising tool in drug discovery because it encouragingly provides new molecules with better affinity and selectivity29. Herein, we have performed the chemical similarity approach and collected small molecules from the National Cancer Institute (NCI) with features that strongly predispose them to bind with RNA. The similar shape and structure of the molecules help it to bind within the same pocket and concurrently, exhibited similar biological activates. NCI database is an easily accessible database that contains more than 250,000 compounds with broad chemical spaces along with complete stereochemistry specifications. Rapid Overlay of Chemical Structures (ROCS)30 software uses 3D shape algorithm for 3D shape comparison between two molecules. The Gaussian function of ROCS helps to fasten the 3D shape-based calculations due to the involvement of maximal intersections of the volumes of two molecules. The compounds with complete stereochemistry specification and reliable 3D coordinates of stereoisomers were chosen for screening. The chemical similarity search of the compounds was done using Omega 2.3.2 (v 2.02) software31 from OpenEye Scientific Software. Chemical similarity search uses two different matrices: Shape Tanimoto coefficient29 for 3D similarities and color score32 for 3D chemistry alignment. Colour force field (implicit Mills–Dean)32 helps to align hydrogen bond donor, acceptor, a hydrophobic group, cations, anions, and rings. The similarity of two compounds is related to either shape or color score that lies in between 0 to 1, which states no or complete similarity respectively. Subsets of the top 19 compounds x6 RNA  range with 5′CAG/3′GUC RNA, indicating their high selectivity for the 5′CAG/3′GAC RNA  stands for the thermodynamically favorable interaction between two molecules. Therefore, the high association constant values of compounds CP2, CP6, and CP13 with high negative ∆H are also in favor of high negative free energy of reaction and subsequently supporting the spontaneity of the reaction. After performing the successful screening of these compounds, we proceeded for further studies to assess the specificity of these potential lead compounds with 5′CAG/3′GAC RNA.Isothermal Titration Calorimetry (ITC) has been considered as a valuable tool to study the interactions of small molecules with different macromolecules including proteinsRNA Fig. . Out of 42 with a large positive peak near 265–275 and a negative peak near 240 nm. Upon addition of compound in RNA up to DN = 4.0, the positive peak near 270 shifts downward while the negative peak near 240 nm shifted upward in (5′CAG/3′GAC)x6 RNA, which is in accord with the base stacking43 of binding compounds with RNA x20, (5′CAG/3′GAC)x6 and duplex (5′CAG/3′GUC)x6 RNA x20 and ∼3–5 °C (5′CAG/3′GAC)x6 RNA while no significant change was observed with (5′CAG/3′GUC)x6 RNA -3′ was used, and the proton assignment was done from our previous study23. On gradual addition of CP13 to RNA, the change in the shape of proton resonances, as well as the chemical shift, have been observed in RNA . RNA was lined entirely within the grid box, and each of the compounds was blindly docked individually using a Lamarckian Genetic Algorithm (LGA) with defined parameters. Although the compounds were free to bind with any of the bases of the RNA however, compounds preferred to bind with the C4A5G6 triad with lowest binding energy. The most potent conformer of CP2, CP6, and CP13 compounds bind with RNA with the binding energy of −6.76, −6.84 and −7.43 kcal/mole, respectively  for 12 hours each. The same sets of cells were then subjected to microscopic fluorescence analysis in independent experiments to examine the formation of aggregate  RNA which further corroborates the selectivity of these compounds for (5′CAG/GAC3′) RNA , e.g., diclofenac and ibuprofen and analysis of the effect of our screened drugs on proteasome or autophagy may provide the molecular mechanism of action of these compounds in alleviating cells from proteotoxicity of protein aggregates64.In this study, compounds CP2, CP6, and CP13 were tested for their potential in alleviating the cells from proteotoxicity of expanded polyglutamine aggregates of Huntington’s and spinocerebellar ataxias. Different experiments performed during the study, substantiate the effect of the compounds mentioned above in the removal of the cytotoxic proteinaceous inclusions from the cell. A different strategy for evaluating the effect of these drugs on protein aggregation may involve analyzing changes in the level of components of proteins quality control such as E3 ubiquitin ligases like CHIP, MGRN1, ITCH that were previously shown to be effective in modulating accumulation of protein65. PolyQ mediated aggregates have been studied using ThT assay previously66, therefore, to determine whether studied compounds can alleviate the poly Q mediated aggregation, we have performed the ThT assay for protein extracted from HD patient-derived cells (GM04281)  has been very widely used to probe the protein aggregation in various neurological disorders. ThT has characteristic emission profile with emission maxima at 485 when excited at 440 nm, which significantly increases upon binding with protein aggregates81) Fig. . In ThT Moreover, the RFU of cells treated with compounds decreases on increasing the concentration of compounds. All three compounds decrease the RFU of ThT however, the pattern of reducing the RFU was different for all three compounds which could be due to the different mode of binding of the compounds or the mode of action and need to be studied in future Fig. . Lastly,exp have been implicated in various trinucleotide repeat expansion diseases (TREDs) including HD and SCAs. Although the causative agents of these TREDs have been studied many years back, the alleviation of the pathogenesis is a key obstacle. Therefore, therapeutics that could reverse or slow the progression of the pathogenesis of the TREDs is a crucial medical need. The emergence of small molecule-based therapeutics for these TREDs have shown promising avenues. Herein, we have used the shape-based similarity approach for our previously reported compound Myricetin  to delve other potent compounds with high affinity and specificity for (CAG)exp RNA.RNA has always been studied vastly, but its role as a therapeutic target was eclipsed for many years. However, recently, a tidal wave of indispensable discoveries towards RNA as a therapeutic target has provided novel avenues in medicine. Trinucleotide repeat RNAs like (CAG)exp RNA. In addition to these preliminary studies, our lead compounds also exhibited the potential to alleviate the polyQ mediated pathogenesis in HD cellular models and cells derived from HD patients. Conclusively, our lead compounds could provide new hope as therapeutics for incurable diseases like HD and SCAs. However, further research involving the animal models and elucidation of the mechanism of action of these compounds is still needed.Moreover, the biophysical studies, including ITC, CD, UV melting, and NMR spectroscopy studies suggested that the compounds were a potent and selective binder of (CAG)2PO4, Na2HPO4, (HPLC Grade), D2O, DMSO, Sodium Chloride and other cell culture reagents such as MTT were obtained from Sigma-Aldrich Chemicals Ltd. . Tissue culture plates and consumables were purchased from Tarsons Pvt. Ltd. (India) and Thermo Fisher Scientific Inc., (USA). The synthetic RNA oligo (5′-rCrCrGrCrArGrCrGrG-3′) was procured from Integrated DNA technology (IDT) Inc. Different plasmids mentioned below were purchased from Addgene . Lipofectamine 3000, OptiMEM, and Antifade Reagent with DAPI were procured from Life Technologies (USA). Anti-GFP and anti-β-actin antibodies were procured from Santa Cruz Biotechnology, Inc., (USA).All Chemicals and solvents like NaHPlasmids: pEGFP-C1-Ataxin3Q28 (Addgene-22122), pEGFP-C1-Ataxin3Q84 (Addgene-22123)36. Briefly, synthetic DNA templates either amplified by PCR or cloned in a plasmid, were transcribed using T7 RNA polymerase, and transcribed products were purified by denaturing 15% PAGE. After UV shadowing, RNAs were extracted using 0.3 M NaCl by tumbling down for overnight at 4 °C.RNA sequences used for biophysical studies and gel retardation assay were prepared by runoff transcription method using synthetic DNA template as previously reported67. Briefly, virtual screening was done using OMEGA (version 2.5) and ROCS (version 3.2.0.4) software (OpenEye Scientific Software). Discovery studio 4.0 was used to energy minimized (MMFF94 force field). 1000 compounds based on TanimotoCombo scores were ranked as potential hits.Virtual screening was done as previously reported36. 10.0 μM of RNAs were serially diluted with the last well taken as a blank (No RNA). Each sample was tested in duplicates at RT with a 5′CAG/3′GUC pair served as control. The data was analyzed as per the following equation using SigmaPlot 12.0 software , which states for two receptor binding sites with two different affinities kd1 and kd:2Fluorescence titration experiments were performed with multi-mode plate reader as previously describedmax = maximum number of binding sites.Bd = equilibrium binding constant.K36. RNAs were prepared in 10 mM Na2HPO4 buffer . RNA was used in the cell, and the compounds were titrated using a syringe with an initial injection of 0.4 μL with 60 s initial delays. The sample was stirred with 750 rpm, and the reference power was set to 8 μcal/s during whole experiment. The two site binding model was used for data fitting to determine the dissociation constant using origin software version 7  provided with the instrument.MicroCal iTC200  was used to perform all the ITC studies as previously described36. Briefly, RNA (20 μM) was titrated with increasing concentration of compounds in 10 mM Na2HPO4 buffer . All the CD studies were performed in a quartz cuvette with a 0.2 cm path length. Spectra were recorded at 0.1 nm intervals from 200 nm to 300 nm with a 1 nm-slit width and averaged over three scans. Finally, data were normalized by subtracting the buffer CD spectra from RNA CD spectra as well as RNA-Drug complex CD spectra.Circular Dichroism (CD) experiment was performed on J-815 Spectropolarimeter  as reported previously36. using Perkin Elmer Lambda 35 spectrophotometer equipped with a Peltier temperature programmer (PTP 6 + 6) and water Peltier system PCB-1500. The change in absorbance at 260 nm at a rate of 1 °C/minute in the absence and presence of compounds at D/N ratio of 2.0 and D/N ratio of 3.0 was monitored upon heating of RNA to 25 °C to 95 °C. The change in thermal denaturation was calculated by normalizing the changes in absorbance at 260 nm vs. temperature using the SigmaPlot 12.0 software.Thermal denaturation studies were performed as previsouly published method5 µM of each RNA (AA and AU pair) was incubated with decreasing concentration of compounds (1000.0–0.0 µM) and kept for 30 minutes at room temperature. 3% agarose gel stained with ethidium bromide was used to assess the inhibition in movement of bands. Finally, the inhibition of movement in bands was analyzed using ImageQuant LAS 4000 .36. Briefly, the Template for AA × 6 (5′- GGA GAG GGU UUA AUC AGC AGC AGC AGC AGC AGU ACG AAA GUA CAG CAG CAG CAG CAG CAG AUU GGA UCC GCA AGG - 3′) and complementary sequence (5′- GGC CGG ATC CTA ATA CGA CTC ACT ATA GGG AGA GGG TTT AAT - 3′) were procured from Sigma-Aldrich Chemicals Ltd. . The reaction was performed in a master mix consisting 1X PCR buffer, 0.33 mM dNTPs, 4.25 mM MgCl2, 2 μM oligonucleotide, 2.5 units Taq DNA polymerase  and a dose of titration of ligands. The thermal cycling conditions were as follows: 95 °C for 5 min, followed by 25 cycles of 95 °C for 30 s, 50 °C for 30 s, 72 °C for 1 minute and finally held at 4 °C following completion. The amplified products were mixed with 6X DNA loading dye and resolved on 3% agarose gel stained with ethidium bromide. The gel image was analyzed using ImageQuant LAS 4000 .PCR stop assay was performed as previously reported by our group23. RNA samples were prepared in an appropriate buffer, and 3 - (Trimethylsilyl) propionic-2, 2, 3, 3-d 4 acid sodium salt (TSP) was used as a reference. H2O + D2O solvent at a 9:1 ratio was used for all titration studies and 64 K data points were recorded for 1D proton NMR spectra. Topspin (3.5 versions) was used to process, integrate, and analyze the data.Nuclear Magnetic Resonance (NMR) experiments for RNA duplex with a single CAG motif, 5′-rCrCrGrCrArGrCrGrG-3′ were performed on a high-resolution AVANCE III 400 or 700 MHz BioSpin International AG, Switzerland as reported previously36. The previously reported structure of CAG duplex RNA (PDB code:2MS547) was utilized as the starting model. The structure was refined further using CHARMM force field performed on Discovery studio3.5  for the necessary replacements, optimization of structure and ligands structures and the addition of residues. Autodock 4.048  was used for the docking study with RNA as a rigid body. Default values were used for all other paramters. RNA and ligand structures were converted to AD4 format files, and Gesteiger charges were assigned to the atoms. The grid was arranged so that it covers complete RNA structure so that ligand can explore the whole conformational space. The Lamarckian genetic algorithm was used for the search, and the results were analyzed based on binding energy.The molecular docking of RNA and compounds was described earlierThe following cell lines GM07492 and GM04281 were obtained from the NIGMS Human Genetic Cell Repository at the Coriell Institute for Medical Research. The Coriell Institute maintains the written consent forms and privacy of the donors of the samples, and the authors had no contact or interaction with the donors.All the Fibroblasts cells were procured from the Coriell Cell Repositories . An unaffected individual (GM07492 line) expresses normal repeat tract, r(CAG)17 while HD patient (GM04281), expresses expanded repeats, r(CAG)69. All the cell lines were grown in appropriate medium supplemented with 10% FBS and 1x antibiotic-antimycotic solution.36. Breifly, COS-7 cells were transiently transfected with the desired plasmid. The medium was replaced with fresh medium containing with either DMSO or compounds of interest, after 6–8 hour post-transfection. The cells were washed with PBS and fixed with 4% paraformaldehyde for 15–20 minutes and further permeabilized with 0.5% Triton-X 100 for 5 minutes for immunofluorescence assay. Afterward, 2–3 washing with PBS and mounting with DAPI was done and finally, fluorescence microscope was used to capture the images. Aggregates counting were done by using few of the same set of cells at lower magnification.The immunocytochemistry and aggregation counting assays were performed as previously reported68. For Cell viability assay, cells viability assay was performed in triplicates via MTT assay as described in a previous publication69.Briefly, COS-7 cells were transiently transfected with desired plasmids  and ataxin-3(Q84)). Each set of transfected cells was treated either with DMSO or compounds of interest for 12 hours each. Immunoblot analysis was done with same set of cellsThioflavin T assay fluorescence was performed for total protein extracted from HD patient-derived cells treated with compounds of interest. Cells were seeded in 24 well plates for, and media containing a compound of interest was added at 80% confluency of the cells. After 24 hours, the cells were lysed using RIPA lysis buffer and total protein were  isolated. At the day of the experiment, 2.5 mM Thioflavin-T (ThT) stock solution was prepared in phosphate buffer and filtered through 0.22 µm filter (Millipore). ThT dye  was applied to untreated (control) and treated sample. ThT alone, as well as the sample containing ThT dye, was excited at 440 nm and emission spectra were recorded at 470–700 nm. SigmaPlot 12.0 software  was used to plot the emission spectra of each sample.Supplementary Information


It remains formidable to predict what unreported RNA pairs can form new fusion transcripts. By systematic mapping of chromatin-associated RNAs and their respective genomic interaction loci, we obtained genome-wide RNA–DNA interaction maps from two noncancerous cell types. The gene pairs involved in RNA–DNA interactions in these normal cells exhibited strong overlap with those with cancer-derived fusion transcripts. These data suggest an RNA-poise model, where the spatial proximity of one gene’s transcripts and the other gene’s genomic sequence poises for the creation of fusion transcripts. We validated this model with 96 additional lung cancer samples. One of these additional samples exhibited fusion transcripts without a corresponding fusion gene, suggesting that genome recombination is not a required step of the RNA-poise model. Fusion transcripts are used as biomarkers in companion diagnoses. Although more than 15,000 fusion RNAs have been identified from diverse cancer types, few common features have been reported. Here, we compared 16,410 fusion transcripts detected in cancer  with genome-wide RNA–DNA interactions mapped in two normal, noncancerous cell types . Among the top 10 most significant RNA–DNA interactions in normal cells, 5 colocalized with the gene pairs that formed fusion RNAs in cancer. Furthermore, throughout the genome, the frequency of a gene pair to exhibit RNA–DNA interactions is positively correlated with the probability of this gene pair to present documented fusion transcripts in cancer. To test whether RNA–DNA interactions in normal cells are predictive of fusion RNAs, we analyzed these in a validation cohort of 96 lung cancer samples using RNA sequencing (RNA-seq). Thirty-seven of 42 fusion transcripts in the validation cohort were found to exhibit RNA–DNA interactions in normal cells. Finally, by combining RNA-seq, single-molecule RNA FISH, and DNA FISH, we detected a cancer sample with EML4-ALK fusion RNA without forming the EML4-ALK fusion gene. Collectively, these data suggest an RNA-poise model, where spatial proximity of RNA and DNA could poise for the creation of fusion transcripts. Fusion transcripts are associated with diverse cancer types and have been proposed as diagnostic biomarkers –3. Compa5Despite the large number of gene pairs in identified fusion transcripts, it remains formidable to predict what unreported pair of genes may form a new fusion transcript. Recent analyses could not identify any distinct feature of fusion RNA forming gene pairs . Here, wChromatin-associated RNAs (caRNAs) provide an additional layer of epigenomic information in parallel to DNA and histone modifications . The recSI Appendix, Fig. S1A). The proximal and distal interactions were defined as the intrachromosomal interactions where the RNA end and DNA end were mapped to within and beyond 200 kb, respectively. Following Sridhar et al. . Within each chromosome, the number of iMARGI read pairs negatively correlated with their genomic distances .We compared iMARGI with the MARGI technology previously described . iMARGI SI Appendix, Table S1). diMARGI generated many fewer valid interchromosomal (∼0.5 million) and distal  read pairs.First, we compared the distribution of the read pairs. iMARGI and pxMARGI produced similar amounts of valid interchromosomal (∼19 million) and distal (1–4 million) read pairs . diMARGI revealed severalfold fewer caRNAs in every RNA type , consistent with its many fewer usable read pairs.Second, we compared by the numbers of discovered caRNAs. Under the smallest possible threshold , iMARGI revealed a similar amount of caRNAs to that of pxMARGI, with mRNA, lincRNA, pseudogene RNA, and antisense RNA as the most abundant types of caRNAs . The correlation increased with the bin size . diMARGI data exhibited much weaker correlation to iMARGI data , likely also due to its very small amount of usable read pairs.Third, we compared by the “RNA attachment level”  on everySI Appendix, Fig. S4A). Investigating this gene pair, we found the reporting of FHIT-PTPRG fusion transcripts from kidney, liver, head and neck, lung, and prostate cancers . Fusion transcripts from this second-ranked gene pair were reported from liver and prostate cancers . The 2D distribution was not uniform, with more intrachromosomal than interchromosomal gene pairs . A total of 8,891 and 6,253 Futra pairs were intra- and interchromosomal, respectively. Chromosomes 1, 12, and 17 harbored the largest amounts of intrachromosomal gene pairs . Chromosomes 1, 11, 12, 17, and 19 contribute to the largest amounts of interchromosomal gene pairs . Higher density of gene pairs appeared on the diagonal line of the fusion map, suggesting enrichment of gene pairs within chromosomes or large chromosomal domains . These enriched chromosomal domains ranged from approximately 1/10th to 1/3rd of a chromosome in lengths, which are ∼10–20 times the typical sizes of topologically associated domains (TADs)  of Futra pairs were interchromosomal, whereas less than 15% of chromosomal conformation capture-derived read pairs were interchromosomal , 14. The,144 of FSI Appendix, Fig. S1D) and observed pronounced similarities . For example, a set of 34 Futra pairs was enriched in an ∼7-Mb region on chromosome 9 (chr9) , 1-Mb . Four fusion transcripts, KMT2C-AUTS2, KMT2C-CALN1, KMT2C-CLIP2, and KMT2C-GTF2IRD, were formed between the KMT2C mRNA near the 152-Mb location of chromosome 7  and four mRNAs that were approximately 80 Mb away  . Correspondingly, a total of 73 RNA–DNA iMARGI read pairs were mapped to KMT2C and the four fusion partners in HEK cells .We asked to what extent Futra pairs may coincide with genome-wide RNA–DNA interactions. We carried out a visualized comparison of the 2D distribution of Futra pairs with that of RNA–DNA interactions (C), 1-Mb A–C, and C), 1-Mb D resolutP value <2.2 −16, χ2 test). Among the 8,891 intrachromosomal Futra pairs, 7,427 (83.5%) overlapped with RNA–DNA interactions in either HEK or HFF cells . These data pointed to a common feature of cancer-derived Futra pairs, which is their colocalization with RNA–DNA interactions in normal cells.We quantified the overlaps between Futra pairs and iMARGI-identified RNA–DNA interactions. Among the 6,253 interchromosomal Futra pairs, 3,014 (48.2%) overlapped with RNA–DNA interactions in either HEK or HFF cells , whereas both assays detected fusion transcripts in a NSCLC cell line (H2228) . Taken together, these data suggest that, although cancer-derived Futra pairs colocalized with RNA–DNA interactions in normal cells, the fusion transcripts found in cancer are not present in the normal cells.A model that may explain the colocalization of RNA–DNA interactions and Futra pairs is that RNA–DNA interactions in the normal cells poise for creation of fusion transcripts. Recognizing that this model cannot be tested by perturbation due to the very small likelihood for a fusion transcript to occur in a cancer sample, we carried out two other tests. First, we tested whether the cancer-derived Futra pairs were detectable in normal cells. We reanalyzed the merged RNA-seq datasets of more than 75 million 2 3T cells  and ran 3T cells  on these3T cells , and the3T cells A. NeitheNext, we tested whether the RNA–DNA interactions in normal cells are predictive of fusion transcript formation in cancer. To this end, we analyzed a validation cohort comprising 96 new lung cancer samples from patients who were not part of the TCGA cohorts. We also analyzed a NSCLC cell line (H2228). RNA was extracted and targeted RNA-seq was carried out with Illumina’s TruSight RNA Pan-Cancer Panel. Of these 96 samples, 27 did not yield sufficient RNA for sequencing, whereas the other 69 samples produced a sequencing library and yielded on average 3.9 million uniquely aligned read pairs per sample A. STAR-FP value <2.2 −16, χ2 test). We asked whether only intrachromosomal Futra pairs colocalized with RNA–DNA interactions. Nineteen of the 42 (45%) detected Futra pairs were interchromosomal  colocalized with RNA–DNA interactions in the assayed normal cells, supporting the idea that RNA–DNA interactions in the already assayed normal cells are predictive of Futra pairs in cancer . The FISH probes were designed to hybridize to the consensus exons shared among all 28 variants of EML4-ALK fusion transcripts that have been identified to date  that were known to express EML4-ALK fusion transcripts  , consistent with the lack of such a fusion transcript in HEK293T cells .To identify other cancer samples that express EML4-ALK fusion transcripts, we reanalyzed our collection of 96 lung cancer samples with FuseFISH, a single-molecule fluorescence in situ hybridization (sm-FISH)–based method for the detection of fusion transcripts , 20. We  to date . Followi to date , 20, fusFig. S8) . In contIn our collection of 96 tumor samples, only 57 had remaining tissues for FuseFISH analysis. These 57 samples included 39 that yielded RNA-seq data and 18 that did not yield RNA-seq data B. The FuOur RNA FISH and DNA FISH analyses revealed a cancer sample that contained a fusion transcript without the corresponding fusion gene. Such an example, although not often seen in the literature, may not be a rare case . The lacFusion transcripts can be created by two processes. The better-recognized process is through transcription of a fusion gene that was created by genome rearrangement. The less-recognized process is by RNA splicing errors, where two separate transcripts were spliced together (transsplicing) . TransspThe RNA-poise model fills this theoretical gap. The preinstallation of gene 1’s transcripts on gene 2’s genomic sequence positions gene 2’s nascent transcripts spatially close to gene 1’s transcripts, allowing for the possibility of transsplicing. Furthermore, the majority of splicing events are cotranscriptional. The availability of transcripts of gene 1 during gene 2’s transcription allows for the opportunity to perform cotranscriptional transsplicing.Remote RNA–DNA interactions could be created by at least two means. First, the caRNA can target specific genomic sequences, which could be mediated by tethering molecules  were used throughout all data analyses.HEK293T RNA-seq datasets were downloaded from the NCBI BioProject database (accession nos. SRR2992206–SRR2992208 under project no. PRJNA305831) . The thrwww.tumorfusions.org)  . Tier 1 ons.org) , Futra pons.org)  with Bioons.org)  and Inteons.org) .Heatmaps of the count matrix were plotted using Bioconductor package ComplexHeatmap . Genomic6 cells were used for the construction of an iMARGI sequencing library. Cells were cross-linked in 1% formaldehyde at room temperature (RT) for 10 min with rotation. The cross-linking reaction was quenched with glycine at 0.2 M concentration and incubated at RT for 10 min. Cells were pelleted, washed using 16 in each tube. To prepare nuclei, cross-linked cells were incubated in 1 mL of cell lysis buffer (10 mM Tris2).Approximately 5 To prepare the RNA and DNA ends for linker ligation, nuclei were incubated in 200 To reverse cross-linking, nuclei were washed twice with 1https://github.com/Zhong-Lab-UCSD/iMARGI_methods). Briefly, they include three main steps. First, the read pairs were cleaned by in-house scripts. According to the library construction design, read pairs were filtered out if the 5′-most two bases of their DNA end (Read 2) were not CT. In addition, the first two bases of the RNA end (Read 1) were removed as they are random nucleotides. Then, the cleaned read pairs were mapped to the human genome (hg38), using bwa mem (version 0.7.17) with parameters “-SP5M” (https://github.com/mirnylab/pairtools) and in-house scripts were used to parse, deduplicate, and filter the mapped read pairs. The valid read pairs that were mapped to genomic locations within 200 kb of each other were defined as proximal interactions, which were excluded from our analysis. GenomicRanges (The detailed iMARGI data-processing methods can be found in our GitHub repository ( “-SP5M” . FinallyicRanges  and InteicRanges  were useHeatmaps of the count matrix were plotted using Bioconductor package ComplexHeatmap . GenomicAn iMARGI read pair was regarded as overlapping with a Futra pair when the RNA end was strand-specifically mapped to the gene body of one gene in the Futra pair and the DNA end was mapped to the gene body Oligonucleotide probes were designed to hybridize to exons 2–6 of EML4 RNA and exons 20–23 of ALK RNA. These exons were chosen because they were present in all of the observed variations of EML4-ALK fusion genes. These probes were 35–40 nt in length, with similar GC contents and melting temperatures.Oligos were modified on the 5′ end with a primary amino group and a spacer of 30 carbons to minimize steric hindrance of probe–RNA hybridization. These probes were conjugated with quantum dots through the amino group using EDC reaction . The proProbe hybridization in H2228 cells was carried out as previously described , 21. BriProbe hybridization in tissues was carried out as previously described . BrieflyCells or tissues were imaged in 1escribed . BrieflyRNA was extracted with Trizol from an H2228 cell line and lung cancer tissue samples of the approximate size 3 mmrameters . Fusion rameters , requiriSupplementary File


Streptomyces lividans is an attractive host for production of heterologous proteins and secondary metabolites of other Streptomyces species. To fully harness the industrial potential of S. lividans, understanding its metabolism and genetic regulatory elements is essential. This study aimed to determine its transcription unit (TU) architecture and elucidate its diverse regulatory elements, including promoters, ribosome binding sites, 5′-untranslated regions, and transcription terminators. Total 1,978 transcription start sites and 1,640 transcript 3′-end positions were identified, which were integrated to determine 1,300 TUs, consistent with transcriptomic profiles. The conserved promoter sequences were found as 5′-TANNNT and 5′-TGAC, representing the −10 and −35 elements, respectively. Analysis of transcript 3′-end positions revealed the presence of distinctive terminator sequences and the RNA stem structure responsible for the determination of the 3′-boundary of a transcript. Functionally related genes are likely to be regulated simultaneously by using similar promoters and being transcribed as a poly-cistronic TU. Poly-cistronic TUs were further processed or alternatively transcribed into multiple TUs to fine-regulate individual genes in response to environmental conditions. The TU information and regulatory elements identified will serve as invaluable resources for understanding the complex regulatory mechanisms of S. lividans and to elevate its industrial potential. Streptomyces, possesses a long linear chromosome with high GC content (approximately 70%) and is characterized by complex life cycle, accompanied by morphological and physiological changes  and computational tools revealed additional uncharacterized, biosynthetic gene clusters for novel secondary metabolites present in the genomes of Streptomyces, further elevating its industrial and clinical potential , is a prominent host for recombinant protein production, since S. lividans displays reduced extracellular proteolytic activity compared to other Streptomyces species  at 30°C. The composition of R5 − medium is as follow: 5.73 g TES (pH 7.2), 103 g sucrose, 10 g glucose, 5 g yeast extract, 10.12 g MgCl2⋅6H2O, 0.25 g K2SO4, 0.1 g casamino acids, 0.08 mg ZnCl2, 0.4 mg FeCl3⋅6H2O, 0.02 mg CuCl2⋅2H2O, 0.02 mg MnCl2⋅4H2O, 0.02 mg Na2B4O7⋅10H2O, and 0.02 mg (NH4)6Mo7O24⋅4H2O in 1L distilled water. Cell growth was determined with biological triplicates. For dRNA-Seq, Term-Seq, RNA-Seq and Ribo-Seq, cultures were prepared as biological duplicates and sampled at 9.5, 14, 16, and 20 h after inoculation for early, mid- and late-exponential, and stationary phases, respectively. For Ribo-Seq, cultures were treated with thiostrepton for 5 min before harvesting the cells.The mycelium of 2), and resuspended with 500 μL of lysis buffer . The cell suspension was frozen with liquid nitrogen, and the frozen suspension was physically lysed by grinding using mortar and pestle. The lysate was centrifuged at 4°C for 10 min at 16000 × g and the supernatant was saved and stored at −80°C until used for RNA extraction. For RNA extraction, 100 μL of the supernatant was used. The volume of the supernatant was adjusted to 350 μL with DEPC-treated water and mixed with equal volume of phenol:chloroform:isoamyl alcohol = 25:24:1 solution. The mixture was then centrifuged and RNA was extracted from the upper aqueous phase with ethanol precipitation. To remove any DNA contamination, the RNA samples were treated with DNase I . To remove ribosomal RNA (rRNA), Ribo-Zero rRNA Removal Kit Bacteria  was used following the manufacturer’s instructions. The rRNA-depleted RNAs were visualized with 2% agarose gel electrophoresis for quality control. RNA-Seq libraries were constructed using TruSeq Stranded mRNA Library Prep Kit , following the manufacturer’s instructions.After harvesting, the cells were washed with 500 μL of polysome buffer  was ligated to the purified RNA with T4 RNA ligase  in 1 × RNA ligase buffer and 0.1 mg/mL BSA by incubating at 37°C for 90 min. The adaptor-RNA ligate was then purified using Agencourt AMPure XP beads  according to the manufacturer’s instructions. The purified product was reverse-transcribed with SuperScript III Reverse Transcriptase (Invitrogen) and purified using Agencourt AMPure XP beads. The purified cDNA was divided in half and amplified and indexed using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) for Illumina sequencing. The amplification step was first monitored using a CFX96 Real-Time PCR Detection System  with SYBR Green I Nucleic Acid Gel Stain (Invitrogen). The remaining half of the cDNA was amplified and stopped 1 cycle before the signal becomes totally plateau. Finally, the amplified library was purified using Agencourt AMPure XP beads, and the concentration of the library was measured with Qubit 2.0 fluorometer (Invitrogen). The size distribution of the library was checked by gel electrophoresis on 2% agarose gel.Approximately 2.5 μg of DNase I treated RNA from the 4 sampling time points were mixed, and the rRNA in the RNA mixture was depleted using Ribo-Zero rRNA Removal Kit Bacteria (Epicentre). Approximately 700 ng of rRNA-depleted RNA was incubated in 1 × RNA 5′ polyphosphatase  reaction buffer and 1 U of SUPERase-In , the RNase inhibitor, with [TAP(+)] or without [TAP(−)] TAP at 372, and 33.5 μg/mL thiostrepton) and resuspended with lysis buffer . The cell suspension was frozen with liquid nitrogen and lysed by grinding using mortar and pestle. The cell lysate was centrifuged at 4°C for 10 min at 16,000 × g and the soluble supernatant was recovered. Ribosome-unprotected RNA was digested by incubating with RNase I (Invitrogen) at 37°C for 45 min. After RNase I digestion, RNase reaction was inactivated by treatment with SUPERase-In and monosomes were recovered using a Sephacryl S-400 column . Ribosome protected RNA fragments were recovered using phenol:chloroform:isoamyl alcohol = 25:24:1 solution and rRNA was depleted with Ribo-Zero rRNA Removal Kit Bacteria (Epicentre) according to the manufacturer’s instructions. After rRNA depletion, RNA was resolved on a 15% TBE-urea gel and 26–34 nt RNA fragments were size-selected. The size-selected RNAs were eluted in 300 mM sodium acetate pH 5.2, 1 mM EDTA and 0.25% SDS. The eluate was ethanol precipitated and libraries were constructed with NEB Next small RNA library prep set (New England Biolabs) according to the manufacturer’s instructions. The constructed libraries were divided in half and amplified and indexed using Phusion High-Fidelity DNA Polymerase for Illumina sequencing. The amplification step was first monitored on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories) with SYBR Green I Nucleic Acid Gel Stain (Invitrogen). The remaining half of the library was amplified and stopped 1 cycle before the signal becomes totally plateau. The amplified libraries were further size-selected on 2% agarose gel with MinElute Gel Extraction Kit .Before harvesting, cells were pre-treated for 5 min with thiostrepton to inhibit translation elongation. The harvested cells were washed with polysome buffer  and eluted with 9 μL DEPC-treated water. The RNA-adaptor ligates were then fragmented using fragmentation buffer  by incubating at 72°C for 90 s. After fragmentation, the products were purified with Agencourt AMPure XP beads and eluted with 8 μL DEPC-treated water. The fragmented RNA was reverse transcribed using 1 μL of 10 μM reverse transcription primer (5′-TCTACACTCTTTCCCTACACGACGCTCTTC-3′) with SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions. After reverse transcription, the cDNA was purified with Agencourt AMPure XP beads and eluted with 5 μL DEPC-treated water. The purified cDNA was subjected to another adaptor ligation cycle as above, with increased incubation time (8 h) and different amino-blocked adaptor sequence (5′-p-NNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3′). After adaptor ligation, the product was purified using Agencourt AMPure XP beads and indexed by PCR for 10 cycles with Phusion High-Fidelity DNA Polymerase using forward (5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTA CACGACGCTCT-3′) and reverse (5′-CAAGCAGAAGACG GCATACGAGATNNNNNN (6 nt index) GTGACTGGAGTT CAGAC-3′) primers.Term-Seq libraries were constructed as previously described with some modifications . 1.25 μgS. lividans TK24 genome (Accession number CP009124). For RNA-Seq and Term-Seq, reads were reversely mapped to reference.All libraries were sequenced using Illumina HiSeq 2500 platform with either 1 × 100 bp (RNA-Seq and dRNA-Seq) or 1 × 50 bp (Ribo-Seq and Term-Seq) read length. The reads were trimmed and mapped to the TSSs were identified as previously described . The 5′-in vivo address of 3′-end position of each RNA transcript, resulting from transcription termination or post-transcriptional processing including programed mRNA cleavage and RNA decay. Only the 3′-end position of Term-Seq reads located within intergenic regions including up to 10 bp downstream of the gene were considered as potential transcript 3′-end positions (TEPs). The positions were clustered together based on the distance from adjacent positions (<10 bp). Within each cluster, the read count of each position was assumed to follow normal distribution and read count enriched positions were deduced by calculating the modified z-score as previously described with some modifications (Each 3′-end position of Term-seq read indicates the ications .whereZ(x) is the modified z-score at positionx, r(x) is the read count of evaluated position x. μ(r(x))and σ(x) are the mean and standard deviation of read counts of other positions in the cluster except the evaluated position, respectively. N(x) is the length of the cluster containing position x and C(x) is the set of positions within the cluster containing position x.z-scores less than 3 were discarded. Among the remaining potential TEPs, the reproducible positions with the highest read count within the intersecting region of clusters from two biological replicates were selected as TEPs. For example, if genomic positions from 3 to 25 were clustered together for one replicate and genomic positions from 13 to 42 were clustered together for another replicate, the potential TEP with the highest read count within the genomic positions from 13 to 25 was selected as the TEP.The positions with read counts of less than 3 or modified P-value was less than 0.05, were regarded as the motif. After finding each sequence element, 21 nt sequences of −10 elements (5′-N8TANNNTN7) and 16 nt sequences of −35 elements (5′-N5TGACN7) were extracted and the sequence motifs were written by using Weblogo  motif was found by using the method for −35 element detection with up to 25 nt 5′-UTR sequences upstream of start codons of genes, whose 5′-UTR is longer than 10 nt. The −10 element sequence upstream of start codons of leaderless genes was found by the same method for RBS detection with 25 nt sequences upstream of start codons. For terminator sequence analysis, sequences from 41 bp upstream to 20 bp downstream of each TEP were collected and used for sequence alignment and motif discovery, and upstream 41 bp sequences were used for ΔG prediction. The sequence alignment and motif were created using Weblogo, and ΔG was predicted using RNAfold with temperature parameter of 30°C .Briefly, adjacent TSSs and TEPs were paired together for determination of the TUs. In case of S. lividans TK24 genome. Since Streptomyces display polymorphous growth and dynamic gene expression, we sampled at four different growth phases  to determine TSSs  approach, we experimentally identified TSSs of ine TSSs . In prinine TSSs . From thine TSSs . First,  RNA-Seq . The tra RNA-Seq . The RNA RNA-Seq .The genomic position of TSS leads to information on regulatory elements for transcription initiation. Thus, we analyzed the sequence elements affecting transcription initiation.Streptomyces genomes. The alternate occurrence of purines at template strand and non-template strand across the TSS is consistent with the beneficial effect of base stacking interaction for binding of the first and second nucleotides  can be utilized for TSS (+1 site), but in most cases (more than 85%), transcription starts from either A or G, which are purines . On the leotides .Streptomyces typically encodes a remarkable number of σ factors in the genome and 62 σ factors are present in the S. lividans’ genome  and especially, the promoter sequence of SCO4895 is identical to SLIV_13900  (Important genetic information is found in the 5′-untranslated region (5′-UTR), that is the sequence between the TSSs and the start codon. We thus identified the 5′-UTR for of each gene. 5′-UTR contains a key regulatory sequence for translation and, in addition, unique structures in the 5′-UTR can mediate co-transcriptional or post-transcriptional regulation under specific conditions . The lenon (21%) . The lacon (21%) .S. lividans  revealed the presence of a conserved sequence, 5′-RRGGAG, upstream of the start codon which acts as the RBS of lividans . Howeverlividans . The laclividans . The AUGlividans .The transcription levels of certain genes often do not coincide with their actual abundance as fully functional proteins and one major cause is the presence of translational regulation . RegulatWe first evaluated the start codon dependence of the TE. For both leaderless and leadered mRNAs, TE was higher for genes exploiting AUG start codon, compared to other start codons, supporting the hypothesis that selection of start codon affects translation . The expStreptomyces lividans is genetically close to the model actinomycete, S. coelicolor, however, the two organisms differ in their metabolism , whose 5′-UTR length is shorter than 10 nt  in S. coelicolor’ genome, 27 BGCs are well conserved in S. lividans   were down-regulated during late-exponential phase, while their homologs of S. lividans  were up-regulated. Similarly, SCO7221 of germicidin BGC was down-regulated during mid-exponential phase, while S. lividans’ homolog, SLIV_03190, was up-regulated. Meanwhile, the overall gene expression pattern of each BGC is synchronous, suggesting that the master regulator for each BGC is differentially expressed in the two organisms. Interestingly, two gene homologs in actinorhodin BGC were differentially regulated during stationary phase, while other genes in the actinorhodin BGC were up-regulated in both S. coelicolor and S. lividans. It is noteworthy that the two differentially regulated gene homologs, SLIV_12965 and SLIV_12970 of S. lividans and SCO5084 and SCO5083 of S. coelicolor, are related to the transport of actinorhodin. Inactivation of the actinorhodin transporter leads to decrease in actinorhodin production and the differential regulation of actinorhodin transporter during stationary phase in the two organisms may lead to difference in the actinorhodin production of for the two organisms (S. lividans was low in our culture condition (estimated by the color of media). Then we analyzed the underlying genetic regulatory element that leads to differential expression of the transporter genes. There are about 80 single nucleotide variances (SNVs) and a 12 nt deletion in the 21 kbp actinorhodin BGC. Interestingly, 15 SNVs are enriched in the 137 bp intergenic region between SLIV_12970 and SLIV_12975, where the promoter of SLIV_12970 is located   . For coerganisms . Indeed,V_12970) . Among tcis-regulatory (C) TEP. A total of 136 TEPs were assigned to this category. It is noteworthy that the minimum length between a primary TSS and cis-regulatory TEP was set as 60 nt for proper transcription termination. Again, the diversity of genomic positions of TEPs and the number of TEPs in each category suggest the presence of complex regulatory mechanisms for gene expression.Though the precise functional role of 3′-termini of bacterial transcripts remains poorly understood compared to the 5′-termini, transcript 3′-termini affects gene expression in various manner . To map S. lividans (Streptomyces (>70%), the frequency of U is higher than that observed in randomly selected intergenic positions. This supports the hypothesis that U-rich TEPs are the product of Rho-independent transcription termination .In the sequence alignment of all TEPs, GC-rich region was found upstream of TEPs and may form a stable RNA stem structure by the strong interaction between G and C. The RNA stem structure formation is critical for determination of the 3′-end of a transcript for both Rho-dependent and Rho-independent transcription termination . For RhoStreptomyces genomes may result in low ΔG values for randomly selected positions, and thus we analyzed the RNA structure itself, rather than the ΔG value of RNA that indirectly represents RNA structure. RNA structure was predicted for 101 nucleotides upstream of TEPs or randomly selected positions and the frequency of base interactions between the two positions was calculated. The base interactions were enriched around 20 nt upstream of TEPs for both U-rich and U-lacking TEPs . COG analysis showed that the genes located in the same poly-cistronic TU were functionally related to each other with great significance compared to randomly chosen gene sets   . TogetheS. lividans genome encodes overlapped TUs sharing one or more genes within the TUs, suggesting the presence of complex regulation coordinated by the alternative use of TSSs or TEPs  is composed of 2 TUs  and TAP(−) libraries) (ectC and ectD was not determined since the 5′-end present in the intergenic region between ectB and ectC was not determined as a TSS). And the corresponding RNA-Seq profile changes across the intergenic region, resulting in biased expression of ectC and ectD from ectA and ectB. The inconsistent expression across the ectoine biosynthetic TU cluster is highly likely due to different stability between the transcript bearing ectA and ectB and the transcript bearing ectC and ectD. Taken together, such complex TU architecture defined by a set of TSS and TEP serves as an efficient strategy to fine-tune the gene expression and balance the stoichiometry of individual gene in response to diverse environmental conditions.A total of 732 TU clusters were determined where most genes were transcribed independent of other genes and the number of genes comprising a TU cluster was generally proportional to the number of TUs comprising the same TU cluster . The incof 2 TUs . One TEPbraries) . The preStreptomyces have complex life cycle and undergo drastic morphological and physiological changes with the transition from primary to secondary metabolism  under accession number of B-KC designed the study. YL, YJ, NL, SH, and WK performed the experiments. YL, SC, and B-KC performed the data analysis. YL, SC, BP, and B-KC wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Great Amazon Reef (GARS) is an extensive mesophotic reef ecosystem between Brazil and the Caribbean. Despite being considered as one of the most important mesophotic reef ecosystems of the South Atlantic, recent criticism on the existence of a living reef in the Amazon River mouth was raised by some scientists and politicians. The region is coveted for large-scale projects for oil and gas exploration. Here, we add to the increasing knowledge about the GARS by exploring evolutionary aspects of the reef using primary and secondary information on radiocarbon dating from carbonate samples. The results obtained demonstrate that the reef is alive and growing, with living organisms inhabiting the GARS in its totality. Additional studies on net reef growth, habitat diversity, and associated biodiversity are urgently needed to help reconcile economic activities and biodiversity conservation. Biogenic reefs  are mostly composed by crustose calcareous algae, with sparse areas covered by scleractinian corals, particularly Madracis decactis4.The GARS is currently considered as one of the most important mesophotic reef ecosystems of the South Atlantic7, as well as by industrial sectors and politicians in favour of oil exploration in the region. The main argument is that there are no large biogenic structures built by living corals and/or other reef-building organisms , particularly in the northern portion of the GARS, due to larger influx of sediments from the Amazon River across the continental shelf7, which may have led to a relict reef environment, developed during MIS28. Because of such scepticism, a more detailed description of the GARS was provided by4. These authors showed that the GARS is composed of large and extensive biogenic structures build mainly by living calcareous algae  , which are surrounded mostly by siliciclastic sediments15. Brazilian biogenic reef ecosystems are recognised for being well adapted to high turbidity levels due to terrestrial input (via large rivers discharge) and sediment resuspension16.The predominance of terrigenous sediments, particularly in the inner and mid-shelf portions of the Amazonian shelf, is not a limiting factor for the existence of biogenic reefs, as there is enough light available for photosynthetic organisms across most of the Amazon shelf, even in the north sector4. The area is substantially influenced by the Amazon River, with a mean annual water discharge on the order of 6.0 1012 m3 and annual suspended-sediment load on the order of 1.2 109 tons17, affecting all physical and chemical aspects of the water column  over the shelf including the GARS. Most of the sediment travels north in a plume driven by the northwest-flowing North Brazil and Guiana Currents, which are forced by local wind patterns18.The GARS is located mostly at the middle, and outer shelf of the Foz do Amazonas and Pará-Maranhão marginal sedimentary basins within a depth range of 70 to 220 m19. There are four distinct zones based on light regimes reaching the bottom, three of them with constant light regimes  and one zone with seasonal changes in benthic light regimes (northern mid-to-outer shelf)10.The Amazon River plume has a very dynamic character and even though it is generally driven to the northwest, seasonal variations of winds and currents on the shelf lead to some southeastward expansion of the plume, as a result of displacements caused by the Inter-Tropical Convergence Zone (ITCZ), with substantial rainfall and NE Trade Winds between January and June. In contrast, between July and December rainfall is reduced, and SE Trade Winds prevail20. Besides the oceanic currents and waves, tidal water level variation and tidal currents are also substantial in the area, with tidal amplification on the shelf resulting in macrotidal conditions at the coast21.Waves reach the region mainly from the east and northeast, because of the NE-SE trade winds, with dominant offshore wave heights between 1 and 3 m. The most energetic waves approach the region between December and March, with strong NE Trade Winds offshoreIn this paper, we aim to summarise published and unpublished radiocarbon dating from carbonate samples from the GARS in order to: 1) evaluate its modern or relict nature and 2) contribute to the understanding of its evolution by proposing a theoretical model from the end of Marine Isotope Stage 2 until modern ages.Table i.22, with three exceptions, the first corresponding to an age of ca. 41,800 cal BP, and two others, with ages corresponding to the Younger Dryas, as delimited by23. The oldest ages correspond to the dating of oolites, published by8;Contains samples exclusively from the northern sector, at water depths between 104 and 150 meters, encompassing samples with ages from the MIS2, as defined byii.Encompasses samples from the North and Central sectors, and presents a period from ca. 7,100 cal BP (Mid-Holocene), to historical ages (non-modern radiocarbon ages). Water depth from these samples varies from 50 to 95 meters, and;iii.24 and contains samples from the three sectors. The water depth range of these samples varies from 23 to 100 meters; the shallowest samples are located in the Southern sector.Corresponds to samples with modern radiocarbon ages, as defined byRadiocarbon ages can be divided into three main groups Fig. :i.Contai25, the area of the potential occurrence of mesophotic reefs during the Last Glacial Maximum (LGM) was rather narrow and restricted to the continental slope. As sea level rose quickly from LGM towards Mid-Holocene, there was a period of restriction of areas within the mesophotic depth range, before the drowning of the shelf.Cross-shelf profiles of sediment samples obtained between 2017 and 2019 show that the morphology and the sedimentary characteristics from the central and southern sectors of the GARS support the ages compiled here Fig. . Conside26.The bathymetric profile from the central sector shows a substantially wider shelf, within a deeper outer shelf Fig. , favouri27 confirms the intertidal and shallow subtidal formation of most of the marine oozes but emphasizes the complexity of the biogeochemical processes involved in their formation. When comparing the position of the oolites dated by8 with the sea-level change curve iii.4.Finally, modern ages are found in samples from the three sectors, indicating the spread of the reef complex, from Northwest to Southeast. Also, reef constituents are found in areas as shallow as 20 meters (in the southern sector), and as deep as 220 metersConsidering these ages and the sea-level curve shown in Fig. 234. Also, it was clear from these previous studies, that rhodoliths have a fraction of living carbonate cryptofauna and/or epifauna, including Foraminifera (Homotrema rubrum), Polychaeta , Bryozoa, and Mollusca (Vermetidae) phyla33.Our radiocarbon dating study is coherent with previous studies performed on rhodoliths from the Pacific and the Atlantic oceans where metagenomics demonstrated that the majority of living material corresponds to microbes (bacteria) capable of growing in a variety of extreme environmental conditions for carbonate precipitation4. Reef growth and high structural complexity are presently concentrated in the central and southern sectors, which are shallower and with higher light incidence over the bottom wealth. Net reef growth estimates  are scarce even for shallow reefs. Only recently, this question started to be addressed for mesophotic reefs36. Similar studies are urgently needed for the GARS, particularly considering its ecological importance and the threats posed by large scale oil and gas exploration in the region4.The GARS is considered an ecotone of biodiversity between Brazil and the southern Caribbean, possibly acting as an ecological corridor between the South and North Atlantic. It comprises a high diversity of habitats and large areas dominated by healthy reef-building organisms The analysis of previously published and new radiocarbon data from the GARS allowed us to recognize a Northwest-Southeast growing trend of the reef complex. Older ages correspond to samples from the northern sector, in areas located below the present plume of the Amazon River. These ages refer to the carbonates that sustained the reef during MIS2 and to the beginning of the last deglacial. After a gap, occurred between ca. 12,100 and 7,100 cal BP, the reef extended to the Central and Southern sector of the area. This gap corresponds to the time interval of acceleration of sea-level rise and plume development, after the Younger Dryas (Melt Water Pulse 1B).Modern radiocarbon ages, obtained in rhodoliths and sponges, are present in the three sectors, indicating that differently than suggested, living organisms inhabit the GARS in its totality.3. Besides our new radiocarbon data, we gathered dating information from other sources37 in order to evaluate the modern or relict character of the reef complex. When available, all of the conventional radiocarbon datings were recalibrated using software Calib 7.138, using the Marine13 calibration curve39 and a global reservoir effect. Ages published in37 are presented “as is” , since we did not have access to the original data. We also dated ten other samples  as stated by22.In order to compare our data with sea-level changes, and due to a lack of a reliable Late Quaternary sea-level curve for northern Brazil, we used the global curve proposed byThe location of the samples is shown in Table