A growing literature investigates health effects of the recent economic crisis. This study examines how different economic mechanisms affected low self-rated health (SRH) in Europe over the crisis period (2008–2011). We measure changes in low SRH over 2008–2011 and analyze how they are accounted for by changes in household income levels and income distribution , labour market developments , and non-income poverty .We use balanced panel data for 2008–2011 covering 26 European countries and 43,456 participants. The data come from longitudinal 2011 European Union Statistics on Income and Living Conditions (EU-SILC) database. Increases in low SRH incidence over time are decomposed into the contributions of changes in the distribution of covariates and changes in returns to the covariates. Main covariates include household income and its distribution, labour market developments, and non-income poverty . The decompositions are performed using a detailed non-linear multivariate regression-based decomposition methodology.Low SRH incidence increased in Europe during the crisis by almost 2 percentage points, and by 3.7 percentage points in case of the Baltic countries. Decomposition analysis shows that: 1) decreasing household incomes and changing income distribution had no impact on low SRH incidence, 2) rise of material deprivation accounts for a significant portion (12%) of the overall growth in low SRH rates , 3) decreasing levels of full-time and part-time employment as well as transitions to unemployment, economic inactivity, disability, or retirement account jointly for about 21% of the rise in low SRH in Europe .Together, the recession-related economic factors account for about 33% of the increase in low SRH incidence in Europe during the crisis, and for about 100% of the increase in the Baltic countries. Public health policy during recessions should focus also on reducing material deprivation through free or subsidized access to public services, public housing, and other means.The online version of this article (10.1186/s12939-019-0926-1) contains supplementary material, which is available to authorized users. The recent economic crisis that started in 2008, often called the Great Recession (GR), was the worst global economic recession since the Great Depression of the 1930s. For many European countries, reduced economic activity has brought decreasing household incomes, increased unemployment or labor market inactivity, and growing material deprivation. Most of the countries experienced loses in average household incomes over 2008–2013 with Iceland (28% decline) and Greece (40%) being the biggest losers. Unemployment rates grew over 2008–2013 in all European countries except Germany. Income poverty anchored at 2008 poverty line increased over 2008–2013 in almost half of European countries and more than doubled for Greece and Iceland. Severe material deprivation rate, which captures the proportion of people whose living conditions are severely affected by lack of resources also grew in most of the European countries, and it more than doubled for Iceland, Luxembourg and Malta. Several governments responded to the crisis by implementing policies of austerity or fiscal consolidation, which could have further amplified negative impact of the crisis on health outcomes. For example, governments in Greece, Ireland, Latvia and Portugal have made large cuts to public health spending [Theoretical considerations suggest several pathways leading from worsening economic conditions to health deterioration , 3. LoweSeveral previous empirical studies have investigated the impact of the GR on various health outcomes such as suicide rates , fertiliThe existing empirical literature has so far analysed the impact of the GR on health mostly through labour market developments including growing unemployment rates. However, health during economic crises can be also affected through other channels such as declining household incomes and growing income and non-income poverty . Research has shown that income and its distribution as well as material deprivation can have a significant effect on health –24. The characteristics  and a component attributable to differences in the returns to these characteristics. The former component is often called an “explained” part of the difference in the outcome, while the latter component is called an “unexplained” one. The Oaxaca-Blinder methodology has been applied to analyse various issues in the health literature [k), where k is the chosen cut-off level separating individuals with low SRH from individuals with medium or high level of SRH. In our empirical analysis below, we define k as “fair” SRH status. Under a logit probability model, the likelihood of a person i having a low level of SRH in time t, F is the logistic probabilistic cumulative distribution, i in period t, and X includes our choices for variables related to the recession, control variables, and country dummies (see next section for details).To identify factors that are associated with changes in low SRH over time, we use a variant of multivariate regression-based Oaxaca-Blinder decomposition technique , 26, whiterature , 29, 30.mentclass2pt{minimt, Ht, is equal to the average predicted probability of having low SRH:F is the logistic probabilistic cumulative distribution, i in period t, and t. Using property (2), the difference between the low SRH rates observed in two periods t = 0 and t = 1,E, and aggregate coefficients or “unexplained” effect, C:For the logit model, the low SRH rate in period Eg and Cg, g = 1, …, G, representing the contributions of an individual gth element of X to, respectively, the aggregate effects E and C:W are the appropriate weights obtained by approximating the value of the average of function X, X.The aggregate “explained” and “unexplained” effects can be further decomposed into individual effects equation  around \nt}X¯0β0 , 31, 32.We use data on SRH and its correlates from the longitudinal 2011 European Union Statistics on Income and Living Conditions (EU-SILC) database. These data refer to the period 2008–2011, which covers most of the crisis period in Europe. We use a balanced four-year panel sample of individuals that were interviewed in each year between 2008 and 2011. The sample covers 26 EU countries; no longitudinal information for Germany and Ireland was available for the 2008–2011 period. In all analyses, we apply EU-SILC individual longitudinal weight to account for survey non-response and longitudinal attrition. Standard errors for all estimates are clustered at the country level.The wording of the SRH question in EU-SILC is as follows: “How is your health in general?”. The answers are ranked on a 5-point scale: 5 = “very good”, 4 = “good”, 3 = “fair”, 2 = “bad”, and 1 = “very bad”. We define low SRH as reporting “fair” or worse health. Our analysis is performed for individuals between 18 and 64 years of age. We excluded proxy interviewees (respondents who had someone else answer the questions for them). Table In our decomposition analyses aiming at identification of factors that are associated with changes in low SRH, we use several variables that can affect SRH. The standard controls include age, sex, educational attainment, marital status, degree of urbanization, and country dummies. We expect that the impact of the economic crisis on low SRH can be captured using three types of economic variables: income and its distribution , 33, labTable Decompositions of increases in low SRH rates over 2008–2011 for all EU countries and the Baltic countries are presented in Table Table All individual characteristics effects related to labour market status are highly significant. Full-time and part-time employment rates in the EU have jointly fallen over 2008–2011 by 4.3 percentage points. These labour market perturbations contributed to the rise in low SRH rate by about 0.5 percentage point . For the Baltic countries, declines in employment rates account for 1.1 percentage point increase in the low SRH incidence. The results suggest also that increasing rates of unemployment during the crisis had a small (0.1 percentage point), statistically significant effect on reducing low SHR rates in the EU . This counterintuitive result is due to weakening of the association between unemployment and the probability of reporting low SRH during the recession  incomes and income inequality on health is empirically small or even negligible , 40, 41.An important novel insight of the present paper is that growing material deprivation can be an important factor worsening health during recessions. This calls for both more research on the relationship between material deprivation (or other measures of non-income poverty) and health, as well as for reconsideration of policies directed at reducing material deprivation. Such policies could include progressive taxation, free or subsidized access to public services, active labour market interventions, public housing, and others .This study has a number of limitations. First, it relies on self-reported health data, which are subjective and can have limited comparability across time and various groups in the society . Second,In this paper, we used balanced panel data from 2008 to 2011 EU-SILC database and regression-based decomposition methodology to understand how different economic mechanisms  have affected changes in the incidence of low SRH in Europe during the recent economic crisis. To our knowledge, this is the first study to estimate the separate impact of distinct recession-related economic factors on SRH in Europe. We have found that low SRH incidence increased during the GR by almost 2 percentage points for all EU countries, and by 3.8 percentage points for the Baltic countries. The recession-related economic factors together account for about 33% of the increase in low SRH incidence in Europe, and for about 100% of the increase in the Baltic countries. Our novel finding is that non-income poverty  has been an important factor worsening health during the recession as it accounted for 12% of the overall growth in low SRH in Europe . Future studies should investigate whether the link between increasing material deprivation and poorer SRH can also be found in case of other recessions.Additional file 1:Table A1. Descriptive statistics for the sample of Baltic countries  (DOCX 42 kb)


Bacillus subtilis, so far only type I and type II TA systems were found, all encoded on the chromosome. Here, we review our present knowledge of these systems, the mechanisms of antitoxin and toxin action, and the regulation of their expression, and we discuss their evolution and possible physiological role.Toxin–antitoxin (TA) systems were originally discovered as plasmid maintenance systems in a multitude of free-living bacteria, but were afterwards found to also be widespread in bacterial chromosomes. TA loci comprise two genes, one coding for a stable toxin whose overexpression kills the cell or causes growth stasis, and the other coding for an unstable antitoxin that counteracts toxin action. Of the currently known six types of TA systems, in  Toxin–antitoxin (TA) systems are genetic modules that are widespread in bacterial genomes and plasmids. They consist of a gene pair encoding a stable toxin that impedes cell growth by interfering with fundamental processes, and an unstable antitoxin that counteracts the toxin activity. Currently, six types of TA systems are known. Whereas type I and III TA systems employ RNA antitoxins, the other TA systems use proteinaceous antitoxins. In type I TA systems, antitoxins interact with the corresponding toxin messenger RNAs (mRNAs) . In typeAlthough TA systems are ubiquitous in bacterial genomes and their molecular activities were investigated for many years, the biological function of the majority of them is still elusive. To date, the following major biological functions were demonstrated: Post-segregational killing (PSK), abortive infection, persister formation/antibiotic tolerance, and stress response (reviewed in Reference ). Bacillus subtilis, both type I and type II TA systems were discovered and characterized. In this review, we provide an overview of the known B. subtilis TA systems with special focus on their molecular mechanisms, the control of toxin and antitoxin expression, the modes of toxin action, and their biological role.In B. subtilis and designated txpA/RatA [txpA/RatA [bsrG/SR4 [bsrE/SR5 [yonT-yoyJ/SR6 [In 2005, the first type I TA system was discovered in xpA/RatA . SubsequxpA/RatA  and bioixpA/RatA  revealedxpA/RatA , five ofxpA/RatA ,10, bsrGbsrG/SR4 , bsrE/SRbsrE/SR5 ,13 and yyoyJ/SR6 —were invyoyJ/SR6 , are yetB. subtilis SR4 is the first bifunctional antitoxin; it both impedes translation of bsrG mRNA and facilitates its degradation [Two principal mechanisms of action of type I antitoxins are known: inhibition of toxin translation or promotion of degradation of the toxin mRNA. radation   sequence to inhibit toxin translation directly , or it prevents translation of a leader peptide translationally coupled to the toxin . AlternsB/IstR1 ).B. subtilis type I TA systems studied so far , the RNyonT/SR6 ,11,12,14nslation . NeitherxpA mRNA  nor antisrE mRNA  was suchyonT/SR6 TA system, no secondary structures were probed to investigate this issue. Interestingly, SR6 controls a second toxin gene, yoyJ, using its 5′ region to base-pair to the yoyJ ribosome binding site (RBS) [yonT mRNA degradation, it neither affects the amount nor half-life of yoyJ mRNA [yoyJ translation. For the te (RBS) . WhereasoyJ mRNA . Therefocis-encoded sense/antisense systems, and binding kinetics of sense and antisense RNAs were measured  are in the range of 1 × 105 to 1 × 106 M−1·s−1. High kapp values are indicative for efficient pairing between two complementary RNAs. For bsrG/SR4, a kapp value of 6.5 × 105 M−1·s−1 was determined [bsrE/SR5, a kapp value of 4 × 106 M−1·s−1 [txpA/RatA and for yonT/SR6, kapp measurements are yet to be performed. To narrow down the minimal regions required for efficient complex formation with their toxin mRNAs, truncated SR4 or SR5 species were employed in interaction studies with their toxin mRNAs [bsrE RNA, since replacement of either of them or of both loops 2 and 4 decreased binding 6–7-fold [Binding pathways were studied in detail for many eference ). On avetermined  and, for M−1·s−1 . For txpin mRNAs ,15. Both6–7-fold . fst/RNAII [hok/Sok [Initial contacts between a base-pairing small regulatory RNA (sRNA) and a target mRNA can either take place between two complementary loops  or betw[hok/Sok ). In botbsrG/SR4, binding starts between the so-called recognition loops, the terminator loop L4 of SR4 and L3 of bsrG RNA, followed by base-pairing between L3 of SR4 and a stretch within the main helix of bsrG RNA. Eventually, SR4 L2 and the bsrG L3 interact, although this interaction is not required for inhibition [bsrE/SR5  are involved in the first loop–loop contact, whereas only one U-turn motif participates in the bsrG RNA/SR4 interaction. Thirdly, in contrast to SR4, SR5 does not induce structural changes around the bsrE SD, i.e., the stem topped by L1 is not extended.In the case of hibition   mRNA was demonstrated to be decisive for a rapid RNA interaction with the Sok antisense RNA [N-ethyl, N-nitrosourea (ENU) footprinting. Surprisingly, in the case of bsrG/SR4, two potentially important 5′ YUNR motifs were detected. However, whereas the motif in L3 of bsrG RNA is implicated in the initial contact with L4 of SR4, the 5′ YUNR motif in L2 of SR4 that interacts with bsrG L4 was not required, as the entire SL2 was not essential for the inhibitory function of SR4 [bsrE/SR5, two loops (L4 of bsrE RNA and L4 of SR5) each containing a U-turn motif are involved in the initial rapid interaction  can potentially form a U-turn with a sharp bend in the RNA backbone that presents the next three or four bases in a stacked, solvent-exposed configuration. This provides a scaffold for rapid RNA/RNA interactions. Via one or two contacts across the bend, the backbone change is stabilized. U-turn motifs were first discovered in yeast transfer RNA (tRNA) and the hammerhead ribozyme—and confirmed by crystal structure analysis—and later in 45 sense or antisense RNAs (reviewed in Reference ). E. colense RNA . Later, ense RNA . Only inn of SR4 . In bsrEraction  in four or five GC base-pairs [bsrG, in vitro translation products could be only obtained after mutations were introduced to open the double-stranded region in the mRNA [txpA and yonT, whose RBSs display more than 11 bp complementarity to the anti-SD in 16S rRNA. Such perfect RBSs are predicted to allow efficient recruitment, but only slow release of ribosomes , alternse-pairs ,13,15, weference ). B. subeference ).trans-encoded base-pairing small regulatory RNAs (sRNAs) or to promote the interaction with their target mRNAs [E. coli TA system ralR/RalA with a short (16 bp) stretch of complementarity between ralR mRNA and its antitoxin RalA. Here, indeed, a requirement of Hfq was discovered [E. coli antitoxins nor the B. subtilis antitoxins like SR4, SR5, or SR6 [B. subtilis that the widespread RNA chaperone CsrA whose recognition sequence comprises a 5′GGA motif in a single-stranded or loop region can also restructure an mRNA to facilitate the interaction with its regulatory sRNA [Hfq is an abundant RNA chaperone that was found—among other things—to either stabilize et mRNAs ,28. The scovered . Neither, or SR6 ,12,14, wory sRNA . Therefocis-encoded sRNAs, RNase III, the double-strand specific RNase present in all bacteria, cleaves the antisense/sense RNA duplex to initiate target RNA degradation resulting in inhibition of gene expression. This was discovered 30 years ago for antisense RNAs encoded on plasmids, transposons, and bacteriophages (reviewed in Reference [hok toxin mRNA/Sok antitoxin duplex from E. coli plasmid R1 [txpA/RatA [bsrG/SR4 [bsrE/SR5 [E. coli OrzO [B. subtilis SR4 [E. coli antitoxins Sok (reviewed in Reference [In the majority of eference ). In typasmid R1 , and >20xpA/RatA , bsrG/SRbsrG/SR4 , and bsrbsrE/SR5  duplexesbsrE/SR5 ), and E.oli OrzO . By contilis SR4 , as welleference ), SymR (eference ), IstR1 eference ), and Sieference ). ApparetxpA/RatA and bsrG/SR4 from B. subtilis corroborates this hypothesis. SR4 is a bifunctional antitoxin that not only facilitates bsrG RNA degradation, but also impedes bsrG translation [bsrG ORF were observed in an rnc knockout strain [bsrG mRNA 14 nt downstream of the 3′ end of complementarity with SR4 at position 185. By contrast, RNase III cleavage of txpA RNA in its duplex with RatA is essential to prevent toxin expression [txpA mRNA conformation that further inhibits translation. In the absence of RNase III, txpA RNA levels were six-fold enhanced, whereas those of RatA were not affected. RNase III is also essential for yonT RNA degradation, which shows a seven-fold higher stability in the absence of RNase III [bsrE/SR5, two- to three-fold higher amounts of bsrE RNA were found in the absence of RNase III in the logarithmic and stationary phase, respectively [bsrE stop codon [bsrE overexpression inhibits cell growth and causes cell lysis, RNase III was not found to be essential [A comparison between nslation . Neithert strain  demonstrpression . RatA onpression , it doesNase III . For bsrectively . Three dop codon . Howeverssential . B. subtilis type I TA systems. SR5 [rnc mutant strain [yonT mRNA in complex medium and minimal medium without glucose in all growth phases. Only in minimal medium with glucose, from late log phase toward stationary growth phase, were 3:1 to 1:1 SR6/yonT RNA ratios determined [bsrG RNA only in complex medium from the transition phase until late stationary phase [The role of RNase III for antitoxin stability differs between the various ems. SR5  and SR6 ems. SR5  behave lt strain . Apparent strain  and SR5 t strain  are in etermined . By contry phase . B. subtilis bsrH/as-bsrH, the levels of bsrH toxin mRNA are neither affected by RNase III nor by RNase Y, but by RNase J1, an endonuclease and 5′–3′ exoribonuclease. Degradation was proposed to be rather exoribonucleolytic [bsrE mRNA [bsrG RNA and SR4 was only 1.5-fold [Surprisingly, in the case of leolytic . RNase JsrE mRNA , whereas1.5-fold ,35.bsrG RNA was found. Whereas bsrG RNA was two-fold less stable in the absence of RNase Y, SR4 showed a two-fold higher stability [bsrE RNA was similar to that on bsrG RNA, while RNase Y did not affect SR5 [bsrG and bsrE RNA in the absence of RNase Y might be due to indirect effects. RNase Y also initiates degradation of RatA. Afterwards, the 5′ part of RatA alone is degraded by 3′–5′ exoribonuclease PnpA and the 3′ part by 5′–3′ exoribonuclase J1 , an RNase [The vast majority of the currently known type I toxins are small hydrophobic peptides of less than 60 amino acids (aa) that have a similar predicted secondary structure containing an α-helical transmembrane domain see , which ieference ). Exceptan RNase , and Ralan RNase , which aB. subtilis type I toxins TxpA, BsrG, BsrE, and YonT reviewed here are smaller than 60 aa and contain transmembrane domains .B. subtilis bsrG, bsrE, and yonT RNAs [bsrG and bsrE RNA decreases after heat shock at 48 °C or 55 °C, whereas the amount of yonT RNA increases. Both antitoxins SR4 and SR5 are not affected by heat shock, but the amount of SR6 decreases concomitantly with the increase of its toxin mRNA [bsrG RNA more accessible to single-strand-specific RNases J1 and Y [bsrE RNA has a similar secondary structure to bsrG RNA  and manganese stress were observed (four- to five-fold decreased amounts). Lower effects were found for yonT RNA and SR6 upon salt stress, iron limitation, and oxidative stress [sigB-dependent, suggesting a transcriptional regulation. Most probably, bsrH mRNA is also multistress-responsive, as riboprobes used for the detection of bsrE mRNA also allowed bsrH mRNA detection in Northern blots .Both  systems ,14. The  RNase Y . In addipression . For yonB. subtilis SR4 [No difference between antitoxin expression in rich or minimal medium was observed for ilis SR4  and SR5 ilis SR4 . By contilis SR4 .hok/Sok in E. coli  or fst/faecalis , is largbsrG/SR4, a 6–8-fold excess of toxin mRNA over antitoxin in transition and early stationary growth phase in complex medium was calculated, suggesting a function in growth adaptation [bsrG toxin mRNA is temperature-sensitive and could play a role during temperature changes [bsrE toxin mRNA is in excess over its antitoxin SR5 [yonT RNA and SR6, strong effects of vancomycin  and manganese stress were observed. However, no relative ratios of SR6 and yonT RNA were calculated under stress conditions. In a transcriptomics study, the amounts of txpA and bsrH mRNA were found to increase after exhaustion of glucose [Some chromosomally encoded type I TA systems are only induced under certain metabolic or environmental stress conditions. Therefore, they could play a role in metabolic or stress adaptation by, e.g., killing the cells when nutritional sources or oxygen are exhausted. For aptation . Further changes . By contoxin SR5 , while,  glucose , suggestBacillus subtilis strains display a variety of aa exchanges in BsrG, only maintaining the transmembrane domain. Thus, identification of homologous type I toxins is difficult.It is difficult to analyze sequence conservation among type I toxins, because, in these systems, not the toxin protein, but the toxin mRNA and the antitoxin interact with each other. Mutations in the toxin mRNA will not affect base-pairing with the cognate antitoxin if the latter is transcribed from the complementary DNA strand. Such type I TA systems should be more prone to mutations than the type II TA systems. Conservation analysis  search) of BsrG supported this hypothesis. Even closely related bsrG/SR4 loci in other Bacillus species, but some of them encoding BsrH/E instead of BsrG. This might be due to an exchange of the BsrG and the BsrE/H group toxin genes by mobile genetic elements via horizontal gene transfer. Moreover, the striking similarity of the secondary structures of bsrG mRNA and bsrE mRNA, as well as SR4 and SR5, implies a common ancestor of these two TA systems, although the toxicity and perhaps the mechanism of toxin action are different. BsrG encoded on prophage SPβ belongs to the same family as TxpA encoded on the skin element, and both cause lysis on agar plates in the absence of the antitoxin. By contrast, BsrE encoded by P6 is grouped with BsrH encoded on the skin element in one family, and only overexpression of bsrE—and most probably bsrH—is toxic.Interestingly, BLAST search also identified conserved txpA/bsrG-like TA systems can only be found in a few Bacillus species, including B. subtilis, B. halotolerans, B. amyloliquefaciens, and also a Paenibacillus species. By contrast, bsrE/bsrH-like systems could be identified in a variety of Bacillus, Lactobacillus, Halobacillus, and Enterococcus species. Moreover, BsrE is also encoded on B. subtilis plasmid pBS72, but its effect on post-segregational killing is so far unknown. The genes encoding toxins YonT and YoyJ are also widespread in various Bacillus species, and sometimes truncated variants can be found, albeit mainly with conserved transmembrane (TM) domain regions.The B. subtilis type I TA systems by mobile genetic elements such as phages or plasmids, for which they might have originally been addiction modules.Together, these findings provide evidence for an interspecies distribution of Bacillus subtilis was SpoIISA (YkaC)/SpoIISB/SpoIISC in 2001 [E. coli, although a high similarity to E. coli plasmid-encoded PemK/PemI was already shown in 1998 [The first type II TA system discovered in  in 2001 . In 2005 in 1998 . In 2012 in 1998 . Except Bacillus subtilis encodes a variety of further toxins or antimicrobial substances and corresponding immunity genes, e.g., SunA/SunI [unA/SunI , WapA/WaunA/SunI , SkfA, aunA/SunI . HoweverR38Q also inactivates the otherwise toxic SpoIISA, suggesting an oligomeric state for active SpoIISA [spoIISA leads to disintegration of the cytoplasmic membrane, as well as detachment from and disruption of the cell wall [The toxin SpoIISA is a 248-aa protein with three N-terminal transmembrane (TM) domains (aa 1–87) and a cytoplasmic C-terminal domain (aa 88–248) ,53. Trun SpoIISA . Expressell wall .spoIISA in E. coli showed a similar phenotype, suggesting a function directly in the plasma membrane and independent of further interaction partners [Expression of partners . It is apartners , supportpartners ,54. E. coli homolog MazF [ndoA in the absence of ndoAI inhibits growth of both E. coli and B. subtilis. Toxin action is rather bacteriostatic than bactericidal [The toxin NdoA is a 116-aa dimeric endoribonuclease that folds like the log MazF ,58,59,60log MazF . NdoA cllog MazF . Overexpericidal ,61. E. coli [B. subtilis, YqcG displays exclusively DNase activity [E. coli [B. subtilis, YqcG is bactericidal and rapidly kills the vast majority of cells. The toxins of the YqcG group are similar to each other. They consist of 531–669 aa with a well-conserved N-terminal and a more variable C-terminal domain (last 120–150 residues), both similar to contact-dependent growth inhibition (CDI) toxins in Gram-negative bacteria . The C-tE. coli , in B. sactivity . The N-tactivity . The C-t[E. coli . In B. s2·SpoIISB2 heterotetramer [SpoIISA is inactivated by two similar antitoxins: SpoIISB 56 aa) and SpoIISC (45 aa) ,56. SpoI aa and Stetramer  2–(NdoA)2 complex [E. coli antitoxin MazE is not able to inactivate NdoA [NdoA is neutralized by binding of its antitoxin NdoAI. Two NdoAI proteins form a homodimer via their N-terminal RHH motifs. Each subunit displays an extended C-terminal helix that binds an NdoA dimer and blocks the RNA binding groove, resulting in an inactive and stable hexameric (NdoA) complex   is counteracted by their cognate antitoxins . AlthougToxin action has to be tightly controlled by the cell to avoid undesired damage. Since toxin action is only aspired under very specific circumstances, most of the time, the antitoxin has to bind and neutralize the toxin. How this is achieved is detailed below.spoIISABC system, three promoters could be identified .In the entified . The fir spoIISB . Both PArulation . At leasactor σK . This exrulation . The thiormation ,56, likespoIISABC system remains unknown. Although toxin expression seems to be highest during sporulation, antitoxin expression is also elevated at the same time. One study used a combined ndoA/spoIISA deletion strain and observed 25% less programmed cell death and an altered morphology in biofilms [The physiological role of the biofilms . AlthougndoA/ndoAI system is transcribed as a bicistronic ndoAI-ndoA mRNA under control of one or two ndoAI promoter(s) which are yet to be further characterized. As the gene alr located upstream of ndoAI lacks a detectable terminator, read-through transcripts from the alr promoter cannot be excluded [E. coli mazE/mazF are autoregulated [ndoAI/ndoA in B. subtilis, although a palindromic sequence at the ndoAI promoter suggests autoregulation [B. subtilis crude extracts [The excluded   irradiation [B. subtilis cells as a quorum-sensing molecule, activates NdoA by blocking antitoxin, but not RNA binding [ndoA deficiency suppresses sporulation [E. coli MazF generates leaderless mRNAs, as well as ribosomes with cleaved 16S rRNAs, thus establishing a modified translational machinery to expedite expression of specific genes [B. subtilis 16S rRNA lacks a recognition site for NdoA, rendering this mechanism unlikely.The physiological role of eference ). Since eference . In the adiation , suggest binding , furtherrulation . E. coliic genes . HoweveryqcG homologs is highly similar and identical to their cognate antitoxin genes. Expression of both toxins and antitoxins is elevated during sporulation, under starvation, and at high cell density [yqcG transcription during sporulation was shown to be dependent on Spo0A via the SinI/SinR pathway and further via DegU [yobL/yobK, yeeF/yezG, and yqcG/yqcF, toxin and antitoxin genes are transcribed bicistronically. By contrast, yxiD/yxxD, yokI/yokJ, and ywqJ/ywqK are located in in larger operons and are, therefore, translated from multicistronic transcripts [Transcription of all six  density . Additiovia DegU ,72. Consnscripts .Most likely, these six highly similar systems fulfil comparable functions, although their C-terminal toxin domains differ. The shared putative N-terminal secretion signal implies a function outside of the cell. YqcG was shown to eliminate defective cells and to reduce the number of living cells in starved cultures and biofilms . This coLike type I TA systems, type II TA systems are thought to originate from plasmids, phages, or other mobile genetic elements where they function in post-segregational killing. However, type II TA systems are also found in well-conserved regions of the core genome, where they were not eliminated during evolution and, most likely, adopted by the cells for own regulatory fine-tuning.spoIISABC system is part of the core genome in many Bacillus species and also found in some non-Bacillus species, likely adopted by horizontal gene transfer [Bacillus subtilis and close relatives, the gene locus is adjacent to the PBSX prophage, but does not seem to be involved in phage maintenance. A few other homologs were also found on plasmids [E. coli, the system was not able to maintain plasmid stability by post-segregational killing [spoIISABC systems display a high dissimilarity with only four invariant and eight conserved SpoIISA residues [The transfer . Howevertransfer . In Baciplasmids . However killing . All knoresidues . The secresidues .ndoAI/ndoA (mazE/mazF) system is widely distributed among Gram-positive and Gram-negative bacteria. However, B. subtilis ndoA shares much more similarity to mazF homologs of other Gram-positive bacteria than to those of Gram-negative bacteria [E. coli MazF cleaves 5′–ACA–3′ sequences [B. subtilis NdoA needs a more specific and less abundant 5′–UACAU–3′ sequence [E. coli, and NdoA from B. subtilis are well conserved [The bacteria . In addiequences , B. subtsequence . Despiteonserved , suggestBacillus species. In addition, similarities to the widespread CDI genes from Gram-negative bacteria are apparent. Both toxin domains, the well-conserved putative N-terminal translocation signal and the toxic C-terminal domain harboring nuclease activity, are similar . Howevength CDI , but theB. subtilis and investigated to some degree. Although valuable information about toxin action could be obtained for BsrG, the targets of all type I toxins are still unknown. On the one hand, they might be encoded by essential genes as seems to be the case for BsrG. On the other hand, it is challenging to purify sufficient amounts of these membrane-bound toxins for biochemical studies, even with tightly controlled expression systems, as only a few molecules suffice to kill the host cells. Furthermore, experiments are needed to elucidate whether the additional nine potential systems, whose putative toxins were grouped into the YhzE family [To date, only type I and type II TA systems were discovered in E family , are indE. coli MazF is known to act as interferase to degrade specific mRNAs, its physiological role is still unknown. YqcG and some of its homologs act as DNases and RNases, but their sequence specificity has to be analyzed and their location confirmed. Although the crystal structures of SpoIISA and NdoA were solved, information regarding regulatory and physiological function is sparse and hard to obtain. Currently, we cannot exclude that B. subtilis and other Bacillus species might also encode type III, IV, or V TA systems. Moreover, more type I and type II systems or even combined systems could exist. More bioinformatics and subsequent experimental work is required to answer these questions.For the type II systems, the picture is different. SpoIISA seems to act like a phage holin, although more data, e.g., on its effects on the membrane potential, are needed to confirm this assumption. Whereas NdoA that resembles


ABCB6 gene have been linked to a variety of pathophysiological conditions ranging from transfusion incompatibility to pigmentation defects, its precise cellular localization and function is not understood. In particular, the intracellular localization of ABCB6 has been a matter of debate, with conflicting reports suggesting mitochondrial or endolysosomal expression. ABCB6 shows significant sequence identity to HMT-1  proteins, whose evolutionarily conserved role is to confer tolerance to heavy metals through the intracellular sequestration of metal complexes. Here, we show that the cadmium-sensitive phenotype of Schizosaccharomyces pombe and Caenorhabditis elegans strains defective for HMT-1 is rescued by the human ABCB6 protein. Overexpression of ABCB6 conferred tolerance to cadmium and As(III) (As2O3), but not to As(V) (Na2HAsO4), Sb(V), Hg(II), or Zn(II). Inactivating mutations of ABCB6 abolished vacuolar sequestration of cadmium, effectively suppressing the cadmium tolerance phenotype. Modulation of ABCB6 expression levels in human glioblastoma cells resulted in a concomitant change in cadmium sensitivity. Our findings reveal ABCB6 as a functional homologue of the HMT-1 proteins, linking endolysosomal ABCB6 to the highly conserved mechanism of intracellular cadmium detoxification.ABCB6 belongs to the family of ATP-binding cassette (ABC) transporters, which transport various molecules across extra- and intra-cellular membranes, bearing significant impact on human disease and pharmacology. Although mutations in the The online version of this article (10.1007/s00018-019-03105-5) contains supplementary material, which is available to authorized users. ATP-binding cassette (ABC) transporters constitute one of the largest protein families in prokaryotes and eukaryotes. ABC transporters are integral transmembrane proteins that function as active transporters, channels or regulators. Active ABC transporters harness the energy of ATP to move a diverse array of substrates in or out of cells, or into cellular vesicles. There are 48 human ABC transporters, many of which are linked to severe inherited diseases, such as cystic fibrosis, or X-linked adrenoleukodystrophy . WhereasABCB6 is widely expressed in many tissues, especially in the heart, liver, skeletal muscles , the redSchizosaccharomyces pombe), nematode (Caenorhabditis elegans) and the fruit fly (Drosophila melanogaster) fulfill a conserved role in conferring heavy metal resistance  in whole blood stored at, or below room temperature (RT), without additional hematological abnormalities . XW1957: qxIs110 (pges-1::mCHERRY::RAB-5); XW1962: qxIs111 (pges-1::mCHERRY::RAB-7); XW9119: qxIs213 (pges-1::mCHERRY::RAB-10) strains were a kind gift from Dr. Xiaochen Wang .d source . The fol2. Cells were periodically tested for mycoplasma contamination with the MycoAlert mycoplasma detection Kit .The SNB-19 glioblastoma cell line was obtained from DSMZ (Germany), HeLa cells were from ATCC. Cells were grown in high glucose DMEM (Gibco 521000-47) supplemented with 10% FBS, 2 mmol/L glutamine, and 100 units/mL penicillin and streptomycin (Life Technologies) at 37 °C in 5% COE. coli strain Top10  grown at 37 °C in liquid Luria–Bertani (LB) medium supplemented with appropriate antibiotics. Hemagglutinin-tagged S. pombe hmt-1 (Z14055) cDNA was synthesized by GenScript . Site-specific mutation was engineered using the QuikChange site-directed mutagenesis kit ; the mutation was confirmed by sequencing. The cDNAs encoding hmt-1 and ABCB6 variants were subcloned into the pREP1 fission yeast expression vector; pEGFP-N1  was used for the N-terminal EGFP-tagging of the transporters.Plasmid constructs were amplified in hmt-1::ABCB6::gfp and phmt-1::ABCB6::mCherry reporter, codon-optimized ABCB6 cDNA was synthesized by GenScritpt . After restriction digestion with SphI and XmaI, ABCB6 was subcloned in frame with the gfp sequence of the pPD95.75 vector. A 5′ regulatory region of hmt-1 (2.8 kb immediately upstream of the start of the hmt-1 coding sequence) was PCR-amplified using primer pairs designed to introduce SphI and HpaI restriction enzyme recognition sites at the 5′ and 3′ ends, respectively. The PCR-amplified hmt-1 promoter was subcloned into the pPD95.75-ABCB6-EGFP vector. In the case of phmt-1::ABCB6::mCherry, the GFP reporter sequence of pPD95.75-ABCB6-EGFP was changed to the sequence encoding mCherry.To generate the pA600nm (OD600) of 1, cells were grown in EMM containing minimal glucose (5 g/L). Cells were transformed using the standard lithium acetate procedure  co-transformation marker plasmid DNA were bombarded onto unc-119(ed3) mutant adult hermaphrodites. Non-Unc transgenic animals exhibiting GFP or mCherry-mediated fluorescence were selected, and the stable integrated strains TTV634: eluIs310 [phmt-1::ABCB6::gfp + unc-119(+)]; unc-119(ed3) and TTV677: eluEx383[phmt-1::ABCB6::mCherry + unc-119(+)]; unc-119(ed3) carrying the array extrachromosomally were selected for further analysis. TTV 634 was crossed with hmt-1(gk161) to generate TTV 635: hmt-1(gk161); eluIs310 [phmt-1::ABCB6::gfp + unc-119(+)].Transgenic r et al. . BrieflyABCB6 knock-down and overexpression were achieved using a self-inactivating lentiviral system, as described previously in ; qxIs110(Pges-1mCHERRY::RAB-5), TTV702 unc-119(ed3)III; eluIs310[phmt-1::ABCB6::gfp + unc-119(+)]; qxIs111(Pges-1mCHERRY::RAB-7), TTV703 unc-119(ed3)III; eluIs310[phmt-1::ABCB6::gfp + unc-119(+)]; qxIs213(Pges-1mCHERRY::RAB-10), TTV705 unc-119(ed3)III; gfIs1; qxIs110(Pges-1mCHERRY::RAB-5), TTV706 unc-119(ed3)III; gfIs1; qxIs111(Pges-1mCHERRY::RAB-7), TTV707 unc-119(ed3)III; gfIs1; qxIs213(Pges-1mCHERRY::RAB-10).Transgenic strains were grown in normal growth conditions at 20 °C. To test the subcellular co-localization of ABCB6 and CeHMT-1, the ribed in . To dete markers , resulti2 fragment conjugated to Alexa Fluor 647 (#4410) and secondary goat anti-rabbit IgG (H + L) F(ab′)2 fragment conjugated to Alexa Fluor 647 (#4414) were from Cell Signaling Technology. Secondary goat polyclonal antibody to human IgG conjugated to DyLight 488 (ab96907) was purchased from Abcam. Hoechst 33342 (R37605) nuclear counterstain was from Thermo Fisher Scientific. The OSK43 antibody was a kind gift from Dr. Yoshihiko Tani . SNB-19 cells expressing ABCB6 variants were plated in an Eppendorf 8-well imaging coverglass (#0030742036). Hoechst 33342 was applied to the cells for 20 min prior to fixation; subsequently, cells were rinsed in PBS and fixed for 30 min in 4% Paraformaldehyde/PBS at RT. Fixed cells were quenched for 10 min in PBS/100 mM glycine (quenching buffer), washed with PBS and blocked and permeabilized in PBS containing 0.2 mg/mL BSA/0.1% Triton X-100/10% Normal Goat Serum (blocking buffer). Primary antibody was diluted in PBS containing 0.2 mg/mL BSA/0.1% Triton X-100/3% normal goat serum . Cells were incubated with the primary antibody overnight at 4 °C in a humified chamber, washed five times in IB, and incubated with the corresponding secondary anti-human, anti-rabbit and anti-mouse antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 647 diluted in IB for 90 min at RT. Samples were washed five times with PBS and subsequently imaged. Confocal images were obtained using a LSM 700 confocal laser scanning microscope  equipped with a Plan-Apochromat 63×/1.4 NA Oil DIC M27 objective. Images were acquired in three channels (blue (Hoechst33342), green (Alexa Fluor 488), red (Alexa Fluor 647)), blue emitting Hoechst 33342 was excited using the 405 nm laser line, green emitting Alexa Fluor 488 was excited using the 488 nm laser line and infrared emitting Alexa Fluor 647 was excited using the 633 nm laser line. Noise reduction and deconvolution of the images were performed with Huygens Essential (Scientific Volume Imaging B.V.).Monoclonal antibodies, dyes and their sources were as follows: Rabbit monoclonal Anti-AIF [D39D2] antibody (#5318) to apoptosis inducing factor, rabbit monoclonal Anti-EEA1 [C45B10] antibody (#3288) to early endosome antigen 1, rabbit monoclonal Anti-LAMP1 [D2D11] antibody (#9091) to lysosome-associated membrane protein 1, secondary goat anti-mouse IgG (H + L) F(ab′)Supplementary material 1 (DOCX 391 kb)Below is the link to the electronic supplementary material.


Coral reefs worldwide are increasingly damaged by anthropogenic stressors, necessitating novel approaches for their management. Maintaining healthy fish communities counteracts reef degradation, but degraded reefs smell and sound less attractive to settlement-stage fishes than their healthy states. Here, using a six-week field experiment, we demonstrate that playback of healthy reef sound can increase fish settlement and retention to degraded habitat. We compare fish community development on acoustically enriched coral-rubble patch reefs with acoustically unmanipulated controls. Acoustic enrichment enhances fish community development across all major trophic guilds, with a doubling in overall abundance and 50% greater species richness. If combined with active habitat restoration and effective conservation measures, rebuilding fish communities in this manner might accelerate ecosystem recovery at multiple spatial and temporal scales. Acoustic enrichment shows promise as a novel tool for the active management of degraded coral reefs. Healthy coral reefs have an acoustic signature known to be attractive to coral and fish larvae during settlement. Here the authors use playback experiments in the field to show that healthy reef sounds can increase recruitment of juvenile fishes to degraded coral reef habitat, suggesting that acoustic playback could be used as a reef management strategy. Taking active steps to maintain healthy fish communities will be vital in reversing reef degradation; fishes perform a diverse suite of important functional processes7, meaning that damaged reefs have a higher chance of recovery if they have healthy fish populations9. Reef fish populations are sustained by recruitment, whereby young fish that spend their larval stage in the open ocean use a range of sensory cues to detect, orient toward, and settle to reef habitat11. However, degraded reefs smell and sound less attractive to juvenile fishes13, and receive lower levels of fish settlement than healthy systems15. Artificially reversing degradation-associated sensory changes might restore habitat attractiveness, promote the settlement and retention of functionally important fish species, and enhance local-scale recovery processes. Acoustic cues are particularly amenable to artificial restoration, due to their use by a wide range of settlement-stage fishes17 and their ease of manipulation in field conditions using underwater loudspeakers17.Climate change and local anthropogenic stressors are causing unprecedented damage to coral reefs globallyHere, we investigate acoustic enrichment as a novel management tool for aiding the mitigating and reversal of coral reef degradation. We use loudspeakers to broadcast healthy soundscapes on experimental coral-rubble patch reefs for 40 days during a natural recruitment season (November–December 2017) on Australia’s northern Great Barrier Reef. We compare the developing fish communities on these acoustically enriched reefs with those on two categories of acoustically unmanipulated control reefs (with and without dummy loudspeaker rigs). We find that acoustic enrichment enhances fish community development within an important reef fish family, across a range of specific trophic guilds and at the level of the whole community. Rebuilding fish communities in this manner shows promise as a novel tool that might complement existing techniques for the active management of degraded coral reefs.18) and individuals can be visually surveyed accurately with minimal disturbance to the developing fish community. Compared to both no-loudspeaker reefs and dummy-loudspeaker reefs, acoustically enriched reefs attracted damselfishes at a faster rate in the early stages of the experiment and maintained higher abundance throughout the 40 days  were repeatedly surveyed across 6 weeks; this family was chosen because they are non-cryptic, highly abundant  was occasionally observed during surveys in the later stages of the experiment (T.A.C.G. pers. obs.); this is consistent with other studies that document high natural mortality rates of juvenile coral reef fishes20. An absence of overall declines in abundance during this phase of the experiment therefore suggests that ongoing recruitment was compensating for density-dependent predation of juvenile fishes.The asymptotic trajectories of damselfish abundance on reefs in the second half of the experiment Fig.  likely rχ2 = 7.499–43.473, df = 2, p = <0.001–0.024  in four of the five trophic guilds (linear mixed models (LMMs) and GLMMs, loudspeaker treatment: χ2 = 21.107, df = 2, p < 0.001; Fig. χ2 = 12.848, df = 2, p = 0.002; Fig. χ2 = 5.990, df = 2, p = 0.050; Fig. Consistent differences across multiple trophic guilds resulted in a positive effect of acoustic enrichment at the community level. Acoustically enriched reefs had twice the total abundance of juvenile fishes of dummy-loudspeaker and no-loudspeaker reefs, which were not significantly different from each other  created temporally stable population increases in the most abundant taxonomic group of reef fishes , leading to widespread ecosystem change33.This study was carried out during October–December 2017 in the lagoon to the south-west of Lizard Island Research Station , and particle-acceleration recordings were taken using a triaxial accelerometer  connected to a digital 8-track recorder . Recordings were all taken at a sampling frequency of 48 kHz, and analysed using the paPAM34 (particle acceleration) and PAMGuide35 (sound pressure) packages on MATLAB, across a frequency range of 10–4000 Hz as the likely hearing range of many juvenile fishes37, with a Hamming window, a 50% overlap and an FFT size of 2048.Prior to establishing experimental sites, the potential detection distance of reef-sound playback by juvenile fishes was determined using recordings at the study site. Recordings were taken from the deepest experimental reef (4.5 m mid-tide depth), to provide a conservative estimate of acoustic isolation; lower frequency (longer-wavelength) sounds attenuate relatively quickly in shallow watern = 20, SPL: χ2 = 2.785, df = 1, p = 0.103; PAL: χ2 = 0.447, df = 1, p = 0.508). Thus, recordings of reef-sound playback taken at a distance of 50 m were unable to distinguish the playback signal over the natural ambient conditions at the study site. Based on this finding, all experimental patch reefs were placed a minimum of 100 m from each other . Reefs consisted of 70 l of dead coral rubble, collected from a single degraded reef near the study site  from the nearest natural reef in 2–4.5 m water depth . There was no significant difference in depth between the three treatment groups , or a real loudspeaker system playing back healthy reef sound overnight (acoustic-enrichment treatment). Loudspeaker treatment was allocated pseudorandomly to reefs, avoiding allocation of the same treatment to reefs that were spatially adjacent. Acoustically enriched reefs were fitted with an underwater loudspeaker  powered by an amplifier  and a 12 V 12 Ah sealed lead-acid battery, connected to an MP3 player  that was powered by an external battery pack . Loudspeakers were fixed to the centre of reefs and oriented upwards, to ensure even distribution of sound in all directions laterally. Amplification systems were housed in a barrel floating directly above reefs, attached by a rope to a breeze block. Dummy-loudspeaker reefs consisted of a concrete tile of the same size, shape and colour as the loudspeakers, fixed to the reef in the same manner as the loudspeakers, attached by a rope to a floating buoy of the same dimensions as the barrel used in loudspeaker systems.33. Five different recordings were used to reduce pseudoreplication, with recordings being allocated to acoustically enriched reefs at random . Recordings used in playback were full-night recordings, taken using an omnidirectional hydrophone  connected to a digital recorder . The hydrophone was freely suspended 1 m above the seabed in water depth of 3.5 m, from a rope-anchored barrel that contained the recorder.The acoustic-enrichment treatment consisted of playback of recordings taken at a healthy reef in the middle of the study site in November 2015; this was before the study site experienced two severe mass bleaching events in 2016 and 2017 that caused extensive coral mortality and widespread ecosystem change throughout the region39; playback therefore started 0.5–1.5 h before sunset and stopped 2–5 h after sunrise the following morning. Playback was also matched by time of night to within 45 min of the original recording, such that playback tracks were playing approximately ‘real-time’ throughout the night. For an illustrative waveform and spectrogram of the reef-sound playback, see Supplementary Fig. Reef-sound playback was conducted overnight. Fish settlement is predominantly a nocturnal behaviour18) facilitates adequate statistical power to test for differences in community development, and their non-cryptic nature allows accurate surveying with minimal disturbance. Visual surveys by a SCUBA diver (T.A.C.G.) were used to monitor communities of juvenile damselfishes . The observer and dive buddy remained at least 1 m from the reef during surveys, in order to minimise disturbance to the community. Each reef was surveyed 10 times throughout a 40-day period that started immediately following construction, with 3–9 days between consecutive surveys. The start of the survey period on each reef was staggered across a total duration of 10 days , to allow a single observer (T.A.C.G.) to complete surveys at the same experimental time point. Surveys took up to 1 h per reef; staggering was therefore necessary to allow for surveying of 33 patch reefs at the same experimental time points. Deployment order was counterbalanced such that the same number of reefs within each treatment were constructed and surveyed on each day.The family Pomacentridae (damselfish) was surveyed regularly throughout reef deployment. The high abundance of damselfishes on coral reefs  checked each piece of rubble thoroughly, using dilute clove oil and a hand net to capture all juvenile fishes on the reef. The dive buddy kept a continuous watch for ‘stray’ fishes that attempted to escape across the sand flat or burrow into the sand during the survey process. All reefs were double-checked for missed fishes after surveys were completed; to our knowledge, no fishes were missed from surveys. All fishes were identified to species, except in cases where uncertainty meant that identification was only possible to family or sub-family level. In these cases, fishes that looked similar were assigned as the same species (e.g. ‘Unknown goby 1'); this was the case for 4% of species, whose members together constituted 9% of the total abundance. Adult fishes were excluded from analyses, as their larger home ranges mean that they do not exhibit fixed associations with reef habitat to the same extent as juvenilesVisual surveys throughout the 40-day period were used to create accumulation curves for juvenile damselfishes (family Pomacentridae). A generalised additive mixed model (GAMM) with a negative binomial distribution included time (days since patch reef creation) as a smooth term, loudspeaker treatment  and patch-reef ID as parametric coefficients, and playback-track ID as a random term. A first-order autoregressive structure was used to account for temporal autocorrelation, and visual examination of diagnostic plots and comparison of model outputs to raw data were used to confirm goodness-of-fit.41). Trophic classification was based on published literature45 and FishBase46; for full details, see Supplementary Table p ≤ 0.05).Comprehensive surveys at the end of the 40-day period were analysed using LMMs and Poisson-distributed GLMMs. Separate models were run for abundance of juvenile damselfishes (family Pomacentridae), herbivores, omnivores, planktivores, invertivores and piscivores, as well as total community abundance, species richness and effective Shannon diversity , James Cook University , and the University of Exeter (2013/247).Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Myoparr is a promoter-associated long non-coding RNA (lncRNA) that is expressed from the promoter region of myogenin gene. Myoparr is essential for the proper differentiation of skeletal muscle cells; it accomplishes this by activating the expression of myogenin and myogenic microRNAs (miRNAs). In this study, we provide the RNA-seq data describing the changes in gene expression induced by knockdown of Myoparr, myogenin, and two Myoparr-binding proteins (Ddx17 and hnRNPK) during skeletal muscle differentiation in C2C12 cells. Raw data files were deposited in Sequence Read Archive in DNA Data Bank of Japan (DDBJ) under the accession number DRA005527. These data are related to the research article “Myogenin promoter-associated lncRNA Myoparr is essential for myogenic differentiation” Hitachi et al., 2019. Myoparr, myogenin, Ddx17, or hnRNPK, respectively.The data were deposited in DDBJ Sequence Read Archive  and hnRNPK  were the tests. The target sequence of Stealth RNAi for Myoparr is as follows; 5′- GATGGACCCTGTCTGATGCTCTTAA -3'. The target sequence of Stealth RNAi for Ddx17 was previously described The mouse myoblast cell line, C2C12, was grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C and 5% COThe intact Poly(A)+ RNA was isolated from 1 μg of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module  and used for RNA-seq library construction using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's protocol.2.2https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Trimmed reads were mapped to the mouse reference genome (mm10) using the Hisat2 ver. 2.0.5 software with the default parameters The libraries were sequenced using Illumina HiSeq 1500 at Fujita Health University. We sequenced two biological replicates for each sample (100 bp single-end reads). The bcl2fastq 1.8.4 software was used for base calling. The raw data for each sample were deposited in DDBJ Sequence Read Archive . Quality


Dendrobium catenatum, was designed to generate 39 mature-leaf-tissue RNA-seq sequencing datasets with over two billion reads. These datasets were validated by a series of quality assessments including RNA sample quality, RNA-seq read quality, and global gene expression profiling. We believe that these comprehensive transcriptomic resources will allow a better understanding of the drought-resistant mechanisms of orchid epiphytes.Orchid epiphytes, a group containing at least 18,000 species, thrive in habitats that often undergo periodic drought stress. However, few global gene expression profiling datasets have been published for studies addressing the drought-resistant mechanism of this special population. In this study, an experiment involving the effect of continuous drought treatments on an epiphytic orchid,  Most studies concerning drought stress mechanisms have been performed in Arabidopsis thaliana and other drought-intolerant C3 plants2. Studying a highly resistant plant that has been shaped by natural selection is the most direct and effective way to extract crucial genes and determine the main metabolic pathways of the drought stress procedure.In response to prolonged water deficit stress, plants have evolved coping mechanisms to increase their drought tolerance through physical adaptations, molecular regulations, and environmentally suitable metabolic pathways5. Due to the poor moisture supply in these habitats6, these plants usually suffer periodic water shortage7. While adapting to harsh habitats, some orchid species have evolved succulent storage-organs, such as pseudobulbs9, thick leaves10, and crassulacean acid metabolism (CAM)11, a photosynthetic pathway with high water-use efficiency12. Morphological and anatomical studies show that orchid plants possess desirable qualities for mitigating drought stress14. By measuring physiological indexes and secondary metabolites of Dendrobium moniliforme15, Wu et al. found that increasing antioxidant enzyme activities and osmolytes play an important role in protecting plants under drought stress. Although several physiological traits might provide clues for the mechanism of drought resistance, there is no large data set that allows holistic understanding. Unfortunately, few comprehensive transcriptomic profiling studies that address drought resistance have been published.In the wild, most epiphytic orchids, a prosperous group containing over 18,000 species, take root on the surface of tree bark or rocks17, Phalaenopsis equestris and Dendrobium catenatum, the latter possesses more Heat-shock protein 70 family members and R genes17, which suggests that D. catenatum can tolerate a much wider variety of environments and has superior qualities for adverse resistance. A previous study demonstrates that D. catenatum uses the facultative CAM pathway as a drought-enduring process11. Hence, this species can be considered as drought-resistant material useful for elucidating mechanisms of mitigating drought stress in epiphytic orchids. Previous studies show that the circadian clock modifies responsiveness to environmental input and stress according to the time of day18–21. With regard to the correlation between CAM and circadian rhythm22. the conventional sampling tactics that focus on a single time point per day should be abandoned as, if the daylight sampling time is fixed, some important clues to key resistance genes could be missed.Comparing the two published genomes from epiphytic orchid speciesD. catenatum plants were subjected to continuous drought treatments by simulating their natural environment under controlled conditions. Sampling time points were set for both day and night during the drought procedure. A dataset containing 39 RNA-seq with over 41 million sequence reads per sample was generated using the Illumina HiSeq 2500 platform. We assessed RNA sample quality, RNA-seq read quality, and the global gene profile  with sphagnum moss as the matrix. Eight-month-old plants were transferred into a phytotron chamber  and adapted to the controlled conditions for 10 days before being used for the follow-up experiment. The experiments were conducted on initially healthy individuals (~12 cm height). Plants were irrigated on the first day and then water was withheld to mimic drought stress. We collected leaf samples when the volumetric water content of the base material declined to ~30–35%, ~10–15, and ~0%, respectively, at both 09:00 h and 21:00 h (2 and stored at −80 °C.Clones of  21:00 h . The fouhttp://www.tiangen.com/) according to the manufacturer’s protocols. RNA purity was estimated using a NanoPhotometer® spectrophotometer . RNA quality was assessed using an RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system . RNA samples of acceptable quality were used to construct non-strand-specific sequencing libraries with the TruSeq RNA Sample Prep Kit . These libraries were sequenced using the PE150 mode on an Illumina HiSeq2500 platform at Novogene Corporation .Total RNA was extracted from the samples mentioned above  using th23. Sequencing adapters, low-quality bases, viral sequences, and rRNA sequences were cleaned. The criteria for this filtering procedure were set as follows: (1) RNA 5′ and 3′ adapters were set as [5′-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′] and [5′-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC (index) ATCTCGTATGCCGTCTTCTGCTTG-3’] (the indexes are listed in 24 were set as recommended according to Fastq_clean instructions. The statistics of clean reads are listed in http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and then summarized using MultiQC v1.325.The raw data  were filtered using Fastq_clean v2.0D. catenatum genome17 (GenBank Assembly ID ASM160598v2) using Hisat226 with default parameters. Salmon v0.9.127 was used to estimate gene abundance as read counts in the alignment-based mode. The raw read counts were imported into the R package DESeq228 for normalization. We used the package ReSQC29 to assess RNA-seq read coverage skewness over the gene body based on the above mapping results.The clean reads were mapped to the 30 was used for the calculation of Poisson distance, and the R package Pheatmap (https://cran.r-project.org/web/packages/pheatmap/index.html) for visualization. A principal component analysis (PCA) was also employed to assess sample relationships based on rlog-transformed values of raw read counts.A heatmap for cluster relationships among samples representing Poisson distance were generated with raw read counts. The R package PoiClaClu31 was used to detected gene clusters (modules) on normalized read counts (Data Citation 2) using the WGCNA v1.6333 package in R. This analysis generated a topological overlap matrix plot (http://www.omicshare.com/tools/Home/Soft/gogsea). The edge information of each gene cluster and the results of both GO annotation and GO enrichment are stored in the Figshare repository (Data Citation 3).To determine the highly correlated genes in this prolonged drought experiment, weighted gene co-expression network analysis (WGCNA)rix plot  that illThe R scripts for reads count filtration and normalization, heatmap illustration, PCA and WGCNA are available in Figshare (Data Citation 4).The RNA-seq raw data of 39 samples are deposited at the NCBI Sequence Read Archive (Data Citation 1).Supplementary materials are available on the Figshare data management platform . Data Citation 2 provides expression profiles of raw read counts and normalized read counts; Data Citation 3 contains WGCNA results, GO annotation for all genes, and GO enrichment for gene clusters. Data Citation 4 is dedicated to the R scripts in this study.34, which means low RIN would lead to a bias in gene expression profiles. In this study, RNA samples with a RIN value >6.5 were employed for RNA-seq library construction, which meant that high-quality reads were obtained for subsequent studies. The quality values for RNA samples, including RIN, are listed in The quality of total RNA is a critical parameter for the construction of sequencing libraries and the follow-up quantitative analyses. In particular, RNA integrity (RIN) is positively correlated on uniquely mapped reads in RNA-SeqThe high clean data rate , rangingBoth the heatmap  and PCA How to cite this article: Wan, X. et al. Transcriptomic profiling for prolonged drought in Dendrobium catenatum. Sci. Data. 5:180233 doi: 10.1038/sdata.2018.233 (2018).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Our findings confirmed that the presence of a 5′-m7G cap enhances template switching efficiency. We also profiled the composition of the nontemplated addition in the absence of TSO and observed that the 5′-end of RNA template influences the terminal transferase activity of the RT. Furthermore, we found that designing new TSOs that pair with the most common nontemplated additions did little to improve template switching efficiency. Our results provide evidence suggesting that, in contrast to the current understanding of the template switching process, nontemplated addition and template switching are concurrent and competing processes.Single-cell RNA-Seq (scRNA-Seq) has led to an unprecedented understanding of gene expression and regulation in individual cells. Many scRNA-Seq approaches rely upon the template switching property of Moloney murine leukemia virus (MMLV)-type reverse transcriptases. Template switching is believed to happen in a sequential process involving nontemplated addition of three protruding nucleotides (+CCC) to the 3′-end of the nascent cDNA, which can then anneal to the matching rGrGrG 3′-end of the template-switching oligo (TSO), allowing the reverse transcriptase (RT) to switch templates and continue copying the TSO sequence. In this study, we present a detailed analysis of template switching biases with respect to the RNA template, specifically of the role of the sequence and nature of its 5′-end (capped  Deep RNA-Seq has emerged as an important genomic tool as it provides a snapshot of the transcriptomes of tissues and cells under physiological and pathological conditions . Single-Template switching is an innate property of certain reverse transcriptases (RTs) to switch from one RNA template molecule to another –12. It i–13–Despite its widespread use, the underlying mechanism by which template switching occurs remains elusive. The prevailing assumption is that MMLV-based reverse transcriptases add three nontemplated deoxycytidines (+CCC) to the 3′-end of the cDNA strand –19 via a–et al. . We profiled the number and composition of the nontemplated nucleotides incorporated to the 3′-end of the cDNA strand and demonstrated that the makeup of the RNA template 5′-end plays a key role in swaying the RT terminal transferase activity. We confirmed that the presence of a 5′-m7G cap enhances the efficiency of template switching and that the cap itself can act as a template for the reverse transcriptase. Furthermore, we provide evidence that template switching and nontemplated addition are concurrent and competing processes. Our results give a new perspective on the template switching process and underscore the contribution of the RNA template sequence as a source of bias during library preparation.In light of the lack of in-depth experimental evidence on the origin of biases in template switching, we set to investigate how template switching is affected by both the RNA sequence and nature of its 5′-end (capped p), or 5′-m7G cap). By utilizing a defined RNA sequence, we minimized the dependence of template switching on internal sequence variation. We carried out the template-switching reverse transcription by using a 5′-FAM–labeled DNA primer and a TSO that contained three terminal guanosine ribonucleotides at the 3′-end (rGrGrG-3′ TSO) (A). As it has been reported that MMLV-based reverse transcriptases add multiple nontemplated cytidines to the 3′-end of the cDNA strand during reverse transcription (B).To determine whether the 5′-end of RNA affects template switching efficiency, we designed a set of 25mer RNA oligonucleotides, varying the identity of the first nucleotide  and the modification at the 5′-end (5′-hydroxyl (5′-OH), 5′-monophosphate (5′--3′ TSO) A. As it cription , the rGrcription B.C). For RNA oligonucleotides with the same 5′-end modification, template switching was most efficient when the first nucleotide was G followed by C or A. RNA oligonucleotides starting with U had the lowest template switching efficiency. Template switching was also affected by the modification of the 5′-end of the RNA. For RNAs with the same first nucleotide, m7G-capped oligonucleotides showed significantly greater template switching efficiency than 5′-p or 5′-OH oligonucleotides. In addition, we examined the template switching bias on m7G-capped and 5′-p uncapped RNA oligonucleotides with six MMLV-based reverse transcriptases, including MMLV RT, Template Switching RT, SuperScript II, SuperScript IV, Maxima H Minus, and SMARTScribe in both their manufacturers' suggested buffers  and in a commonly used template switching buffer (Smart-seq2 buffer) . Although the overall template switching efficiencies varied significantly when the RTs were used in their own buffers, similar efficiencies were observed when the reverse transcription reactions were performed in the Smart-seq2 buffer . More importantly, all the RTs investigated in this study showed the same pattern of template switching products with respect to the RNA template sequence, with RNAs starting with G being the most effective and those with U being the least effective. Additionally, capped RNA showed higher template switching efficiencies than uncapped RNA regardless of the RT.Template switching was significantly affected by the first nucleotide of the RNA template C. For RN buffer)  . The relative composition of the first four ribonucleotides (N1:N2:N3:N4) in the RNA template was determined by deep sequencing to account for any nucleotide misrepresentation introduced during RNA synthesis  . We then performed the template-switching reverse transcription on these RNA oligonucleotides and constructed Illumina libraries to determine the extent of biases (B–D). The sequencing data from template switching experiments were normalized to the relative composition of the first four ribonucleotides in the respective synthetic templates. The results suggested that regardless of whether the 5′-modification was capped or uncapped, there was a strong preference for guanosine at position N1 (B–D), which is consistent with the template switching efficiency results described in Fig. 1C. Uncapped RNA templates also favored guanosine at position N2. The nucleotide preference decreased along each of the remaining randomized nucleotides with N4 showing minimal contribution to the overall bias.To further analyze the effect of the internal RNA sequence on the bias of template switching, we synthesized RNA oligonucleotides with the first four nucleotides randomized. Again, the 5′-end of the RNA oligonucleotide was either a 5′-OH, 5′--m7G cap A. The reig. S2A) . We founf biases , B–D. Thition N1 , B–D, whA and AS3). It is important to note that these experiments were carried out in the absence of a TSO.The RT terminal transferase activity is critical for the process of template switching. In an attempt to explain the origin of the biases observed for templates starting with a guanosine, we profiled the number and identity of the nontemplated nucleotides added by the RT to the 3′-end of the cDNA across different RNA template 5′-ends. Two independent methods, deep sequencing and MS, were used to determine the nontemplated addition profile A and S3AA). We found that the number of nontemplated nucleotides varied significantly depending on the 5′-end RNA modification (B). Whereas m7G-capped templates led to cDNA products with the addition of three nontemplated nucleotides as the major species, the 5′-OH and 5′-p RNAs gave rise to products with primarily one nontemplated nucleotide. These results confirm earlier evidence that an MMLV-based reverse transcriptase will add a different number of nontemplated nucleotides depending on whether the RNA is capped or not (C). Surprisingly, unlike what has been reported previously . In general terms, a higher terminal transferase activity evidenced by an increase in nontemplated additions correlated with a higher template switching efficiency (C and 1C).For deep sequencing, a library of RNA templates with four randomized nucleotides at the 5′-end was created using ssDNA ligation A. We foufication B. Wheread or not . Next, wd or not C. Surprieviously , 27, m7Gficiency C and 1C.7G-capped RNAs was almost always dC, pointing toward a contribution of the guanosine cap on “templating” the first addition, which has been suggested before . No significant difference in the nontemplated addition profile was observed by the varying nucleotides at the N1 position of the RNA template with the exception of N1 = U, which favored +A and rarely added more than +2 nucleotides. This apparent discrepancy between sequencing and MS results for N1 = U may be attributed to the fact that the MS analysis is semiquantitative and was carried out with a single template , whereas the deep sequencing results were averaged from a randomized pool comprising all potential nucleotide combinations at each of the three positions N2, N3, and N4.Interestingly, the first nontemplated nucleotide incorporated across all md before . However7G-capped RNA (N1 = G) with two additional enzymes, MMLV and SuperScript II reverse transcriptases, both in their manufacturer's recommended buffer and in the Smart-seq2 buffer. We found that the +CAA product was the major species formed with either enzyme in the Smart-seq2 buffer , which is consistent with the results obtained with Template Switching RT. In line with the observed low template switching efficiencies , the reverse transcription reactions performed in the manufacturer's recommended buffers resulted in cDNA products containing only one nontemplated addition, favoring the formation of the +C species .To further confirm that the observed nontemplated addition profile generally applies to other MMLV-based reverse transcriptases, we repeated the reverse transcription reaction of one mp templates, the most abundant cDNA products contained +A, +C, and +G additions except in the case of RNAs with N1 = U where +A was observed predominantly (C and BS3). In uncapped templates where N1 = G, besides +A and +C, two-nucleotide additions, +AA and +GA, were also observed, corroborating the hypothesis that the ability of MMLV-based reverse transcriptase to template switch is directly associated with its terminal transferase activity. It is also noteworthy that 5′-OH and 5′-p RNAs led to a considerable amount of cDNA products with no nontemplated nucleotides added (denoted by “−”). The general preference for a single nontemplated addition across all uncapped RNAs was also confirmed by MS analysis .For uncapped RNA templates, there was much less discrimination against the incoming nucleotide during the nontemplated nucleotide addition. With both 5′-OH and 5′-minantly C and S3B7G-capped RNAs. An rGrUrG-3′ TSO was designed as a variant of the rGrGrG-3′ TSO to account for the additional +CA motif observed in the nontemplated addition, and an rUrUrU-3′ TSO was designed to account for the prevalent +A addition, particularly for 5′-OH and 5′-p RNA templates. All template switching experiments with the new TSOs were compared with the standard rGrGrG-3′ TSO (A).As it has been reported that template switching relies on the successful annealing between the TSO 3′-end and the cDNA 5′ protruding nontemplated nucleotides , we desiG-3′ TSO A.7G-capped RNAs in the absence of a TSO, the matching rUrUrG-3′ TSO showed minimal template switching and was markedly inefficient compared with the rGrGrG-3′ TSO (D). The poor performance of rUrUrG-3′ TSO suggests that the +CAA addition may not play a significant role during the template switching process. Interestingly, the rGrUrG-3′ TSO provided template switching efficiencies comparable to the rGrGrG-3′ TSO. The fact that, in the absence of a TSO, +CAC/+CCC were noticeably rare additions and that the highest template switching efficiencies were observed for rGrUrG-3′ and rGrGrG-3′ TSOs led us to consider that a TSO-mediated extension of the nascent cDNA seemed to be competing with an outright TSO-independent terminal transferase process.Surprisingly, although +CAA was the most common nontemplated addition observed with mG-3′ TSO D. The pop) with the newly designed TSOs were also largely inferior to those obtained with the rGrGrG-3′ TSO. Only the rGrUrG-3′ TSO showed efficiencies comparable to the rGrGrG-3′ TSO. Interestingly, the rUrUrU-3′ TSO did not trigger any appreciable template switching even though uncapped RNAs (in particular when N1 was uridine) showed significant amounts of +A incorporation by the RT terminal transferase activity alone (B and C). We speculate that the abundant +A nontemplated addition in 5′-U RNAs would result in weaker hydrogen bonding relative to a C–G base pair and thus lead to weaker TSO/nontemplated addition interactions and less efficient template switching.Similar to what was observed for capped RNAs, the template switching efficiencies for uncapped RNAs , and the identity of this nucleotide was almost always a deoxycytidine (B). In contrast, uncapped RNAs overwhelmingly (∼90% of the reads) led to no extra nucleotides in the junction (A).Given the conflicting results observed between the nontemplated addition profile in the absence of a TSO and the template switching efficiencies in the presence of corresponding TSOs, we decided to investigate the nucleotide sequence in the cDNA product at the interface between the RNA template and TSO strands . The sequencing results from the randomized library in Fig. 2junction A, and thcytidine B. In conjunction A.C). Similar to what was observed by deep sequencing, MS analyses indicated an extra +C present at the cDNA–cTSO junction with all m7G-capped RNAs . We also detected minor incorporations of +A, +CA, and +AA; however, we cannot rule out by MS alone whether some of these additions (particularly of +A) may have occurred at the 3′-end of the cDNA–cTSO strand (across the biotin group) and not at the cDNA–cTSO junction. When uncapped RNAs were subjected to the same template switching conditions, no nontemplated +C addition was observed at the cDNA–cTSO junction .To independently verify the sequencing profile at the cDNA–cTSO junction, we carried out a slightly different experiment using MS. Because mass accuracy decreases with the increase in size of the cDNA product, we designed a TSO with a 5′-biotin modifier to limit concatemer formation  and at tFig. 57G cap structure itself may act as a template for the RT during cDNA synthesis, making the first nontemplated addition of +C, in reality, templated. It is unclear at this point whether this extra cytidine may be responsible for seeding the hybridization with the incoming rGrGrG-3′ TSO. The lack of other nucleotides at the cDNA–cTSO junction suggests that template switching may immediately occur as soon as the first +C is added (B). Hence, once the template switching starts, the nucleotide incorporation at the 3′-end of cDNA strand is dictated by the sequence of the TSO. Although this does not necessarily explain why other TSOs do not give rise to efficient template switching, we can only presume that the stability of the TSO/cDNA interaction may be a key factor in this process.The major addition of a single +C at the cDNA–cTSO junction conveys the idea that the mis added B. Hence,A). Not surprisingly, under these conditions, the template switching efficiency was reduced by 2–5-fold (A and 4A). The results indicate that the process of template switching happens concurrently with that of nontemplated addition. If the two processes happened sequentially and independently, the template switching efficiencies may have remained the same regardless of the timing of the TSO addition.To further study the relationship between nontemplated addition and template switching, we performed the template switching reaction with a delayed addition of TSO with the goal of permitting TSO-independent nontemplated addition to take place prior to the strand switching process A. Not su2–5-fold A and 4A.B). Even for the 5′-m7GpppU RNA, which otherwise exhibited poor template switching, there was a dramatic increase in efficiency, bringing it to a level comparable with the other capped RNAs. As expected, nontemplated addition in the absence of a TSO and with 10× dCTP resulted in a marked increase in deoxycytidine incorporation . Interestingly, these were the only conditions under which the +CCC extension alone was observed with any abundance. These results align with our hypothesis that the nontemplated deoxycytidines likely enhance template switching by creating a stronger, more stable interaction with the TSO.Based on the premise that a +C addition favors template switching whereas a +A disfavors it, we sought to enhance the overall efficiency of the switching process by altering the availability of dCTP and dATP during the reverse transcription reaction. Concordantly, when we utilized 10-fold excess dCTP (10× dCTP), the template switching efficiency increased by nearly 2-fold for all capped RNAs B. Even fB).Next, we tested the reverse transcription reaction with an unbalanced dNTP mixture containing ped RNAs B.C). In the presence of a capped RNA template and a DNA primer, a cDNA strand is generated to the end of the RNA template. As soon as the first nontemplated deoxycytidine is added, template switching can occur. If further nontemplated additions take place, most commonly of deoxyadenosines, template switching is suppressed. Therefore, we propose that nontemplated nucleotide addition and template switching happen concurrently, not sequentially, in the context of the reverse transcription reaction.Taking into consideration the cumulative results of this study, some new light can be shed on the mechanism of template switching by MMLV-based reverse transcriptases C. In the7G-capped RNAs; however, considering that mRNA transcription start sites usually comprise G or A and rarely U, the overall bias effect for single-cell RNA-Seq is not as drastic as with uncapped RNAs. Fundamentally, understanding the mechanisms by which RTs template switch is imperative to minimize RNA-Seq data distortion, particularly in low-input sequencing.Uncovering and understanding inherent biases of template switching–based methods is critical for RNA-Seq at the single-cell level. In view of the well-documented biases found in traditional ligation approaches for the introduction of 5′-sequencing adaptors –33, many–It has been generally accepted that three to four nontemplated 3′-terminal deoxycytidines are added to the nascent cDNA strand during reverse transcription , 26. Thret al. . The RNA template 4rN-25mer-RNA-FAM was synthesized using the “hand-mix” option with an equimolar ratio of all four bases (Integrated DNA Technologies). 5′-Triphosphate RNAs were synthesized as described previously (Table S1.All reagents were from New England Biolabs (NEB), Ipswich, MA, unless otherwise stated. 5′-OH and 5′-eviously . RNA tempppNUAGAACUUCGUCGAGUACGCUCAA-3′) was performed at a 500-μl reaction volume using the Vaccinia Capping System. Briefly, 10 μm 5′-triphosphate RNA, 1× Capping Buffer , 30 μm GTP, 200 μm SAM, 5 units of pyrophosphatase , and 500 units of Vaccinia Capping Enzyme  were incubated overnight at 37 °C. Capped RNAs were purified by phenol/chloroform extraction using Phase Lock Gel tubes  followed by PAGE. Purified capped RNAs were resuspended in 50 μl of water, and the final oligonucleotide concentration was estimated with a NanoDrop spectrophotometer .Capping of 5′-triphosphate 25mer RNAs . The TSOs used in this study comprised a common 39-nucleotide DNA sequence at the 5′-end and a variable three-nucleotide RNA sequence at the 3′-end. TSO and primer sequences can be found in Table S1. Unless otherwise noted, Template Switching RT, an MMLV-based reverse transcriptase with reduced RNase H activity , SuperScript IV , Maxima H Minus , SMARTScribe , M-MuLV reverse transcriptase , and Template Switching RT . For the reverse transcription reactions, a 20mer 5′-FAM V5 primer was designed to be complementary to the 3′-end of the 25mer RNA templates , 1× Template Switching RT Buffer , 2 mm dNTP solution mixture , 0.5 μm 5′-FAM V5 primer, and 400 units of Template Switching RT. The reaction was performed at 42 °C for 90 min followed by a 10-min heat-denaturation step at 72 °C. The template RNA was hydrolyzed by adding 1 m sodium hydroxide (10 μl) and 0.5 m EDTA, pH 8.0 (10 μl), to the reaction and heating to 65 °C for 15 min. The cDNA was purified using the Oligo Clean & Concentrator  and analyzed by MS.A nontemplated-addition reverse transcription reaction  contained 1 μm RNA template, 1× Template Switching RT Buffer , 1 mm dNTP solution mixture, 30 nm 5′-FAM V5 primer, 1 μm TSO, and 100 units of Template Switching RT. The reaction was performed at 42 °C for 90 min followed by a 10-min heat-denaturation step at 72 °C. The reverse transcription reaction was directly analyzed by capillary electrophoresis without purification.A template-switching reverse transcription reaction  contained 0.1 μm NaCl, 20 mm Tris-HCl, pH 7.5, and 1 mm EDTA), rinsed with a low-salt buffer , and eluted with a solution of 10 mm Tris-HCl, pH 7.5, and 1 mm EDTA. The eluted RNA:cDNA was then subjected to RNA hydrolysis as described above and purified using the Oligo Clean & Concentrator. The purified cDNA was analyzed by MS.For template-switching reverse transcription reactions using the biotin-rGrGrG-3′ TSO, hydrophilic streptavidin magnetic beads  were used for purification prior to RNA hydrolysis. Briefly, the reverse transcription reaction was incubated with the beads for 10 min, then washed  and analyzed in an Applied Biosystems  3130xl (16-capillary array) or 3730xl (96-capillary array) Genetic Analyzer with a 36-cm-long capillary coated with POP7 polymer. Data were collected via Applied Biosystems Data Collection software and processed with Applied Biosystems Peak Scanner software  using on-line desalting, flow-injection electrospray ionization on a Thermo Fisher Scientific LTQ-XL ion trap mass spectrometer and analyzed with ProMass Deconvolution software. The composition of the nontemplated nucleotide addition to cDNA was determined by comparison with calculated molecular masses.7G-capped derivative by treatment with guanosine 5′-pyrophosphorylimidazolide in a Mn(II)-promoted reaction  contained 2 μm RNA template , 1× ProtoScript II Reaction Buffer, 1 mm dNTP solution mixture, 0.5 μm i7 primer (see supporting information for primer sequence), 10 mm DTT, and 200 units of ProtoScript II . The reaction was performed at 25 °C for 5 min and 42 °C for 30 min. The RNA template was hydrolyzed by adding 2 μl of 1 m NaOH and incubated at 65 °C for 15 min. The cDNA was purified with a Monarch PCR & DNA Cleanup kit  using the oligonucleotide cleanup protocol. A poly(G) tail was added to the 3′-end of cDNA  using 10 units of Terminal Transferase  and 2 mm dGTP. The reaction was incubated at 37 °C for 1 h. After the dG tailing, a pair of primers  was used to amplify the cDNA. After bead purification using NEBNext Sample Purification Beads , PCR products were mixed with 40% PhiX and subjected to Illumina iSeq-100 sequencing (2X45).The 4rN-25mer-RNA-FAM oligonucleotide was 5′-phosphorylated with T4 polynucleotide kinase and then further converted to the mreaction  followedm RNA template , 1× Template Switching RT Buffer, 1 mm dNTP solution mixture, 30 nm i7 primer, 1 μm biotin-i5 rGrGrG-3′ TSO (see supporting information for TSO sequence), and 100 units of Template Switching RT. The reaction was performed at 42 °C for 90 min followed by a 10-min heat-denaturation step at 72 °C. Then 1 μl of the reverse transcription reaction was subjected to PCR amplification with NEBNext Universal and Index primers  using NEBNext High-Fidelity 2× PCR Master Mix . The libraries were cleaned up with NEBNext Sample Purification Beads  and sequenced on an Illumina iSeq-100 as described above.A template-switching reverse transcription reaction  contained 0.1 μm RNA template , 1× Template Switching RT Buffer, 1 mm dNTP solution mixture, 30 nm i7 primer, and 500 units of Template Switching RT. The reaction was performed at 42 °C for 90 min followed by a 10-min heat-denaturation step at 72 °C. After the reaction, 2.5 units of RNase H  and 25 units of RNase If  were added to the reaction and incubated at 37 °C for 20 min to hydrolyze the RNA template. The cDNA was purified using an Oligo Clean & Concentrator. The purified cDNA was incubated in a 20-μl reaction with 750 nm 5′-AppDNA (see supporting information for sequence), 1× NEBuffer 1, 5 mm MnCl2, and 2 μm Thermostable 5′-AppDNA/RNA Ligase  at 65 °C for 60 min to perform 3′ single-strand cDNA ligation. The reaction was inactivated at 90 °C for 3 min. The ligated products were subjected to PCR amplification with NEBNext Universal and Index primers  using NEBNext High-Fidelity 2× PCR Master Mix . The libraries were cleaned up with NEBNext Sample Purification Beads  and sequenced on an Illumina iSeq-100 as described above.A nontemplated-addition reverse transcription reaction  contained 0.1 μpp-DNA adaptor were extracted and sorted according to the first nucleotide of RNA template. All sequencing FASTQ files are available as supporting information. Graphical representation and statistics were performed using GraphPad Prism 8.Custom Python scripts were written to extract Illumina reads with the constant region of the template RNA. For profiling the composition of the four randomized nucleotides in the RNA template and the biases in the template-switching reverse transcription reaction, the four variable positions were extracted and analyzed. For profiling of the nontemplated-addition reverse transcription reaction, the sequences between the constant region of the RNA template and single-stranded AM. G. W., S. M., P. H., and N. D. data curation; M. G. W. formal analysis; M. G. W., I. R. C., and S. G. writing-original draft; Y. B. and N. M. N. resources; I. R. C. and S. G. supervision; I. R. C. and S. G. writing-review and editing.