Psychiatric diseases have a strong heritable component known to not be restricted to DNA sequence-based genetic inheritance alone but to also involve epigenetic factors in germ cells. Initial evidence suggested that sperm RNA is causally linked to the transmission of symptoms induced by traumatic experiences. Here, we show that alterations in long RNA in sperm contribute to the inheritance of specific trauma symptoms. Injection of long RNA fraction from sperm of males exposed to postnatal trauma recapitulates the effects on food intake, glucose response to insulin and risk-taking in adulthood whereas the small RNA fraction alters body weight and behavioural despair. Alterations in long RNA are maintained after fertilization, suggesting a direct link between sperm and embryo RNA. Adverse experiences can have long-lasting transgenerational effects on mental and physical health, and often increase disease risk , 2. TrauC57BL/6 J mice were housed in a temperature-controlled and humidity-controlled facility under a reverse light–dark cycle, and food and water were provided ad libitum. Experimental procedures were performed during the animals’ active cycle. All experiments were approved by the cantonal veterinary office, Zurich (license 55/12 then 57/15).C57BL/6 J primiparous females and males were mated at 2.5–3 months of age. Randomly selected dams and litters were subjected to daily 3 h proximal unpredictable separation combined with unpredictable maternal stress (MSUS) from postnatal day 1 to 14 as described previously  or left Males used for sperm collection did not undergo any behavioural or metabolic testing and were approximately 3 months old at the time of sampling. For sperm RNA sequencing, mature sperm cells were collected from cauda epididymis and purified by counterflow centrifugal elutriation using a Beckman JE-5.0 elutriation rotor in a Sanderson chamber and a Beckman Avanti J-26 XPI elutriation centrifuge as described previously .C57Bl/6 J primiparous females underwent superovulation consisting of intraperitoneal injection of 5 IU pregnant mare serum gonadotropin followed by 5 IU human chorionic gonadotrophin 48 h later. Females were then left to mate with either an age-matched MSUS or control male. After plug detection, zygotes were scraped out of uterine horn and collected directly into Trizol LS solution.Total RNA was prepared from adult mouse sperm using a standard Trizol protocol. Total RNA was prepared from zygotes and four-cell embryos using the Trizol LS protocol. The quantity and quality of RNA were determined by Agilent 2100 Bioanalyser (Agilent Technologies) and Qubit fluorometer (Life Technologies).500 ng sperm RNA pooled from 4 control or 4 MSUS males each were subjected to Agencourt AMPure XP (Beckman Coulter) following the manufacturer’s instructions. Total RNA contained in 50 μl water was mixed with 125 μl AMPure XP bead solution, incubated for 5 min at room temperature then placed on a magnetic stand to allow magnetic beads to bind long RNA. When the solution appeared clear, the supernatant containing small RNA was aspirated and saved for later processing. Beads with bound long RNA were washed twice with 200 μl of 70% EtOH. Long RNA were eluted by addition of 100 μl elution buffer. Samples were placed again on the magnetic stand to capture magnetic beads while aspirating the supernatant containing long RNA and transferring it to a new tube for subsequent processing. Small and long RNA fractions were subjected to EtOH precipitation for purification. Efficiency of size separation was determined using Agilent 2100 Bioanalyzer  after superovulation , and mating with C57BL/6J males. 1–2 pl of 0.5 ng/μl solution of small or long RNA isolated and pooled from sperm from of four adult MSUS or control males dissolved in 0.5 mM Tris–HCl, pH 8.0, 5 μM EDTA were microinjected into the male pronucleus of fertilized oocytes using a standard microscope and DNA microinjection method . InjectiThe experimenter was double-blinded to treatment, meaning the group assignment was coded twice prior to experiments, once by the experimenter preparing RNA samples and once by the experimenter performing RNA injections. Behaviours were videotaped and scored both manually and automatically by tracking software (Viewpoint). All behavioural tests were conducted in adult age-matched male animals.The elevated plus maze consisted of a platform with two open arms  and two closed arms   elevated 60 cm above the floor. Mice were placed in the center of the platform facing a closed arm, and tracked for 5 min. The latency to enter an open arm was manually scored and total distance moved was automatically recorded by a videotracking system.Each mouse was placed in the lit compartment  of a box (40 × 42 × 26 cm) split into two unequal compartments ). Mice could freely move from the lit to the dark compartment during a 10-min period. Time spent in each compartment and latency to enter the dark compartment were measured manually.Mice were placed in a beaker of cold water (18 ± 1 °C) for 6 min. Floating duration was scored manually.Experimenters were double-blinded to treatment, meaning both the experimenter conducting RNA injections and the experimenter conducting phenotyping on resulting offspring was not aware of groups assignment. All metabolic tests were conducted in adult age-matched males.The amount of consumed food was measured for each cage in 4 months old animals every 24 h. Caloric intake was calculated as the mean amount of food intake over 72 h in relation to mean body weight .Mice were fasted for 5 h. For GTT, glucose was measured in blood at baseline and 0, 15, 30 and 90 min after intraperitoneal injection of 2 mg per g body weight of glucose in sterile 0.45% (wt/vol) saline (injection started at 2 pm). For ITT, glucose was measured in blood at baseline, and 0, 15, 30, 90 and 120 min after intraperitoneal injection of 1 mU per g body weight of insulin (NovoRapid Novo Nordisk A/S) in sterile 0.9% saline (injection started at 2 pm). If blood glucose decreased below 1.7 mM/ml, it was rescued by intraperitoneal injection of 2 mg/g of glucose. For both GTT and ITT, glucose level was determined in fresh tail blood using an Accu-Chek Aviva device (Roche).Sequencing was done using an Illumina Genome Analyzer HiSeq 2500 (Illumina) in Rapid run mode for 51 cycles plus 7 cycles to read the indices in two separate runs. Run 1 consisted of libraries representing biological replicates of control sperm RNA each pooled from five males and libraries representing biological replicates of sperm RNA from males exposed to MSUS, each pooled from five males. Run 2 consisted of technical replicates of run 1 plus one additional sample representing sperm RNA from five males exposed to MSUS. RNA libraries for first sequencing run were prepared with the NEBNext Ultra Directional RNA Library Prep Kit  following the manufacturer’s recommendations. Libraries for the second run were prepared using the TruSeq Stranded Total RNA kit according to the manufacturer’s instructions with indices diluted at 1:3. 220 ng of total sperm RNA was subjected to removal of rRNA using Ribozero (first run) and Ribozero gold kit (second run). Efficiency of Ribozero treatment was validated by Agilent 2100 Bioanalyzer. An average of 6.4 ng of zygotic RNA and 3.7 ng of four-cell embryo RNA per sample were not subjected to ribosomal depletion but immediately used for first strand synthesis. After demultiplexing and adaptor removal, an average of 60,948,122 pass filter reads was obtained in each sperm library and 43,346,362.5 in the zygotes libraries.RNAseq reads were trimmed of adapter sequences on the 3ʹ end using cutadapt v1.14 with a 5% error rare and using adapter sequences of the Truseq universal adapter, the Truseq indexed adapters (with sample-specific barcodes) and their reverse complement. Reads with a length of less than 15 bp were discarded. Samples were then directly quantified using Salmon v0.9.1  on an int tests were used to assess statistical significance for behavioural, body weight, caloric intake and GTT measurements. Chi-square test for two binominial populations with Yates continuity correction was used to analyse the proportion of animals rescued on the ITT before 90 min post-injection. When data did not match the requirements for parametric statistical tests , Mann–Whitney U-test was applied. If variance was not homogenous between groups, adjusted p value, t value and degree of freedom (Welch correction) were determined in parametric tests. Data were screened for outliers using prism’s ROUT test  and identified animals were excluded from analysis. All statistics of behavioural and metabolic tests were computed with Prism and SPSS. All reported replicates were biological replicates, or pooled samples from biological replicates in the case of sequencing samples and RNA injection samples. Significance was set at p < 0.05 for all tests. Boxplot whiskers represents the Tukey method.Samples size was estimated based on previous work on the MSUS model , 6–10. Twww.ebi.ac.uk/arrayexpress) under accession number E-MTAB-5834 (sperm), E-MTAB-6589 (zygotes) and E-MTAB-6587 (four-cell embryos). All relevant data are available from the authors. Previously published datasets used for comparisons in this study are available under Gene Expression Omnibus  [Small RNAseq data have been deposited in the ArrayExpress database at EMBL-EBI (SE50132) , 27.Code used to generate results is available from the authors upon request.First, using the MSUS paradigm, we produced a cohort of mice exposed to traumatic stress in early postnatal life and confirmed their behavioural phenotype in adulthood described previously , 10 Sup with incWe then harvested sperm from adult control and MSUS males, extracted total RNA and fractionated the RNA into small (<200 nucleotides (nt)) and long (>200 nt) RNA using Ampure beads. Effective separation was confirmed by Bioanalyzer analysis  Fig. . GO termBased on the assumption that sperm is transcriptionally silent , we inveBesides providing insight into the potential origin of altered sperm RNA, we examined the fate of these RNA after fertilization. We assessed whether RNA differentially expressed in sperm can be detected in one-cell zygotes after mating of control females with MSUS males. Next-generation sequencing of RNA pooled from 7 to 10 zygotes revealed many reads mapping to protein-coding genes and long non-coding genomic regions  in four-cell embryos after injection of fragmented long RNA from MSUS sperm showed no changes compared to controls  is necessary  and suggA previous study in a model of paternal low protein diet suggested regulation of MERVL elements based on data showing downregulation of MERVL targets in the offspring at embryonic stage . Our datLong RNA affected by MSUS in sperm is expressed in round spermatids, suggesting that their alteration can occur earlier during spermatogenesis and persist until mature sperm. Further, some small RNAs are known to be transferred to maturing sperm from exosome-like vesicles called epididymosomes during epididymal transit . Such trSupplementary MaterialSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4


To assess the extent to which adolescents aged 10–14 have communicated about sexual relationships, pregnancy, and contraception and how agency in the form of voice and decision-making along with an enabling socioecological environment are associated with sexual and reproductive health (SRH) communication.Using data from the Global Early Adolescent Study, we included 1,367, 697, and 1,424 adolescents in Kinshasa, Cuenca, and Shanghai, respectively. Patterns of SRH communication and agency levels were described by site and sex. Multivariable logistic regressions assessed odds of SRH communication first in relation to socioecological characteristics and second with levels of agency, after adjustment for social environmental factors. Interaction terms tested sex differences in associations.Experiences of SRH communication ranged from one in ten in Kinshasa to about half in Cuenca. Pregnancy was the most discussed SRH topic. Socioecological factors consistently related to SRH communication included older age and pubertal onset, while others varied by context. In multivariable analyses, voice was linked to all forms of SRH communication in Kinshasa and Cuenca with adjusted odds ratios ranging from 1.6 to 2.2, but not in Shanghai. In Cuenca, decision-making was associated with a 50% and 60% increase in odds of communication about pregnancy and contraception, respectively. In Kinshasa, a stronger association between voice and pregnancy discussions was observed for girls than boys.Developmental characteristics and voice were linked to communication about SRH among young adolescents across two contexts. Results suggest agency may play a role in shaping antecedents, like communication, to sexual behaviors. This study provides novel evidence about the links between empowerment and sexual and reproductive health (SRH) skills in early adolescence by documenting experiences of SRH communication and their associations with agency and developmental factors.In recent years, investment in the sexual and reproductive health (SRH)  of adoleSRH communication is a skill essential to young adolescents' accumulation of knowledge and arrival at partnered sex prepared, aware of risk and both HIV and pregnancy prevention, and able to negotiate their needs, boundaries, and desires. Existing research has linked young people's formal  and interpersonal  SRH communication to positive behaviors and outcomes, including service utilization , condom One aspect that has not been extensively explored is the degree to which SRH communication relies on young adolescents' empowerment. A body of work has examined relationships between empowerment levels and SRH outcomes among adult women and adolescent girls aged 15 and older. However, few of these studies conceptualize empowerment as a multidimensional construct that lies at the interplay between attributes and external resources that interacts to inform the extent to which individuals are able to achieve their SRH goals. Instead, the vast majority have relied on proxy indicators of agency, ranging from household decision-making to political participation to sexual self-efficacy . A recenTo our knowledge, extant literature examining associations of empowerment-related factors with SRH among adolescents is scarce. Existing analyses on this topic primarily employ measures of perceived behavioral control, reflecting adolescents' anticipated self-efficacy to achieve SRH outcomes like communication ,10. For One of the factors limiting studies of how young people are empowered to voice their needs and to make decisions surrounding SRH is the lack of a cross-cultural indicator assessing agency at this developmental stage. To address this gap and empirically assess the contribution of agency in healthy transitions from early to older adolescence, the Global Early Adolescent Study (GEAS) has developed a multidimensional measure of agency including dimensions of mobility, voice, and decision-making power that are salient to young people's lives across diverse cultural settings. The psychometric properties of the instrument that have been tested among nearly 2,000 adolescents in 15 cities are presented elsewhere . BuildinRecognizing that the exercise of agency is shaped by relational, community, and other environmental factors, indicators are often understood in conjunction with the socioecological environment . Indeed,To address these gaps in knowledge, this analysis employs data from the GEAS, a global study on norms about gender and their relationship to health and well-being beginning in early adolescence. Using baseline data collected in three sites, the objectives of the present study are to (1) describe patterns of SRH communication across sites and by sex, (2) examine associations between adolescents' socioecological environments and SRH communication, and (3) assess whether agency in the form of either voice or decision-making power relates to young people's history of communication about SRH, adjusting for the social environment, and whether this association is consistent by sex and across contexts.This comparative study employs data from three urban low-resource settings in Kinshasa, Democratic Republic of the Congo (DRC); Cuenca, Ecuador; and Shanghai, China. The contexts of these study sites vary considerably. In Kinshasa, a city of over 11 million where adolescents comprise a quarter of the population, young people often grow up in challenging environments ,20. NearAdolescents aged 10–14 were surveyed in disadvantaged urban areas of three cities , as part of the GEAS between June 2017 and March 2018. In Kinshasa, both in-school and out-of-school adolescents were included in the original study due to interest among stakeholders in studying these issues among out-of-school adolescents. Probability and multistaged sampling used to select in-school and out-of-school participants, respectively. Participants in Cuenca were recruited using probability sampling from schools, stratified by age and sex. In Shanghai, all eligible students in grades 6–8 were recruited from three purposively selected public schools.After obtaining parental consent and adolescent assent, adolescents completed the ninety-minute survey using tablets, by face-to-face interview  or computer-assisted self-interview (Cuenca and Shanghai). Research protocols were approved by each site's institutional ethical review committee and approved or deemed exempt for secondary data analysis by the Johns Hopkins School of Public Health institutional review board.The initial samples included 2,842 adolescents in Kinshasa, 704 in Cuenca, and 1,760 in Shanghai. In Kinshasa, we considered only adolescents in the control arm of a broader quasi-experimental study for inclusion , to keep consistency with the other sites and to avoid any introduction of bias due to self-selection into the intervention. We then excluded individuals missing all three SRH communication outcomes  or missing more than 30% of items used to construct the agency subscales . Applying these exclusion criteria, 1.0% of cases were dropped in Kinshasa, 1.0% in Cuenca, and 19.1% in Shanghai. Given the large share of cases excluded in Shanghai, we assessed and noted differences between the included and excluded subsamples by age, sex distribution, educational attainment, caregiver monitoring, and perceived neighborhood safety . After eTwo of the three GEAS cross-cultural domains of agency were considered for this analysis: voice (seven items measuring the extent to which young people can express their opinions and be heard) and decision-making (four items measuring adolescents' ability to make choices autonomously in their daily lives). The items, response options, and internal reliability for each scale are presented in Covariates were self-reported at various levels of the ecological environment. Individual sociodemographic factors included adolescents' age, binary indicators of sex, pubertal onset , and educational attainment (behind in school or out of school vs. at or above expected grade level for age). Family characteristics included binary variables for parental structure (living with both parents vs. with one parent or other relatives) as well as caregiver closeness, monitoring, and migration. Peer and neighborhood-level covariates included a binary variable indicating time typically spent with close friends (no close friends or no time spent with friends weekly vs. saw friends once a week or more), as well as binary indicators reflecting social cohesion (based on an aggregate measure of four items measuring trust and solidarity among neighbors), and whether or not participants feel safe in their neighborhoods.Three individual items were used to assess whether adolescents had ever discussed three sexual and reproductive health topics with anyone: sexual relationships, pregnancy and how it occurs, or contraception.We first conducted exploratory analysis to evaluate patterns of missingness across all items comprising the two agency subscales and excluded observations that met the exclusion criteria  and the three SRH communication outcomes as well as those between agency (voice and decision-making) and SRH communication. Multivariable logistic regressions assessed the independent effect of each ecological factor on SRH communication and subsequently evaluated the effect of agency levels on SRH communication, adjusting for all ecological covariates. Collinearity among covariates was assessed by the variance inflation factor value and no multicollinearity was noted. All analyses were stratified by site to assess similarities and differences in these associations by context. Interactions between agency and sex were also tested in the latter multivariable models in order to assess sex differences in the relationships between agency and SRH communication. kNN imputation was conducted using RStudio ; all other analyses were conducted using Stata/SE 15.1 .SRH communication patterns were examined overall by site and by sex, while agency and ecological factors were described by site, sex, and SRH communication outcomes using chi-squared, Fisher exact, and Student p = .009) and contraception . In Cuenca, more girls than boys had discussed sexual relationships  and pregnancy . No differences by sex were detected in Shanghai.SRH communication patterns varied by site and sex . Adolescp < .001) and comparable by sex in the other two sites. Decision-making mean scores were higher for girls than boys in Shanghai , while no sex differences were observed in Kinshasa and Cuenca.Levels of agency also differed by sex and site . Mean scDistributions of ecological factors by the three SRH communication outcomes are outlined in Additional factors related to SRH communication were heterogeneous across sites in bivariate and multivariable analyses . In Kinsp = .017), with a significantly stronger association among girls than boys . Fewer associations were observed between decision-making capacity and SRH communication. Adolescents with high decision-making scores were more likely to have talked about pregnancy  and contraception  in Cuenca. No significant differences in the associations of SRH communication and decision-making were detected by sex.Bivariate logistic regressions assessing odds of SRH communication by levels of voice and decision-making revealed links between both voice and decision-making and SRH communication in Kinshasa and Cuenca . After aThis analysis found SRH communication among young adolescents was relatively uncommon, associated with developmental factors across sites, and linked in two contexts to adolescents' ability to voice their needs and opinions. Decision-making was related to history of pregnancy and contraception discussions among adolescents in Cuenca. Particularly low levels of SRH communication were observed in Kinshasa, within the context of high fertility  and a loIn Kinshasa and Cuenca, more girls than boys had discussed most SRH topics, a finding that reflects the social and biological consequences of sexual activity that disproportionately impact girls ,31, whicOur analysis also examined the role of the socioecological environment in shaping SRH communication, in recognition its actualization can be promoted or constrained by one's context. Congruent with prior research, increased age  and pubeThis study found additional patterns of individual, family, peer, and neighborhood factors linked with SRH communication that were heterogeneous across sites. Factors predominantly on the individual level in Kinshasa, family level in Cuenca, and family, peer, and neighborhood levels in Shanghai were influential upon SRH communication. These findings indicate the varied cultural, social, and structural factors that impact SRH communication in each setting and indicate differential implications for interventions to create an enabling environment.These varying results across sites also underscore the varied contexts in which such discussions can take place, within family or school settings, or in the neighborhood with peers. Our findings additionally call attention to the agents who may transmit SRH information of varied quality to adolescents, information that ranges in accuracy and in its framing of sexuality and reproductive health. Additional research is needed to more extensively explore the nature of SRH communication interactions in early adolescence and how this communication contributes to SRH knowledge.Beyond the role of ecological factors in shaping SRH communication, a key finding to emerge from our study is the relationship between SRH communication and both increased decision-making power in Cuenca and greater voice in Kinshasa and Cuenca, after adjustment for the socioecological environment. Our findings, if corroborated by additional research, suggest a role for agency-promoting interventions in setting positive SRH trajectories for both boys and girls in the adolescent period. While promoting empowerment is a long-term and complex process, in the short-term, interventions such as comprehensive sexuality education should pay special attention to adolescents who are less able to voice their opinions or influence decision-making to ensure they receive important SRH information even if they are less likely than others to initiate such a conversation. On the other hand, the associations found here may indicate that discussion of SRH may play a role in promoting adolescents' perception of their own power by building foundational negotiation skills and knowledge. Regardless of the direction of this relationship, our study builds upon a small body of prior findings that link SRH-specific agency indicators to SRH outcomes during the adolescent period ,37.This study found considerable variation in agency, experiences of SRH communication, and the relationships between the two across three very distinctive urban contexts. While we found links between all SRH communication and voice in Kinshasa and Cuenca, decision-making was only associated with communication about pregnancy and contraception in Cuenca. In addition, no relationships between these factors were found in Shanghai. Differences in these findings across the three sites could be attributed to context-specific factors such as the influence of religiosity in Cuenca and Kinshasa, the existence of Machismo culture in Latin America, and the impact of the One-Child Policy on girls' empowerment in Shanghai ,39. ThesThe present analysis is also novel in its inclusion of boys and comparison of the examined relationships by sex. While the field has focused on girls' preparedness for sexual activity, the present results challenge the assumption that boys arrive at sexual activity both more empowered and more prepared than girls, as levels of SRH communication were largely comparable by sex in these three settings. While we found that voice was more strongly associated with communication about pregnancy for girls than boys in Kinshasa, notably no other associations between SRH communication and agency differed significantly by sex. Our results also contribute novel evidence by employing a validated measure of overall agency among young adolescents to evaluate such associations, suggesting that a broader concept of agency may relate to SRH skills.Several limitations of this study should be noted. First, findings are not generalizable beyond our study areas. Second, our measure of SRH communication only described whether or not adolescents reported having discussed each SRH topic. These measures lacked specificity about the people adolescents talked to, contexts in which they took place, content, temporality, frequency or quality of these discussions, which likely impact their relationship to agency, and to downstream SRH behaviors and outcomes . IndicatTaken together, our findings suggest that empowerment factors, including the enabling environment and dimensions of agency, are linked to communication about SRH in the early adolescent period in certain contexts. We conclude that further research, with representative samples, longitudinal data and more specific SRH communication items that allow for disaggregation between types of SRH communication, is needed to fully understand the role of empowerment in shaping SRH trajectories during early adolescence.


The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3′ to 5′ direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3′-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging.Sulfolobus solfataricus. A relatively high proportion (17–19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5′ parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3′-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5′-ends of RNAs was detected.To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3′-5′ direction.In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5′-ends of genes suggests simultaneous binding of both RNA ends by the The online version contains supplementary material available at 10.1186/s12864-020-07200-x. Sulfolobus solfataricus is a crenarchaeon with a growth optimum at 80 °C to 85 °C and pH 2 to 4 -ATP were performed. Complexes were resolved on a denaturing neutral-pH SDS-polyacrylamide gel electrophoresis , and transferred to a nitrocellulose membrane. Protein-RNA-complexes were visualized by autoradiography on an X-ray film at − 80 °C. Complexes of adequate size were excised from the membrane and RNA was isolated by proteinase K treatment. iCLIP library preparation was performed as described in [All following steps were performed as described in . In brie3′-RNA linker (L31): P-UGAGAUCGGAAGAGCGGUUCAG-Puromycin.Reverse transcription primers :R1clip P-NNAACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC.R6clip P-NNCCGGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC.R9clip P-NNGCCANNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC.R10clip P-NNGACCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC.R13clip P-NNTCCGNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC.R14clip P-NNTGCCNNNAGATCGGAAGAGCGTCGTGgatcCTGAACCGC.Following oligonucleotides were used:Rrp4 Replicate 1: primer R1 - BC1: NNNGGTTNN.Rrp4 Replicate 2: primer R9 - BC4: NNNTGGCNN.Rrp41 Replicate 1: primer R10 - BC2: NNNGGTCNN.Rrp41 Replicate 2: primer R13 - BC5: NNNCGGANN.Trx Replicate 1: primer R14 - BC3: NNNGGCANN.Trx Replicate 2: primer R6 - BC6: NNNCCGGNN.These reverse transcription primers add the following barcodes to the 5′-end of the CLIP-tag sequences :The control Trx2, which was directly adjacent to the exosome samples on the membrane Fig. d, had soTwo independent bioreactor cultures were used for the iCLIP experiment. Samples from these cultures and from a third bioreactor culture were used for RNA-Seq. No rRNA depletion was performed for any of the samples. Total RNA was sequenced by Vertis Biotechnologie AG, Freising, Germany.http://hannonlab.cshl.edu/fastx_toolkit/index.html) were used for read processing. Due to bad sequencing quality of the first nucleotide, it was removed using fastx_trimmer (−f 2). Reads were subjected to a two-step quality filtering using fastq_quality_filter (−q 5 -p95 and -q 20 -p 90). The random barcode was utilized to remove PCR duplicates via fastx_collapser. 3′ adapter sequences were removed by fastx_clipper (−a TGAGATCGGA) and manually trimming of remaining nucleotides using awk (4.1.3). Fastx_trimmer (−f 3) was used to remove the first random barcode (2 nt). Separation of reads according to the experimental barcode was done with fastx_barcode_splitter.pl (−-bol --exact). Remaining barcodes (6 nt) were subsequently remove using fastx_trimmer (−f 7). Resulting sequences were mapped to the S. solfataricus P2 genome  via bowtie2 (2.3.4) (−-sensitive) [Reads resulting from Illumina sequencing (75 nt) carried a nine nucleotide barcode at the 5′ end. Barcodes were composed of a three-nucleotide random barcode, a four-nucleotide experimental barcode and a final two-nucleotide random barcode. Several tools from the FASTX Toolkit  , 51, sepnsitive)  and bedtnsitive) .S. solfataricus. Therefore the occurrence of circRNAs was investigated. For the detection of circRNAs interacting with the archaeal exosome, we applied a strategy similar to the one of Danan et al. [Sulfolobus, a prokaryotic organism lacking introns in protein-coding genes. The central idea of circRNA detection is the identification of a circularization junction. Sequencing of such a junction will result in a chimeric read, mapping to the target genome in a non-linear pattern. Briefly, detection of circRNAs harbouring a ligation site can therefore be performed by identification of two adjacent blast results in a single read aligning in a permuted way to the reference genome.A major part of the non-aligning reads contained significant amounts of sequence fragments from n et al.  that wasS. solfataricus P2 sequence acting as a reference, whereby a first blast result with a minimum length of 20 nt was obtained. For further analysis blast results were split according to strandedness. Furthermore, only those hits were kept that fulfilled the requirement of a non-aligned sequence within the read 5′ or 3′ to the blast result. Those reads were subsequently subjected to a second blastn search . The blast results obtained had to fulfil two requirements: They were not allowed to be more than 4000 nt apart from the position of the first blast hit and only single positional results were permitted. This allowed for the combination of the second fragments with their firstly identified counterparts.For circRNA detection only unaligned reads were used, as circularization junctions can only be contained in the non-aligning reads. The reads were subject to a blastn (2.2.31+)  search  . CIRI2 rS. solfataricus sequence were identified. Potential circRNAs were excluded.As with the detection of circRNAs, reads harbouring a 3′ attachment synthesized by the exosome can only be found within the set of non-aligning reads. In theory reads carrying a 3′ attachment must consist of a 5′ section, which maps perfectly to the organism’s genome while there 3′ attachment does not, preventing alignment of the entire read. Therefore detection of 3′ attachments shared considerable similarities with circRNA detection. Reads with a 5′ end alignment to the The nucleotide composition of the 3′ attachments of those reads was statistically assessed for all bases as well as for each position within the attachments. Data were summarized by custom Python 3 scripts and plotted using R (3.4.2).S. solfataricus was obtained from the DOOR database  [To evaluate the binding preference of the protein complex, iCLIP crosslink sites had to be determined. To consider only those reads with a high mapping quality and unique mapping position, reads with a MAPQ-score below 20 were removed (samtools view -q 20) . Replica0.07.31) . All genThe coverage ratio between iCLIP and RNA-Seq mappings for all coding sequences (CDS) and intergenic regions (IGRs) was assessed as follows: firstly a bed file containing all IGRs was creating using bedtools complement (v2.27.1)  and the Motif discovery for a predominant crosslink peak was performed using meme (−dna -nmotifs 1 -minw 4 -maxw 10) (4.12.0) . Ten basSulfolobus solfataricus P2 (NCBI Reference Sequence: NC_002754.1) [A-content, AG-content and the length of the longest poly(A) stretch were determined for each coding sequence (CDS) based on the RefSeq genome annotation of 02754.1)  using cuAdditional file 1: Figure S1. Uncropped images. Figure S2. CircRNAs identified in the exosome iCLIP analysis of S. solfataricus.Figure S3. Distribution of bases in RNA-tails detected in the aRrp41-iCLIP of the archaeal exosome. Figure S4. Poly(A) stretches in RNA are bound by the exosome. Figure S5. Global analysis of RNAs enriched by coimmunoprecipitation with the archaeal exosome by iCLIP. Figure S6. Antisense RNAs as preferred substrates of the archaeal exosome. Figure S7. Binding of the archaeal exosome to 5’and 3’parts of selected genes. Figure S8. Low affinity of the archaeal exosome to the abundant mRNA tmoA (SSO_RS06040 gene encoding toluene-4-monooxygenase system protein). Figure S9. Distribution of read counts in the genome of S. solfataricus was analyzed with respect to biotype RNA. Figure S10. Clustering of read counts at the 5′-and 3′-end of protein coding genes. Figure S11. Clustering of crosslink sites at the 5′ and 3′ end of predicted operons. Figure S12. Distribution of crosslink sites around the ends of annotated genesAdditional file 2:. iCLIP and RNA-Seq statistics.Additional file 3:. Mapped crosslink sites.Additional file 4: CircRNAs identified in the iCLIP analysis of the S. solfataricus exosome.Additional file 5: RNA-tails with mapped 5′-sequence, detected by iCLIP of the archaeal exosome.Additional file 6: Nucleotide content of the linear non-mapped cDNA reads in the iCLIP analysis of S. solfataricus.Additional file 7: List of gene products, A-content, AG-content and the length of the longest poly(A) stretch for each coding sequence.Additional file 8: Poly(A) stretches in CDS and in intergenic regions (IGRs), and binding of the archaeal exosome detected by iCLIP.Additional file 9: iCLIP and RNA-Seq counts of annotated genes included in Fig.


Another animal to human transmission of a coronavirus occurred in December 2019 on a live animal market in the Chinese city of Wuhan causing an epidemic in China, reaching now different continents. This minireview summarizes the research literature on the virological, clinical and epidemiological aspects of this epidemic published until end of February 2020. The infections were epidemiologically associated with a seafood ‘wet’ market in the city. ‘Wet’ market means that live and dead animals are sold, raising the suspicion of another zoonotic virus infection that had spilled over into the human population similar to the severe acute respiratory syndrome (SARS) epidemic in 2002, which also started from a live animal market. In SARS, the viral source was traced back to a bat coronavirus with civets as potential intermediate host. The novel Wuhan virus figures under different names , all names indicating that it represents a coronavirus. One might argue whether ‘new’ instead of ‘novel’ coronavirus is not a better term since it is not completely different, but related to the SARS coronavirus, while others find that SARS‐CoV‐2 suggests a too close relations with the SARS virus possibly leading to some confusion  obtained eight complete viral genome sequences from nine patients either by direct RNA isolation and sequencing from bronchoalveolar lavage fluid (BALF) or from classical virus isolation on human airway epithelial cells   coronavirus  such that in China pangolin products, for example are reputed to help against rheumatism; ‘meridian obstruction’; liver disease; and to improve eyesight (Li and Li, None declared.


The co-corresponding author Fei Peng had been removed from the main authorship line at proof by the author erroneously, though Fei Peng was still listed as a correspondence.The authors of the original article “Network pharmacology for the identification of phytochemicals in traditional Chinese medicine for COVID-19 that may regulate interleukin-6” (Biosci Rep (2021) 41(1), The correct authorship list is therefore as follows:1, Feng Wu2, Wen-yue Cao3, Zong-gui Wu1, Yu-Chieh Chao4, Fei Peng5 and Chun Liang1Wen-hao Niu1Department of Cardiology, Shanghai Changzheng Hospital, Naval Medical University, Shanghai 200001, China; 2Department of Cardiology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Affiliated hospital of Shanghai University of Traditional Chinese Medicine, Shanghai 200080, China; 3Department of Ultrasound, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai 200030, China; 4Department of Anesthesiology, Shanghai Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200120, China; 5Department of Nursing, Shanghai Changzheng Hospital, Naval Medical University, Shanghai 200001, China.The live manuscript was corrected 23 February 2021.


Coronaviruses (CoV) are positive-sense single-stranded RNA viruses, harboring the largest viral RNA genomes known to date. Apart from the primary sequence encoding for all the viral proteins needed for the generation of new viral particles, certain regions of CoV genomes are known to fold into stable structures, controlling several aspects of CoV life cycle, from the regulation of the discontinuous transcription of subgenomic mRNAs, to the packaging of the genome into new virions. Here we review the current knowledge on CoV RNA structures, discussing it in light of the most recent discoveries made possible by analyses of the SARS-CoV-2 genome. On one The second layer of information relies on the ability of RNA to fold into stable secondary structures, by base-pairing between regions of internal complementarity, followed by further compaction into tertiary structures. RNA structure elements, as well as their ability to dynamically interconvert between alternative conformations, have been widely proven to be essential players in the life cycle of various RNA viruses. As an example, the HIV trans-activation response (TAR) element, that can switch between two alternative conformations, is essential for the Tat-mediated activation of viral replication, it provides the docking site for the interaction with several host proteins, and it can be further processed into a mature microRNA to repress target host mRNAs .in vivo [In this perspective, recent technological advances based on RNA structure probing approaches coupled to next-generation sequencing (NGS) technologies ,10,11 arin vivo .Coronavirinae, a subfamily of RNA viruses consisting of four genera, namely Alphacoronaviruses , Betacoronaviruses (beta-CoV), Gammacoronaviruses (gamma-CoV), and Deltacoronaviruses (delta-CoV) [In this review, we will focus our attention on lta-CoV) . Alpha-Clta-CoV) . Beta-Colta-CoV) ,16. Corolta-CoV) , with a lta-CoV) . Despitelta-CoV) ,19.The genomic RNA (gRNA) has a 5′ cap and 3′ polyA tail, allowing direct translation of the non-structural polyproteins (nsp) ORF1a, and ORF1b, followed by assembly of the replicase-transcriptase complex (RTC). The RTC drives both genome replication and discontinuous transcription of subgenomic mRNAs (sgRNAs). Discontinuous transcription is mediated by short AU-rich transcription regulating sequences (TRSs), located either right downstream of the 5′ leader (TRS-L), or right upstream of each viral ORF (TRS-B), except for ORF1a and ORF1b . The resOver the past few months, enormous efforts have been put in trying to better understand the biology of the SARS-CoV-2 virus, to find an Achilles’ heel and to confine the COVID-19 pandemic. In a very short time, the structure and function of the SARS-CoV-2 proteome , transcrThe 5′ UTR of CoV is highly structured. A first consensus model of the 5′ UTR was originally proposed by comparative analysis of nine CoV from the three major CoV groups , and identified the three major conserved stem–loops SL1, SL2, and SL4 . All thrThe SL1 has a functionally bipartite structure. On the one hand, the upper part of the SL1 must be folded. Indeed, destabilizing mutations introduced in the SL1 of Mouse hepatitis virus (MHV), a widely used model of beta-CoV, lead to defects in viral replication that can be rescued by compensatory mutations restoring the base-pairing . On the in vitro evolution [4 of the pentaloop is severely impaired, and spontaneous mutants of C1 > A were observed in G4 > U mutants, suggesting that these two bases are close enough in the loop to base-pair [The SL2 is probably the most conserved cis-acting structure, as suggested by multiple phylogenetic analyses ,42,44, hvolution . Likewisase-pair . Mutantsase-pair .in vitro Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE) analysis [in silico prediction and in vitro SHAPE probing of the SARS-CoV-2 genome support folding of the SL3 [in vivo SHAPE and dimethyl sulfate (DMS) probing revealed intermediate reactivities in the stem of the SL3 [in vivo RNA–RNA interactions further supports this model, revealing the formation of a long-range interaction between the unfolded SL3 segment and the 3′ UTR [The SL3 (sometimes referred to as SL-II) is conserved only in a small subset of beta and gamma-CoV . When pranalysis , with thanalysis , in agre the SL3 ,39, in v the SL3 ,39, sugge 3′ UTR .in silico analysis of the SARS-CoV-2 genome suggested the presence of a second additional stem–loop, right downstream of the SL4 [in vitro or in vivo structure probing analyses [The SL4 (sometimes referred to as SL-III) consists of a long bipartite hairpin  located right downstream of the TRS-L, that appears to be conserved in all CoV genera . This el the SL4 , whose eanalyses ,39.nsp1 gene, and is conserved in both alpha and beta-CoV [in vitro probing of MHV [in vitro and in vivo probing [in vivo RNA–RNA interaction capture in SARS-CoV-2 [The SL5 partially encompasses the ORF1ab, including the initial portion of the beta-CoV . It consg of MHV , as well probing ,37,39 anRS-CoV-2 . While tRS-CoV-2 . Indeed,RS-CoV-2 . HoweverRS-CoV-2 , althougRS-CoV-2 .in vitro probing of MHV [in vitro and in vivo probing [in vivo RNA–RNA interaction capture in SARS-CoV-2 [in silico scanning of the SARS-CoV-2 genome in search for structure elements significantly more stable than expected by chance [in vitro and in vivo structure probing analyses conducted in the context of the full-length SARS-CoV-2 genome [in vivo RNA–RNA interaction capture experiments [A number of additional SLs have been proposed downstream of the SL5. Although being located downstream of the start codon of ORF1ab, these additional structural elements are usually regarded as part of the 5′ UTR. SL6 and SL7 have been predicted computationally , and theg of MHV , as well probing ,36,37,39RS-CoV-2 . These tRS-CoV-2 , and tarRS-CoV-2 . ComparaRS-CoV-2 . Howevery chance , nor by eriments , suggestIn vitro SHAPE and cryo-EM analyses of the isolated SARS-CoV-2 FSE confirmed this architecture , and further confirmed in MHV , it has itecture , additiol genome . Indeed,mely low   gene, is the bulged stem–loop (BSL). This element shows very limited sequence, but high structural conservation across beta-CoV, and it has been proven to be essential for viral replication in MHV ,63. The in vitro [in vitro structure probing [in vivo RNA–RNA interactions [in vivo SHAPE probing reveals extensive unfolding of large part of the main HVR stem [in vivo.The P2 stem–loop is part of a large multi-branched structure, known as the hypervariable region (HVR). The HVR shows poor sequence conservation across CoV, except for the octanucleotide GGAAGAGC, that appears to be extremely conserved across all CoV genera ,68, alth probing  and direractions , in vivoHVR stem ,39, suggEven if generally regarded as part of the 3′UTR, the BSL and part of HVR overlap a putative open reading frame (ORF10), whose existence, however, does not appear to be supported by a recent transcriptome analysis of SARS-CoV-2 infected cells .in vitro and in vivo conditions by multiple studies [Escherichia coli 16S ribosomal RNA, suggesting that this structure element might be involved in hijacking the host cell protein synthesis machinery.A highly conserved structure element, stemming from the multi-branched loop of the HVR, is the stem–loop II-like motif (s2m). This stem–loop shows extreme sequence and structure similarity to the second stem–loop from the 3′ UTR of astroviruses and equine rhinovirus , and its studies ,35–39. X studies . Overallin vitro and in living infected host cells, have led to the identification of a plethora of novel highly stable RNA structure elements, spread along the whole genome. DMS probing of infected host cells revealed that seven out of the nine TRS-Bs reside within stem–loop structures , one of the most highly structured viral RNAs to date . Indeed,ructures , top, chve sgRNA . Similarin vivo SHAPE analysis, with previously proposed structure models of Dengue (DENV) and HCV viruses, suggested that the SARS-CoV-2 genome is more prone to form locally-stable secondary structures, with fewer long-range base-pairing interactions, as compared with other positive-sense RNA viruses [in vivo RNA–RNA interaction capture experiments revealed the existence of several long-range interactions, particularly enriched within ORF1a [Comparison of the SARS-CoV-2 genome structure modeled by  viruses . This prin ORF1a   in the SARS-CoV-2 genome . Of the in vivo RNA–RNA interactions showed that the SARS-CoV-2 gRNA establishes several interactions with the small nuclear RNAs (snRNAs) U1, U2 and U4 at the level of ORF1ab, while the remainder of the gRNA is devoid of such interactions. Oppositely, the sgRNAs are enriched for interactions with the U1 and U2 snRNAs within the N gene and the 3′ UTR. Interactions with the U2 snRNA within ORF1a appear to be highly conserved, as confirmed by RNA–RNA interaction mapping in MERS-CoV-infected cells.Apart from intramolecular base-pairings, the SARS-CoV-2 genome appears to be involved also in a number of intermolecular RNA–RNA interactions with the host cell's transcriptome . Particu6A) readers of the YTHDF family, and depleted of interactions with m6A erasers of the ALKBH family, the SARS-CoV-2 genome showed the opposite trend, suggesting that SARS-CoV-2 might escape m6A methylation to increase the gRNA stability [5C) sites might exist in SARS-CoV-2 RNAs [Additionally, gRNA pulldown experiments coupled to mass-spectrometry analyses showed that, during the SARS-CoV-2 life cycle, the gRNA establishes numerous RNA–protein interactions, both with viral and host cell proteins ,31,77. Atability . Direct tability ,85. AlthV-2 RNAs .Recent advances in the RNA field have proven that RNA structure represents an ideal, yet largely underexploited, drug target ,88. In tGiven the fast evolution of RNA viruses, leading to changes in the sequence and structure of viral proteins, the existence of conserved RNA structures in viral genomes provides a unique opportunity to develop more potent and durable antiviral therapeutic strategiesin vitro or in vivo), using orthogonal probing approaches , and reported diverse sets of RNA structures, providing a wide repertoire of potential therapeutic targetsMultiple groups have queried the structure of the SARS-CoV-2 genome under different conditions (While most studies so far only provided a static snapshot of RNA structures in CoV genomes (at a single time-point of infection), future efforts should be aimed at characterizing the dynamics of the CoV RNA structurome to possibly identify crucial transient RNA folds and RNA structure switches, whose targeting might provide the means to effectively inhibit viral replication


However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the “Broccoli” RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, covariation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro.Methods for rapid and high-throughput screening of transcription  A common consensus promoter is TAATACGACTCACTATA followed by one to three guanine nucleotides before the desired sequence for synthesis . As a result of the RNA folded structure, the brightness and stability of Broccoli RNA fluorescence is also partially dependent on temperature, as will be the case for all folded aptamers  of selected samples, transcription of two additional longer RNAs, and comparison across several common buffer conditions. We also performed saturation mutagenesis of the T7 RNAP promoter with this method and found that our results generally coincided well with previous mutagenesis studies. The results of this study provide a resource to researchers interested in the properties of T7 RNAP or that use T7 RNAP for routine synthesis of RNA in the laboratory. The Broccoli RNA aptamer did possess some shortcomings and may have introduced some sequence-dependent biases in template transcription. However, other aptamers with distinct fluorescent properties and folding are now available that may address some of Broccoli’s weaknesses, including Mango, Pepper, and o-Coral , which provides a relative measurement of total RNA production over time. In most experiments, RNA transcripts from fluorescent reaction replicates were pooled and resolved by denaturing PAGE to compare staining on a gel to fluorescence-based detection. Resolution of RNA transcripts by denaturing PAGE sometimes resulted in multiple bands or higher-molecular-weight smearing. It is possible that the strong quadruplex nature of the Broccoli RNA aptamer (A–B).By this method, Broccoli RNA aptamer synthesis and fluorescence are observed in real time B, which  aptamer  resulted aptamer , A–B.2 and ribonucleoside triphosphates (rNTPs), as well as their concentrations relative to one another, has been known to impact in vitro T7 RNAP transcription efficiency . Maximal RNA synthesis at each rNTP concentration appeared to be centered on a MgCl2 concentration that was approximately 10 mM higher than the total rNTP concentration. This was observed at all three rNTP concentrations, suggesting that aptamer reporter dependence on Mg2+ does not contribute significantly to this effect. Nonetheless, although the Broccoli RNA aptamer was demonstrated to only require very low millimolar concentrations of Mg2+ . The RNA yield was severely reduced when rNTP concentrations exceeded MgCl2 concentration. In addition, 50 mM MgCl2 was inhibitory in transcription reactions with 20 mM rNTPs when transcribing 7SK-Broccoli, a ∼450-nt template that encodes the human 7SK RNA sequence fused to the Broccoli RNA aptamer (A). Precipitates of Mg2+ and pyrophosphate are common during in vitro transcription reactions and can potentially alter the effective free Mg2+ concentration available for T7 RNAP. However, precipitation might be neutral or beneficial since the reduction of free Mg2+ would conceivably be proportional to consumption of rNTPs and would also remove potentially inhibitory pyrophosphate from solution. We included inorganic pyrophosphatase (PPase) in fluorescence reactions to prevent cloudiness and maintain accurate reading. Therefore, we investigated the effect of PPase at three different rNTP concentrations. When transcription products were resolved by denaturing PAGE, we did not observe any increase in transcription yield by the addition of PPase (B). Thus, we concluded that PPase or the formation of magnesium pyrophosphate precipitates is unlikely to alter the interpretation of results or cause unforeseen issues in fluorescence-based transcription screens. Together, these results support the importance of balancing and properly covarying rNTP and MgCl2 concentrations. These results are similar to previous reports but suggest that a slightly elevated concentration of Mg2+ during in vitro T7 transcription reactions could be beneficial.Although 30 mM rNTPs should support greater RNA synthesis, the conditions that produced the most consistent yield appeared to be 20 mM rNTPs with 30 mM MgClFig. 1D, . These rto 35 mM A. The RN aptamer A. Precipof PPase B. Thus, in vitro T7 RNAP transcription, which included the concentration of DNA template, T7 RNAP, monovalent ions, and polycations, as well as temperature and pH. Beginning with our standard conditions, we titrated the ssDNA hybrid template for Broccoli RNA transcription. As expected, the amount of DNA template proportionally increased the overall production of RNA transcript when the template was limiting (A). Transcription plateaued around 1.25 μM of template. Of importance, the amount of RNA made at each individual concentration of template also plateaued, indicating that longer incubations will not necessarily result in greater amounts of RNA when the template is limiting. Conversely, excess template did not seem to inhibit the reaction. When transcribing 7SK-Broccoli from a linearized plasmid template we observed a similar proportional increase in transcriptional output up to the final amount of 2 μg (0.2 μg/μl) at which point the total output appeared to begin plateauing (B). This is similar to the concentration of 1 to 3 μg of linearized plasmid per 20 μl reaction that is recommended in common commercial T7 transcription kits, such as the Ampliscribe T7-Flash Transcription Kit (Epicentre/Lucigen). In contrast to the synthetic DNA template encoding Broccoli, transcription of the longer 7SK-Broccoli from a linearized plasmid increased steadily over time at low template concentrations until reaching 1 μg of template (0.1 μg/μl) (B), suggesting that longer incubations may be required to produce more RNA product when plasmid DNA templates are limiting or when longer RNAs are transcribed.We decided to test other conditions fundamental to limiting A. Transcateauing B. This i1 μg/μl) B, suggesB). The transcription reaction reached completion much sooner when higher concentrations of T7 RNAP were present. For example, the reaction appears to have produced its maximum amount of RNA product at 30 min with 5 μM T7 RNAP while the product continues to increase steadily up to 60 min with 2.5 μM T7 RNAP. We also noted that Broccoli fluorescence was quenched at the highest concentrations of T7 RNAP, even though increasing amounts of transcript appeared to be synthesized when visualized by denaturing PAGE. Since Broccoli RNA aptamer fluorescence is sensitive to salts and DFHBI-1T interaction, it is possible that elevated concentrations of protein can partially titrate these or other components in the reaction. Overall, greater amounts of enzyme appear to proportionally increase transcript production independent of other factors. Nonetheless, exceeding 10 μM T7 RNAP under our standard conditions did not produce substantially greater yields and excess RNA polymerase has been reported to inhibit large-scale transcriptions . This was not surprising given that Broccoli RNA, like other folded RNA structures, is more stable at lower temperatures and therefore is expected to bind DFHBI-1T more stably to produce greater fluorescence. When pooled reactions were resolved by denaturing PAGE, transcript production was greatest around the 37.8 and 41.4 °C temperature points. Thus, it appears that transcription is most efficient at slightly elevated temperatures. Although the fluorescence-based melting temperature (Tm) of Broccoli was determined to be ∼47 °C previously and it was shown to be ∼90% folded at 37 °C . The efficiency of the reaction was not dramatically impacted within this pH range but appeared slightly higher around pH 7.9, which is our standard condition and very similar to the optimal pH of 8.1 reported previously  between the polymerase and the promoter (A). These reactions also contained 1 mM KCl to minimally stabilize the Broccoli RNA G-quadruplex structure . The benefits of polyamines like spermidine in transcriptional proficiency have already been established, especially for small, structured transcripts similar to Broccoli RNA. The concentrations of Mg2+ ions and spermidine could offer synergistic effects and may be codependent upon each other, although this was not explored here.Spermidine is usually considered a critical component for eactions , 35. Speeactions  and to iin vitro . In our in vitro B. The bein vitro T7 RNAP transcriptions. Thus, we decided to further investigate several other conditions relevant to our assay and additives often cited to be beneficial to in vitro transcription. Dithiothreitol (DTT) is a reducing agent that protects against protein oxidation and is routinely included in T7 RNAP preparations and in vitro transcription reactions. Early viral RNA polymerase reactions included up to 10 mM 2-mercaptoethanol or 5 mM DTT (A).Our initial screens coincided relatively well with previously established conditions for 5 mM DTT , 22. Ane5 mM DTT A.B). This could be due to improved solubility of DFHBI-1T. However, gel electrophoresis did not show improvements in transcription at DMSO levels up to a final concentration of 10.6%. Of interest, the addition of up to 15% DMSO was previously reported to increase the efficiency of T7 transcription through altered protein structure . Therefore, relatively low concentrations of DMSO were present in our transcription reactions when monitoring fluorescence. This solvent appeared to be helpful in modest amounts (>1.23%), whereas inhibitory at higher concentrations B. This ctructure . These rin vitro T7 transcription yield, either by fluorescence-based measurements or PAGE (A). This result agrees with a previous study . BSA seemed to offer some improvements in transcription kinetics and yield by fluorescence measurements but this was not obvious by gel electrophoresis (C). It is possible that these components would give a more observable effect at reduced concentrations of enzyme or substrate. Likewise, in our standard conditions both PEG (2%) and BSA (50 μg/ml) are normally present. Since they may have a redundant effect, removing only one may not significantly impact the overall transcription.Polyethylene glycol (PEG) and bovine serum albumin (BSA) are common additives that have both been associated with successful  decades , 40, 41. decades , 22. In cription . PEG8000nditions B. BSA sephoresis C. It is 8000 and BSA are rather large molecules , and components not listed were held constant at concentrations noted for our standard reaction conditions. Thus, buffering agent (Tris-HCl), pH, DNA template, NaCl, MgCl2, DMSO, rNTPs, temperature (37 °C), and T7 RNAP were all held constant while the common additives DTT, spermidine, Triton X-100, Tween 20, PEG8000, and BSA were varied. Condition 1 represented our standard control reaction condition that was used to compare with other component variations.A practical outcome of testing the contribution of individual reaction conditions and buffer components is the identification of guidelines for optimal T7 transcription. To determine if our assay could provide more insight, we compared several reaction conditions, including published recipes, with varying additives using PAGE analysis. We transcribed three distinct RNAs from different templates using T7 RNAP to help determine if any trends might be generalizable. The RNAs transcribed were the Broccoli RNA aptamer, a CRISPR-Cas9 single-guide RNA (sgRNA) , and a tvolcanii . Broccolcompared A, and coB), we calculated approximate transcription yields (B). Our standard condition (buffer 1) was excellent for Broccoli RNA production, but synthesis of sgRNA was clearly reduced and tRNATrp production was minimal. Buffer 2, which contained all additives except Tween 20, performed the worst. The overall best-performing condition was buffer 8, which gave consistently robust RNA production for all three templates, calculated to be approximately equivalent to 80 pmol of Broccoli RNA from a 20-μl reaction. Of interest, buffer 8 has no spermidine, Triton X-100, or Tween 20. Spermidine appeared to be beneficial in our individual component screens. It is notable that buffer 5 was absent of Tween 20, PEG, and BSA but gave the second highest RNA production overall.By quantifying stained RNA bands on a denaturing PAGE gel and comparing with our standard curve B, we caln yields B. Our stBuffers 3, 4, 6, and 7 performed similarly, producing as much or more RNA as our standard buffer 1. Buffer 3 contained no spermidine while 4, 6, and 7 did; buffers 3 and 7 contained Triton X-100 while 4 and 6 did not; and buffers 3, 4, and 7 contained no Tween 20 while buffer 6 did. These data support the performance of buffer 8, which provides high RNA production in the absence of each of these components. We note that additives and buffer components can have unforeseen synergistic effects, potentially both positive and negative. Testing of more components, combinatorial conditions, and a more diverse set of templates with fluorescence-based and traditional readouts could further optimize transcription conditions and could be accomplished by fusing the specific sequence of interest to Broccoli with a self-cleaving ribozyme linker, similar to that of Jäschke and co-workers . Nonethein vitro transcribed a set of 60 ssDNA hybrid templates that encoded Broccoli RNA and possessed mutant double-stranded promoters. Each mutant represented a single base-pair change to one of the three other base pairs, which is a mutagenesis method known as saturation mutagenesis (A). The T7 RNAP promoter sequence in common use has been systematically mutagenized previously to identify sequence elements important for activity . Any base pair seemed to be well accommodated at positions −13 and −12. In contrast, position −11 strongly preferred a purine nucleotide on the nontemplate strand, as did position −6. Position −10 is usually an A-T base pair but could accommodate T-A or C-G pairs to some degree. Positions −9 to −7, representing C-G, T-A, and C-G base pairs in the center of the duplex, were essential for promoter activity and could not be substituted with any other nucleotides. The results described above for positions −15 to −6 all matched those of Diaz and co-workers (B). Specific recognition of the G, A, and G bases on the template strand at these base-pair positions is mediated primarily through hydrogen bonding with T7 RNAP amino acids Q758 and R756, with some help from N748 , position +1 preferred an A-T base pair (over G-C), and position +2 performed very well with any other nucleotide other than the canonical G. Frame shifting from +1 to +2 could explain the high transcription efficiency when the +1 position is mutated to a C or T, as suggested by Imburgio and colleagues . HoweverA) despite the observation that dsDNA templates mediated faster transcription kinetics (B). We propose that dependence of G at position +3 for high fluorescence is linked to folding of the Broccoli aptamer since it is a G-quadruplex structure  adjacent to the Broccoli RNA aptamer template sequence. Although our approach uses direct fluorescence-based readout and focuses on one transcription reaction per mutation, the reliance on aptamer reporter sequence, folding, and fluorescence likely contributed to some deviation from previous studies.We have presented a method using the Broccoli RNA aptamer as a direct and rapid fluorescent readout for in vitro transcription reporters include Mango/TO1-biotin, Pepper/HBC, and o-Coral/Gemini-561. Mango, which binds a thiazole orange derivative, causes fluorescence in the green-yellow, orange, or red range, depending on the thiazole orange derivative bound . The cell suspension was sonicated and clarified by centrifugation, and the soluble fraction was purified by affinity chromatography using a 3-ml bed volume of HisPur Cobalt-CMA Resin (Thermo Fisher Scientific). After applying the soluble fraction to the column, the resin was washed with 50 bed volumes (150 ml) of Wash Buffer 1  and 5 bed volumes (15 ml) of Wash Buffer 2  and eluted with 5 bed volumes of Elution Buffer . The purified protein was concentrated with Vivaspin 15 centrifugal concentrators  and the buffer exchanged in the same concentrator using Elution Buffer lacking imidazole to reduce imidazole to 20 mM. One volume of glycerol was added to give a final storage buffer for the T7 RNAP of 20 mM Tris-HCl, pH 7.9, 100 mM NaCl, 1 mM DTT, 10 mM imidazole, and 50% glycerol. The concentration was determined by UV absorbance at 280 nm using a calculated extinction coefficient and Beer’s law. Aliquots of the purified T7 RNAP were flash frozen in liquid nitrogen and stored at −80 °C.BL21 (DE3) cells transformed with a plasmid expressing T7 RNAP (pT7-911)  were culH. volcanii tRNATrp , 5 mM of each ribonucleoside triphosphate , 2.5 μM synthetic DNA template, 3.5 μM T7 RNAP, 0.0025 units of inorganic PPase (Thermo Fisher Scientific), and 60 μM DFHBI-1T (Lucerna Technologies). The presence of KCl is to ensure sufficient quadruplex formation of the Broccoli RNA aptamer . Other reagents were added separately to prepare the master stock. Typically, rNTPs were 100 mM commercial stocks at neutral pH (Thermo Fisher Scientific). Particular reagents being tested were omitted and titrated as appropriate. Once the final master stocks were prepared, these were incubated on ice for 10 min then 9 μl was added to respective wells of the 96-well plate. The plate was equilibrated on ice in a 4 °C refrigerator for 15 min prior to moving to a qPCR instrument for reading.Transcription reactions (10 μl) were assembled in a 96-well qPCR plate on ice. Standard T7 transcription reactions contained 30 mM Tris-HCl, pH 7.9, 30 mM MgCl aptamer . Under oThe prepared plate was placed in a Bio-Rad C1000 Thermal Cycler with a CFX96 Real-Time System where the heating block was set to 4 °C. The reaction was initiated by ramping the temperature up to 37 °C, and the fluorescence intensity of each well was collected every 1 min for 60 min using the SYBR filter. Select representative time points, rather than all 60 time points, were chosen for simplicity of data presentation. Reading times for qPCR instruments vary; however, we found that a 53-s incubation time was necessary to allow for the 7-s read time over each 1-min reading interval when reading the entire plate with a single filter (SYBR) in the CFX-96 instrument. Raw fluorescence (no baseline subtraction) was collected for data analysis. Fluorescence at the first time point was subtracted to begin at zero for each sample. Fluorescent readings were normalized to the max fluorescence reading for the standard amount of the reagent. Thus, fluorescence is reported relative to the standard reaction conditions whenever normalized fluorescence data are presented. When specific buffer components or conditions were varied, they were generally normalized to the typical concentration of that component or condition under standard reaction conditions. Curve fitting and equation development were performed in Prism (GraphPad).Transcription samples were typically pooled from three separate fluorescence transcription reactions. For nonfluorescent RNA products, 20-μl reactions were prepared and the entire reaction was used for PAGE analysis. Samples were typically treated with 1 U of DNase I for 15 min at 37 °C, EDTA was added to a final of 60 mM, 1 volume of Loading Buffer was added , boiled at 95 ˚C for 5 min, then resolved on a denaturing (6 M urea) 15% TBE polyacrylamide gel. The RNA product bands were stained with methylene blue then destained with water. Bands were quantified with ImageJ. Briefly, gel images were saved as .jpeg or .tif files then opened in ImageJ. The gel should be horizontal. The “rectangular” tool was selected and a box was drawn to include the Broccoli RNA bands across all lanes. To quantify the bands, we went to the “Analyze” tab, then clicked on “Gels” in the drop-down menu, then clicked on “Select First Lane.” After confirming first lane selection by clicking “Yes” in the pop-up box, we then followed the same path and clicked “Select Next Lane” and then again clicked on “Plot Lanes.” These last three steps can be accomplished with the shortcut of holding down the control key and pressing the 1, 2, and 3 number keys. Once the lane plot window opened, a line that is approximately horizontal was drawn under the histogram peaks. This line matched with and connected the background levels in the plot. Finally, the peaks are quantified by selecting the “wand (tracing)” tool and clicking in the enclosed area of each peak. The values given in the new “Results” window that opens are taken as the quantification of each peak.Full data sets, including fluorescence time course data and gel images, may be available upon request to the corresponding author.The authors declare that they have no conflicts of interest with the contents of this article.


Synechococcus sp. strain PCC 7002, that both has a unique array of RNases that facilitate RNA degradation and is an industrially relevant strain that could be used to convert CO2 and sunlight into useful products.RNA degradation is an important process that affects the final concentration of individual mRNAs, affecting protein expression and cellular physiology. Studies of how RNA is degraded increase our knowledge of this fundamental process as well as enable the creation of genetic tools to manipulate RNA stability. By studying global transcript turnover, we searched for sequence elements that correlated with transcript (in)stability and used these sequences to guide tool design. This study probes global RNA turnover in a cyanobacterium,  Synechococcus sp. strain PCC 7002 by collecting total RNA samples after stopping nascent transcription with rifampin. We quantified the abundance of each position in the transcriptome as a function of time using RNA-sequencing data and later analyzed the global mRNA decay map using machine learning principles. Half-lives, calculated on a per-ORF (open reading frame) basis, were extremely short, with a median half-life of only 0.97 min. Despite extremely rapid turnover of most mRNA, transcripts encoding proteins involved in photosynthesis were both highly expressed and highly stable. Upon inspection of these stable transcripts, we identified an enriched motif in the 3′ untranslated region (UTR) that had similarity to Rho-independent terminators. We built statistical models for half-life prediction and used them to systematically identify sequence motifs in both 5′ and 3′ UTRs that correlate with stabilized transcripts. We found that transcripts linked to a terminator containing a poly(U) tract had a longer half-life than both those without a poly(U) tract and those without a terminator.RNA degradation is an important process that influences the ultimate concentration of individual proteins inside cells. While the main enzymes that facilitate this process have been identified, global maps of RNA turnover are available for only a few species. Even in these cases, there are few sequence elements that are known to enhance or destabilize a native transcript; even fewer confer the same effect when added to a heterologous transcript. To address this knowledge gap, we assayed genome-wide RNA degradation in the cyanobacterium IMPORTANCE RNA degradation is an important process that affects the final concentration of individual mRNAs, affecting protein expression and cellular physiology. Studies of how RNA is degraded increase our knowledge of this fundamental process as well as enable the creation of genetic tools to manipulate RNA stability. By studying global transcript turnover, we searched for sequence elements that correlated with transcript (in)stability and used these sequences to guide tool design. This study probes global RNA turnover in a cyanobacterium, Synechococcus sp. strain PCC 7002, that both has a unique array of RNases that facilitate RNA degradation and is an industrially relevant strain that could be used to convert CO2 and sunlight into useful products. While general mechanistic models of global RNA turnover have been described and debated , which is among the fastest-growing photoautotrophs (doubling time ∼2.5 h) (–2 and sunlight (–E. coli) and an essential homolog of RNase J   . PCC 700 ∼2.5 h) –18, makisunlight –21. Anotunlight –, but PCCubtilis) . The impt = 10 min). We could not calculate half-lives for 4.4% of transcripts because of null or low expression and for another 4.1% that exhibited a poor fit to the exponential model. For transcripts containing operons, we observed an increase in half-life along the length of the transcript  and 0.5, 1, 2.5, 5, 7.5, and 10 min after rifampin addition. We added synthetic RNA spike-ins to cell pellets immediately before lysis to normalize for potential large differences in mRNA pool sizes. We isolated total RNA and reduced the rRNA content with commercial reagents. We used this pool of enriched mRNA to prepare a library for sequencing via Illumina HiSeq. The number of reads that aligned to the RNA spike-ins was counted and found to linearly correlate with RNA copy number see . The aveanscript  and S3.10.1128/mSystems.00224-20.1FIG S1R2 = 0.996. Download FIG S1, EPS file, 1.2 MB.Transcriptome sequencing (RNA-seq) read counts from RNA spike-ins. The read counts from RNA spike-ins from example replicate B time 0 minutes is shown. There is a linear relationship between the log read count of the spike-ins  and the log spike-in copy number for spike-ins added between 0.1 and 100 copies/cell. The spike-in added at 1,000 copies/cell was retrospectively too high and not used in the normalization calculation. The linear fit for the remaining 5 spike-ins is shown along with the shaded 95% confidence interval, Copyright © 2020 Gordon et al.2020Gordon et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00224-20.2FIG S2FIG S2, EPS file, 1.4 MB.Half-life increases along transcript. The half-lives of genes from eight operons from PCC 7002 are shown with the points located in the middle of the gene. The bottom diagram shows the number of genes, their spacing, and locus tags. The approximate transcriptional start site is +1. Download Copyright © 2020 Gordon et al.2020Gordon et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00224-20.3FIG S3FIG S3, EPS file, 1.2 MB.Inferences gleaned from regression coefficients in the model built using support vector regression with linear kernels for predicting half-lives using 12 sequence- and structure-based features for 2,587 genes in PCC 7002. The model yielded a correlation coefficient of 0.398 between predicted values and actual values and a root mean square error (RMSE) of 0.69 min . Download Copyright © 2020 Gordon et al.2020Gordon et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the t test P values between 9 × 10−6 and 2 × 10−16, Bonferroni adjusted). Only cellular functions that contained greater than 50 genes were included in the analysis. Within the energy metabolism subgroup, transcripts created from the photosynthesis genes had a longer half-life than all other subgroups .We grouped the half-life of protein coding transcripts by cellular function and found that transcripts encoding proteins involved in energy metabolism had a longer half-life than all other groups , but photosynthesis genes seemed to be highly transcribed and their transcripts seemed to be long-lived . Transcripts encoding proteins involved in energy metabolism were more abundant than transcripts encoding proteins involved with other cellular functions , but photosynthesis transcripts were not more abundant than those within the energy metabolism subgroup . Download FIG S4, EPS file, 1.3 MB.Expression level within energy metabolism subgroup. The antenna protein group had a significantly higher expression level than all other groups besides nitrogen metabolism  was predictive of stability of other transcripts. To examine whether different putative terminator forms are significantly associated with transcript stability, we used the Web Genome Scanner for Terminators (WebGeSTer) database (P = 4.8 × 10−13 [L versus I] and P < 2.0 × 10−16 [L versus none]) than transcripts that contained an I-shaped terminator in the 3′ UTR or no obvious Rho-independent terminator . The feE. coli .RNA turnover in PCC 7002 is extremely rapid with a median half-life of coding regions of 0.97 min ∼58 s), faster than any other report of bacterial mRNA decay. Only 15 transcripts were classified as stable . Prior experiments using RNA sequencing to study RNA degradation 8 s, fast–8. Even –We examined if any groups of transcripts that encode different cellular functions had significantly longer half-lives and found both that photosynthesis genes were highly expressed and that their transcripts were highly stable. Moreover, the biophysical feature-based analysis indicated expression level to be one of the top predictors of mRNA half-life  tract. Surprisingly, in many organisms no terminators were found, and it was suggested that they relied on some yet-uncharacterized termination mechanism  that facilitates the folding of the hairpin, irreversibly trapping the RNA polymerase complex and disrupting all key contacts between RNA, DNA, and RNA polymerase . It is p10.1128/mSystems.00224-20.5FIG S5FIG S5, EPS file, 0.1 MB.Role of a gene and occurrence of L-shaped terminators. (A) The category of “energy metabolism” genes has the maximum number of genes with L-shaped terminators (upon excluding the genes with unknown function). (B) Distribution of terminator type among 146 energy metabolism genes. (C) Distribution of terminator type among 38 photosynthesis genes (a subcategory of energy metabolism). Download Copyright © 2020 Gordon et al.2020Gordon et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. colicrp transcript was analyzed, and the removal of the of the G-C hairpin enhanced transcript degradation, but disruption of the U-tract did not affect transcript stability. Additionally, swapping the terminator for other native Rho-independent terminators did not change expression level or transcript stability  strength as predicted by the RBS calculator  were thrE. coli that included an 0.5-min time point also found an extremely low median half-life of 2.8 min in exponential phase and 5.4 min in stationary phase  of ∼0.2, which is between late exponential and early linear phase for the environmental conditions used. We chose this growth phase because we wanted to examine actively growing and dividing cells. This could be why we saw high stability and abundance of mRNA encoded by photosynthesis genes. In this time period, cells were growing rapidly and needed to produce high levels of photosynthesis machinery. Transcript degradation, however, is likely influenced by many factors including growth phase, growth rate, and environmental changes. There is an increasing body of evidence that RNase activity and expression can be influenced by inhibitors  was cultivated in a temperature-controlled environment (37°C) with light provided by cool white fluorescent lights . A −80°C frozen stock of PCC 7002 was streaked on A+ medium , and placed on ice. Rifampin was added at a concentration of 200 μg/ml (in dimethyl sulfoxide [DMSO]), and samples were taken as described above at 0.5, 1, 2.5, 5, 7.5, and 10 min following addition. Samples were spun down in a Beckman Coulter Avanti J-E centrifuge at 7,500 × g for 10 min at 4°C. The supernatant was carefully aspirated to prevent loss of cells, and samples were flash frozen in liquid nitrogen and stored at −80°C until RNA extraction (∼1 week).Two milliliters of each culture was collected for cell counts via hemocytometer and OD730 of ∼0.2). Thus, dilute cultures were harvested via centrifugation. There was some flotation of the cultures which led to pelleting on the side of the vial, but since they were centrifuged in 50-ml vials, the supernatant could be carefully aspirated without disruption or loss of the pellet. Careful harvesting and extraction are crucial aspects of these experiments, and extra care was taken at each step. It is possible that residual RNase activity could affect the measurements. Therefore, we used a quench solution and rapid cooling to prevent residual activity, which would be much more likely before hot-phenol extraction. In addition, we added RNA spike-ins at the extraction step to account for residual RNase activity. The decay curves were fitted to an exponential decay model, and multiple replicates were used to compare samples and provide statistical support to the overall findings. Biological replicates can reduce the error due to potential RNase activity.Processes regulated by circadian rhythms or light-dark cycles could have significant and important influences on gene expression levels and decay rates in addition to overall cellular physiology. To avoid complexity introduced by these variables, continuous illumination was used for precultures and experimental growth conditions. Several tradeoffs were considered during experimental design. First, because light shading in optically dense cultures attenuates growth, we chose to use rapidly growing dilute cultures at late exponential/early linear growth phase  as well as the ϕX174 genome (NC_001422) with Bowtie 2 (v. 2.2.6) and SAMtools (v 1.2). The HTSeq count function (v 0.6.1) was used to count the number of reads that aligned to each feature. A linear relationship between log RNA spike-in copy number and read counts was observed only for spike-ins added at a ratio of 0.01 to 10 copies/cell, so only these were used for normalization  . The inpA variant of random forest regression, often referred to as extremely randomized trees-based regression , was useThe optimal values for hyperparameters, max_features (number of features to consider when looking for best split expressed in terms of fraction) and min_samples_split , were found to be 0.5 and 100 based on a grid search over the range  and , respectively, under 10-fold cross-validation. Number of estimators was set to 100 decision trees.j is the node index, i is the label index, c denotes the cardinality of classes, and f is the fraction of samples belonging to class i.The feature importance scores were computed based on mean decrease in impurity measures, and the distribution of sequence motif in training set was used to determine whether a particular motif is stabilizing or destabilizing in nature. In general, the features that show up at the top of a decision tree are considered more important as they contribute to predictive decision of a larger fraction of samples. The importance of a node in a decision tree can be determined by computing the decrease in impurity metric for the node weighted by the fraction of samples arriving at that node . Feature10.1128/mSystems.00224-20.6FIG S6FIG S6, EPS file, 0.1 MB.Feature importance computation using impurity measures. (A) Toy data set consisting of 5 samples. (B) Schematic decision tree. (C) Feature importance score under regression and classification settings. Download Copyright © 2020 Gordon et al.2020Gordon et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the A set of 19 biophysical features ranging from simple sequence-based features such as length of the transcript and GC content of the coding region to mRNA secondary structure-based minimum free energy for folding were computed for each of the 2,949 genes in the half-life data set . Binary SRP130967). Half-lives of transcripts and per-base-count data have been deposited at the Gene Expression Omnibus (accession number GSE109174).Raw sequencing files have been deposited in NCBI’s Sequence Read Archive (accession number