Circular ribonucleic acid (RNA) molecules (circRNAs) are covalently closed loop RNA molecules with no 5′ end caps or 3′ poly (A) tails, which are generated by back-splicing. Originally, circRNAs were considered to be byproducts of aberrant splicing. However, in recent years, development of high-throughput sequencing has led to gradual recognition of functional circRNAs, and increasing numbers of studies have elucidated their roles in cancer, neurologic diseases, and cardiovascular disorders. Nevertheless, studies of the functions of circRNAs in the immune system are relatively scarce. In this review, we detail relevant research on the biogenesis and classification of circRNAs, describe their functional mechanisms and approaches to their investigation, and summarize recent studies of circRNA function in the immune system. Caenorhabditis elegans using RNA-sequencing data combined with analyses of the human leukocyte database (Circular RNA (circRNA) is a covalently closed loop molecular form of RNA, and was discovered more than two decades ago , 2. Initdatabase . circRNAdatabase –12.Current research on circRNAs focuses on their role in cancer, neurologic diseases, and cardiovascular disorders. In this review, we cover relevant research on the biogenesis and classification of circRNAs and their functional mechanisms and methodological approaches to their study, along with summarizing recent investigations of the roles of circRNAs in the immune system.Rather than canonical splicing, circRNAs are generated through back-splicing , 14 Fig or may oSkipping of exons leads to the formation of a lariat structure containing the skipped exon. If introns are spliced before the lariat is unraveled by debranching enzymes, a stable exon-containing circRNA will be produced , along wIntron pairing-driven circularization is based on reverse complementary matches (RCMs) within flanking introns, and was established independently of the exon-skipping mechanism of circularization. RCMs can induce base-pairing between flanking introns, promoting a hairpin formation, bringing the 5′ and 3′ termini of an exon into spatial proximity, and inducing “head-to-tail” splicing. Intriguingly, the protein adenosine deaminases acting on RNA (ADAR) also participate in this process, together with RCMs. ADAR can unzip double-stranded RNA molecules by converting adenosine residues within them into creatinine molecules, thereby reducing the formation of circRNAs . FurtherDrosophila, the splicing factor muscleblind (MBL) drives the circularization of the second exon of MBL to produce circMBL, by binding specifically to MBL binding sites within introns flanking circMBL sequences  was identified by Conn et al. and shown to promote the production of circRNAs during human epithelial–mesenchymal transition by binding to a motif within circRNA flanking introns  GU-rich element near its 5′ splice site and an 11-nt C-rich element close to the branchpoint to avoid being debranched. Ci-ankrd52 accumulates mainly in the nucleus and promotes transcription of ANKRD52 through a cis-regulatory effect of RNAPol II , and exon–intron circRNA (EIciRNA) Figure . PublishNAPol II .circRNAs are not degraded readily by RNase R, and are more stable than their cognate linear mRNAs, with half-lives exceeding 48 h . circRNA+ leukocytes, CD34+ leukocytes, neutrophils, and HEK293 cells. Analogously, they noted that several nematode circRNAs were expressed in oocytes but absent in 1- or 2-cell embryos 19 embryos .N6-methyladenosine (m6A), which is induced by the methyltransferase METTL3/14 and suppressed by the demethylase FTO, promotes protein translation through recruiting an initiation factor, eIF4G2, and a m6A reader, YTHDF3. Furthermore, they found that m6A-containing circRNAs, with the potential to be translated into proteins, are common in the human transcriptome , can have cis-regulatory effects on gene expression. In 2013, Chen et al. reported that ci-ankrd52, which is generated from the second intron of ANKRD52, accumulates primarily in the nucleus and promotes transcription of ANKRD52 via the cis-regulatory effects of RNAPol II  or short-hairpin RNA (shRNA) targeting circRNA must be directed to the back-splicing site. This strategy limits the design of siRNA sequences, and siRNA or shRNA targeting back-splicing sites will be partially complementary to the cognate linear RNA, raising the possibility of unwanted effects on expression of linear RNA. To solve this problem, Li et al. proposed that the control sequence should be partially replaced (~10 nt) by a siRNA sequence targeting the back-splicing site . SimilarZKSCAN1 to construct a vector, and then overexpressed circ-ZKSCAN1. Then, the flanking sequence was modified, and exons2/3 of ZKSCAN1 was replaced with a polyclonal restriction site to construct an empty vector: pcDNA3.1(+) CircRNA Mini Vector  and circRNA sequences are cloned to generate a eukaryotic expression vector construct. In 2015, Lu et al. found that tRNA introns can form circular RNAs (tricRNAs)   and, in pression . In brieIn general, different circRNAs with different loop-forming characteristics exhibit variation in looping efficiency. Moreover, circRNA production by overexpression vectors is accompanied by the production of linear isoforms, which is an additional challenge for strategies employing circRNA overexpression.in vitro transcription followed by auto-splicing circulation. Surprisingly, they found that in vitro circRNAs induced activation of cellular immune response pathways and inhibited RNA virus infection, which is mediated by retinoic acid-inducible gene-I (RIG-I); however, endogenous circRNAs did not induce this pathway because of their binding to specific RNA-binding proteins  gene, promoted circRNA formation by stabilizing the intron complementary sequence. Upon viral infection, NF90/NF110 was exported from the nucleus to the cytoplasm to inhibit virus replication. Meanwhile, circRNA expression in the nucleus was decreased -inducible circRNA generated from linear RasGEF1B, named mcircRasGEF1B, the expression of which is dependent upon the LPS-Toll-like receptor-4-nuclear factor-kappa B (LPS-TLR4-NF-κB) pathway. mcircRasGEF1B is cell type-specific, exhibits evolutionary conservation between mice and humans, and localizes preferentially to the cytoplasm . Also, iThe relationship between microRNAs and immunity has been well-studied, leading to the hypothesis that circRNA may contribute to immune regulation through interactions with microRNAs. Zhang et al. reported that hsa_circ_0020397 can upregulate expression of PD-L1 (the target gene of microRNA-138) by binding to microRNA-138 in colorectal cancer cells. The consequent increase in PD-L1 levels contributes to tumor escape from immune responses , 75 Fig, implyinin vitro and in vivo experiments. Using the circinteractome database, we found that the host gene of hsa_circ_0032139 is HIF1A, which plays an important part in inflammation via NF-κB and mitogen-activated protein kinase (MAPK) pathways  is a chronic, inflammatory synovitis-based systemic disease of unknown etiology . Zheng e pathway . TherefoType-2 diabetes mellitus (T2DM) is characterized by hyperglycemia, insulin resistance, and chronic inflammation . Fang etSimilarly, Li et al. measured circRNA expression in the T cells of patients with systemic lupus erythematosus (SLE). They found hsa_circ_0045272 to be downregulated significantly. Furthermore, they demonstrated that hsa_circ_0045272 regulated apoptosis and IL-2 production negatively . HoweverNumerous studies have shown that a high proportion of tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment can improve clinical outcomes. Weng et al. showed that high expression of hsa_circ_0064428 is associated with a low proportion of TILs, poor survival, large tumor volume, and tumor metastasis in patients with hepatocellular carcinoma . These oOverall, the roles of circRNAs in immune diseases have been studied based on large-scale microarray and RNA sequencing analyses, by which differentially expressed circRNAs were screened to further verify their functions experimentally. However, the mechanisms by which circRNAs regulate disease development merit further exploration.in vivo and fused into the genome to generate a pseudogene (trans-transcriptional translocation and the effects of pseudogenes derived from circRNAs. In addition, unlike linear mRNA, construction of overexpression vectors for circRNA is challenging because it requires splicing of the two termini of amplified fragments to generate loop structures. Moreover, data in the mouse circRNA database is incomplete, which poses specific obstacles for in vivo experiments. Finally, circRNAs in the cytoplasm, which act as microRNA sponges, have been studied widely, but circRNAs that can function through this mechanism are in the minority. Most circRNAs rarely contain so many miRNA binding sites and have low expression (Although circRNAs have become a hot topic in RNA research in recent years, several important areas merit investigation. For example, Dong et al. showed that circRNA can be reverse-transcribed eudogene . Furtherpression , 28, so Few studies have focused on circRNAs in the immune response. Reminiscent of studies in cancer, circRNA research in immunity also requires measuring circRNA expression, and then elucidating the function of differentially expressed circRNAs and the mechanisms underlying it. Notably, circRNA localization could aid exploration of the mechanisms by which circRNAs regulate immune responses. circRNAs in the cytoplasm are likely to act as microRNA sponges, whereas circRNAs in the nucleus are likely to interact with proteins, or elicit effects by promoting/suppressing the role of their host genes Figure . ExcitinLY the first author, contributed to collection of references and manuscript preparation. JF and YZ contributed to manuscript modifications.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We exposed cultures for one week to lithium chloride or distilled water, extracted total RNA, depleted ribosomal RNA and performed whole-transcriptome RT-sequencing. We analyzed results by RNA length and type. We further analyzed expression and protein interaction networks between selected Li-altered protein-coding RNAs and common AD-associated gene products. Lithium changed expression of RNAs in both non-specific (inverse to sequence length) and specific (according to RNA type) fashions. The non-coding small nucleolar RNAs (snoRNAs) were subject to the greatest length-adjusted Li influence. When RNA length effects were taken into account, microRNAs as a group were significantly less likely to have had levels altered by Li treatment. Notably, several Li-influenced protein-coding RNAs were co-expressed or produced proteins that interacted with several common AD-associated genes and proteins. Lithium’s modification of RNA levels depends on both RNA length and type. Li activity on snoRNA levels may pertain to bipolar disorders while Li modification of protein coding RNAs may be relevant to AD.Lithium (Li) is a medication long-used to treat bipolar disorder. It is currently under investigation for multiple nervous system disorders, including Alzheimer’s disease (AD). While perturbation of RNA levels by Li has been previously reported, its effects on the whole transcriptome has been given little attention. We, therefore, sought to determine comprehensive effects of Li treatment on RNA levels. We cultured and differentiated human neuroblastoma (SK-N-SH) cells to neuronal cells with all- Li may affect cellular signaling processes and promote long-term neuroplasticity3. Li also seems to have neurotrophic properties affecting cell survival and apoptosis mechanisms4, and trace levels may to some extent prevent dementia5, suicide6, and homicide7. The breadth of effects for Li suggest that the metal may be a trace nutrient8. In particular, Li levels in drinking water correlated with reduced age-adjusted Alzheimer’s disease (AD) mortality9 and incidence10. Several investigations of using Li to treat AD or specific symptoms associated with AD are ongoing14. However, Li toxicity produces Parkinson’s disease (PD) - and AD-like outcomes. Li is currently under active investigation for a broad array of psychiatric and neurological disorders , as well as gastrointestinal disease, neoplasia, and other non-neurological conditions. As of June 11, 2019, the US National Institutes of Health (NIH) lists 141 planned, recruiting, or active clinical trials for Li effects on psychiatric and nervous system disorders, along with an additional 104 for conditions that do not involve the nervous system .Lithium (Li) has been used to effectively treat bipolar disorder for more than 60 years16. Three principal hypotheses are its action on i) cyclic AMP18, ii) inositol depletion19, and iii) the inhibition of protein kinases, including glycogen synthase kinase, with subsequent activation of the wnt neurodevelopmental pathway21. Li also increases levels of the anti-apoptotic protein Bcl2 in the frontal cortex of rat brain22. Li displays general neuroprotective effects, including against excitotoxic lesions23 as well as protection against β-amyloid induced cell death24. Li increases N-acetyl-aspartate levels in human brain  during therapeutic treatment, as measured by magnetic resonance spectroscopy25. This increase appears to be related to increases in grey matter volume26. Li, similarly to antidepressants, increases hippocampal neurogenesis27. Nevertheless, multiple clinical effects of Li are not entirely explained by prevailing theories28. Use of neuronally-differentiated pluripotent stem cells from bipolar patients who were responsive to Li vs non-responders revealed that neuronal hyperactivity responded to Li in responders but not in non-responders30. It should be noted that these cells, even though taken from living patients, represent early developmental stages of central nervous system (CNS) cells. Changes in adult neuronal cells from Li-responsive patients may be related to decreased phosphorylation of collapsin response mediator protein-2 (CRMP2) and increased dendritic spine density31.The mechanisms underlying lithium therapeutic activity are still not clear35. However, those reports were array-based assays, not whole transcriptome surveys. If a specific RNA were not a priori included on an array, Li effects on it would not be measured. Thus, we performed whole transcriptome sequencing of Li-treated vs. untreated cells. Neuronal cell lines have the advantage of standardization of biochemical characteristics, ease of adaptation to multiple experimental protocols, and simulation of adult CNS cells’ chemical and physiologic characteristics. We found that Li changes expression of multiple small RNA species, particularly non-coding small nucleolar RNAs (snoRNA), in all-trans retinoic acid (ATRA) differentiated human neuroblastoma neuronal (SK-N-SH) culture, but that small RNA species do not all respond to Li stimulation equally.Since so many pathways are apparently involved in Li treatment of bipolar disorder, and the pathways associated with possible Li treatment of other disorders, such as AD, are yet unknown, a reasonable question is how Li alters the transcriptome. Gene expression changes induced by Li treatment exist in both neuronal cultures and animals36. snoRNA levels differed between schizophrenic and control subjects in the anterior cingulate cortex38. snoRNA may also play a role in some features of autism40. Finally, specific snoRNAs undergo progressive changes with age41, and snoRNA levels vary with progressing Braak stages in AD42. We, therefore, investigated potential connections among Li-perturbed genes and AD-associated genes and proteins. Our results generated interaction networks that can inform future mechanistic research, relevant to AD, bipolar and other neuropsychiatric disorders.The snoRNAs may function in multiple neurobiological disorders and symptoms, including catatoniaNo gross differences in cell morphology or survival were noted.44, cluster centers corresponded to 87 nucleotides (range 22 to 213) and 3047 nucleotides  in length -treated cells in RNA levels, detected by whole-transcriptome sequencing. We found that log lengths of whole transcriptome sequencing had a bimodal distribution. Parameterized Gaussian mixture modelgth Fig. , Table 1es range 2 to 213 gth Fig. , while rgth Fig. . Of the To investigate whether the difference simply reflected RNA transcript length or was also influenced by RNA species/type, we used logistic regression to model presence/absence of Li alteration by type of RNA and sequence length. The optimal model derived was “Li influenced ~ log(Length) + RNA Type”. We discovered an odds ratio (OR) of 0.729 +0.226/−0.171 (p ≤ 0.05) for log RNA length vs. Li influence, irrespective of RNA type, i.e., as RNA length decreased, likelihood of Li influence increased.45 and found three overlapping groups  associated RNAs/snaRNA and vault RNA/vtRNA) were each represented in our sample by a single sequence. We excluded both RNA types from further analysis. When compared to overall levels of perturbation across all RNA types (effect coding), levels of 4 types were significantly more likely  to be altered by Li Table , indepenups Fig. .Table 2RWe also investigated whether a given RNA species would be more likely to be upregulated or downregulated by Li treatment. We found that snRNA, snoRD, snoRA, and scaRNA were all significantly (p < 0.05) likely to have elevated levels after Li treatment (vs. reduced levels), while other RNA species had no significant effect Table .Table 3LAlthough the majority of Li influence concentrated on small, noncoding RNAs, several protein-coding mRNAs influenced by Li in this study play a role in AD Table . The genWe demonstrated two effects of Li treatment on the whole RNA transcriptome of a human neuronal cell line. First, Li treatment is more likely to perturb levels of a given RNA inversely to its sequence length, regardless of RNA type. Second, and more importantly, specific RNA type exerts a significant influence on whether or not Li treatment perturbs its levels. That is, snoRNAs of three types  are all significantly more likely to be perturbed while miRNA is significantly less likely to be perturbed. It is particularly noteworthy that miRNAs, although often of the same approximate length as snoRNAs, were significantly less likely to have levels perturbed by Li treatment.47. The functions of snoRNAs may extend to regulation of alternative splicing, gene silencing, chromatin modification and may even exhibit miRNA-like function54. Understanding the role of snoRNAs in behavior and disease conditions is also emerging. Prader-Willi syndrome is a genetic disorder characterized by intellectual disabilities, hyperphagia, obesity, sleep disorders and behavioral problems; the mechanism involves a paternal deletion of chromosomal region 15q11-q13 harboring snoRNAs SNORD115  and SNORD116 . Mice with a deletion of the Snord116 repeat cluster (Snord116del) show a diurnal disruption in feeding behaviors. Brain transcripts associated with Snord116del show upregulation of diurnally regulated genes, particularly Mtor , Crebp (Creb-binding protein), and Igf2r (insulin growth factor receptor) as well as clock genes, with accompanying greater body lean-ness and increased lipid oxidation during daylight hours55. Clock genes appear to be disrupted in bipolar disorder as well57, although the relationship is complex58; there is evidence implicating circadian rhythm changes in the response to Li59. SNORD115 also regulates maturation of serotonin receptor 2 C (5HT2C) by alternative splicing61.SnoRNAs act as a guide in processing nuclear and ribosomal RNAs62. In addition, alternative splicing is likely to be important in bipolar disorder63.It is notable that snoRNAs are active in chromatin modification. Genome wide association studies (GWAS) implicate histone methylation pathways as the most likely targets of snoRNA chromatin modification64, which may be enlightening to understand potential mechanism in mammalian cells. Specifically, Li appears to inhibit the bisphosphate nucleotidase Hal2p. This results in accumulation of adenosine 3′,5′-bisphosphate (pAp), which is normally broken down by Hal2p, and of pre-snoRNAs. Accumulation of pAp inhibits activities of RNases necessary for processing of snoRNAs. The human homologue for Hal2p is 3′(2′), 5′-bisphosphate nucleotidase 1, or PAP phosphatase (BPNT1). Li inhibits BPNT1 activity in crude human brain extracts65. Paradoxically, BPNT1 protein levels are reduced in the frontal cortex of bipolar patients65. Thus, the specific anti-bipolar activity of Li is unlikely to be a single mechanism.Li influences snoRNA processing in a yeast model66. Paradoxically, miR-10b levels are reduced in PD brain, and greater levels of miR-10b associate with later age of motor onset. However, miR-10b is elevated in Huntington’s disease (HD) brains, and reduced levels of miR-10b associated with later age of motor onset in HD68. miR-24-1 was elevated 42% by Li treatment. This miRNA was downregulated in CSF samples of PD subjects69. It was also significantly reduced in CSF of AD vs. Control subjects70. miR-24 serves to reduce levels of Aβ through regulating expression of nicastrin71. Li regulation of glutamate metabotropic receptor 7 (GRM7) – a gene significantly associated in a GWAS study on bipolar patients – has been shown to act via miR-13473.Although Li influence on miRNA was very limited, some of the specific miRNAs with significantly altered expression play roles in neuropsychiatric disorders. For example, miR-10b was elevated 44% by Li treatment. Elevated expression of miR-10b, in cerebrospinal fluid (CSF), can serve as a diagnostic feature for Parkinson’s disease (PD)When we looked at potential networked effects for Li treatment vs. AD, we first note that none of the “core” AD genes we examined were altered by Li treatment in our experiment. However, when we examined both co-expression and gene product interaction, connections emerged that may be worthy of further investigation, particularly the interaction between SNORD14C and APP. Even though our Li treatment did not alter the levels of the core AD gene products, we did not test effects under more “pathogenic-like” conditions, such as oxidative stress, and it may be the differences between differences (non-stressed vs. stressed cells) that could further elucidate important pathways. Several of the connections we found in our network analysis, such as GSK3B, MAPT, ADAM17, and PSEN1 are also involved in other dementias than AD. However we hesitate to speculate on applicability of Li to multiple dementia disorders, since none of these were directly influenced by Li treatment in our study. Given that we restricted our work to a single differentiated cell line, we anticipate that much more can be learned by using other models, such as human primary brain cultures or induced pluripotent stem cell (iPSC) neuronal cultures.74. Such iron accumulation is accompanied by reduction of levels of APP, which acts as an iron efflux protein75. Iron is only one of several metals that play a role in AD, in part due to the function of APP as an iron efflux protein75 that also responds to magnesium and lead77. Effective use of a metal as a drug may need to take into account interplay of multiple metals’ levels in the cells and environment.Extending the role of Li treatment past bipolar disorder has revealed some challenges for its use. It is well known that, in higher doses, Li can be neurotoxic. It can suppress microtubule-associated protein tau and induce PD-like tremor and iron accumulation in brain78. A larger-scale  retrospective study of Taiwanese patients administered Li reported mixed results5. On the other hand. low/micro-dose treatments improved agitation and stabilized cognitive impairment in AD80. When microdose Li was applied to animal AD models, it prevented memory loss and AD-like pathology81 as well as improved spatial memory and reduced Aβ42-induced neuroinflammation82.Dosing “in the upper therapeutic range” (for bipolar disorder) can associate both with PD and AD-like symptoms and FDG-PET readouts, all of which were relieved by reduction of Li dose83, and BMP4 is elevated in AD84. Furthermore, APP regulates BMP/SMAD signaling in glial differentiation85. Another gene strongly implicated in AD is PPARγ, whose AD-related activities include possible inhibition of β-site amyloid cleaving enzyme 1 (BACE1)86 and SNP-based interaction with apolipoprotein E to alter AD risk87, among many other possible connections. Neurexin 3 (NRXN3) contains a polymorphism (rs17757879) that protects against AD in male subjects88. Furthermore, toxic Aβ oligomers interact with neurexins and reduce NRXN-mediated excitatory presynaptic organization89. Finally, NRXN3 is processed by the same α- and γ- secretases that produce the neurotrophic products of the “non-amyloidogenic” APP processing pathway90, including sAPPα. Other gene products we identified have their own potential associations with AD, as well, such as multiple snoRNAs that vary according to Braak stage42 and were also altered by Li-treatment in our study.Specific mechanisms of Li activity on AD may be explained by examining some of the Li-influenced coding gene products we mention herein. For example, BMP4 reduces hippocampal cell proliferation in animal AD modelsin silico pathways could have been found by examination of multiple databases, any claims connecting Li, snoRNAs, and disease states on that basis would be speculative. What we have experimentally discovered is that Li treatment has (at least) two distinct effects on RNA levels. The first is an RNA species-independent inverse relationship between RNA length and Li influence on RNA levels. The second is different effect levels by RNA type, irrespective of RNA length. The presence of miRNA as the least-influenced RNA type is particularly interesting, since it is in stark contrast against the majority of short RNA species. A different approach to investigating miRNA vs. Li treatment further underscored such “resistance” of miRNA to Li. When Li treatment response phenotypes in bipolar patients was screened by a large  genome-wide association study of pre-miRNA genes plus 20 kb flanking sequence, only one pre-miRNA gene had a nominally significant (p ≤ 0.05) sequence variation association, but when corrected for multiple comparisons, no polymorphisms in pre-miRNA plus flanking sequence showed any association with Li treatment phenotypes91. That study compared symptomatic Li response in patients to genetic sequences rather than response in cell culture transcriptome levels. This is not necessarily in conflict with known associations between miRNAs and bipolar disorder risk, since risk, progression, and treatment response may each be governed by distinct pathways.Our work herein highlights specific RNA species-specific responses to Li treatment, which would fuel mechanistic studies. While countless  Such differences may suggest potentially useful avenues for investigating short RNA species in the multiple disorders currently and potentially treated by Li. What mechanisms could make miRNA less susceptible to Li treatment than RNA types of similar length? Are there genetic variations in miRNA response to Li, and do these correspond to Li treatment resistance or facilitation in patients? Analogous converse questions could be asked regarding snoRNAs and Li. We must point out that our study was on “steady state” RNA levels after one week of Li treatment.35, our study differs from these prior works in one critical and significant way. The preceding studies were limited by using microarray profiling. While the scope of microarrays has become quite impressive, they still each have a fixed number  of pre-defined targets. Our survey was high-throughput sequencing with no a-priori presumptions of the presence (or absence) of any specific RNA transcript.While effects of Li on RNA levels were previously reportedWe finally need to note that, while ATRA-induced SK-N-SH cultures are often accepted as “neuronal”, they are not adult human neurons. Further uses of non-presumptive whole-transcriptome sequencing follow-up to Li treatment should be done in other neuronal cell lines and in systems such as neuronal and mixed neuron/glial induced pluripotent stem cell culture, at the very least. Unfortunately, human neuronal cell models are always approximations of the actual human brain, as are any animal models. ATRA-induced SK-N-SH cultures have important elements of reproducibility and availability, which allows for seminal discoveries to be confirmed and later tested in the more costly and difficult models.All procedures were approved and overseen by the Institutional Biosafety Committee, Office of Research Compliance, Indiana University, and Indianapolis, Indiana, USA.92, then differentiated by ATRA93. Initial propagation was in Eagle’s modified minimum essential medium  supplemented with 10% fetal bovine serum (FBS). For neuronal differentiation, a stock solution of 0.01 M ATRA , was prepared in absolute ethanol and stored in light protected vials at −20 °C and diluted with tissue culture medium right before use. Subsequent dilutions were made in growth medium with a final ethanol concentration of 0.1% (v/v) which did not affect the described system. During differentiation, cells were switched to MEM media with 1% FBS supplemented with 10 µM ATRA for two weeks. Cultures of treated SK-N-SH cells were subsequently treated with 1 mM Li chloride (LiCl) or vehicle (distilled water) for an additional week (n = 6 in each group). This dose is within a range of reported Li treatment of neuroblastoma cells characterized as “low” concentration95. Toxicity of Li treatment of neuroblastoma cells was reported to not occur until concentrations exceeded 10 mM96.Human neuroblastoma (SK-N-SH) cells were obtained from ATCC and cultured as previously describedTM adapters, then subject to reverse transcription with SOLiDTM RT primer mix. cDNA was then amplified and sequenced on a 5500xl Genetic Analyzer (ThermoFisher). Identified transcripts were classified into RNA types using the typology and databases at, specifically pseudogene.org97, HGNC98, and GeneCards99.Total RNA was extracted using the RNeasy mini kit  and ribosomal RNA depleted with Ribo-Zero . Whole transcriptome sequencing was done via SOLiD RNA-Seq kit (ThermoFisher). Briefly, total RNA was fragmented with RNase III, hybridized and ligated to SOLiD100, which explicitly calculated fold differences in specific RNA levels between Li-treated and untreated cells and determined significance  for each difference. We considered those RNAs that had level differences associated with FDR ≤ 0.2 to be “Li-influenced”, regardless of level of influence. We used the mclust43 package to explore possible clustered distribution of sequence lengths. Clustering with mclust used a univariate, unequal variances model to determine cluster borders and cluster centers were not predetermined.We analyzed quantitative transcriptome sequencing by edgeR97, HGNC98, and GeneCards99. The presence/absence of Li influence (significant change in RNA level in Li-treated vs. control cells) was determined by edgeR and coded as 1 or 0, 1 corresponding to Li-induced net RNA level change (either increase or decrease), by individual sequence. Magnitude of change was not considered in this study. Examination of the data showed that only 1.3% of sequences’ expression were influenced by Li treatment. Such a low frequency can introduce unacceptable bias into conventional logistic regression, which can be corrected by penalized log-likelihood estimation, such as via the logistf R package101. However, when we compared conventional and penalized logistic models for bias, we found that conventional methods produced no greater bias than did conventional regression for our data. Final analysis was, therefore, done with conventional logistic regression. A full model of “Li influenced ~ log(Length in bp) + RNA type + log(Length) × RNA type was constructed and assessed by second-order Akaike information criterion (AICc) using exhaustive combinations of predictors102 via the MuMIn R package102. Models were run with Effect contrast coding to explicitly compare Li influence of RNA species vs. the overall mean of Li influence frequency103. The effect coded model was further used for pairwise comparisons of RNA species by estimated marginal means104. Finally, the direction of Li-induced alteration  was modeled by multinomial logistic regression. Network analysis on selected coding gene products was performed vs. human hippocampus co-expression105 or protein-protein interaction106 dataset by NetworkAnalyst107, using the “minimum network” algorithm. Gene ontologies were analyzed by the “Gene Ontology System”108. For these utilities, default parameters were otherwise used.RNA sequences were typologically classified by referring to three databases, specifically pseudogene.orgAll procedures were approved and overseen by the Institutional Biosafety Committee (IBC), Office of Research Compliance, Indiana University, and Indianapolis, Indiana, USA. No animals or human subjects or samples were used in this work.Supplementary Info


In eukaryotes, the separation of translation from transcription by the nuclear envelope enables mRNA modifications such as capping, splicing, and polyadenylation. These modifications are mediated by a spectrum of ribonuclear proteins that associate with preRNA transcripts, coordinating the different steps and coupling them to nuclear export, ensuring that only mature transcripts reach the cytoplasmic translation machinery. Although the components of this machinery have been identified and considerable functional insight has been achieved, a number of questions remain outstanding about mRNA nuclear export and how it is integrated into the nuclear phase of the gene expression pathway. Nuclear export factors mediate mRNA transit through nuclear pores to the cytoplasm, after which these factors are removed from the mRNA, preventing transcripts from returning to the nucleus. However, as outlined in this review, several aspects of the mechanism by which transport factor binding and release are mediated remain unclear, as are the roles of accessory nuclear components in these processes. Moreover, the mechanisms by which completion of mRNA splicing and polyadenylation are recognized, together with how they are coordinated with nuclear export, also remain only partially characterized. One attractive hypothesis is that dissociating poly(A) polymerase from the cleavage and polyadenylation machinery could signal completion of mRNA maturation and thereby provide a mechanism for initiating nuclear export. The impressive array of genetic, molecular, cellular, and structural data that has been generated about these systems now provides many of the tools needed to define the precise mechanisms involved in these processes and how they are integrated. In eukaryotes, the nuclear envelope functions to separate transcription from translation, and this enables transcripts to be modified substantially in the nucleus before they are exported to the cytoplasm where they are translated. During these nuclear steps in the gene expression pathway, pre-RNA transcripts are modified by the addition of 5′ caps and 3′ poly(A) tails, and frequently introns are removed to generate mature mRNAs. A broad spectrum of ribonuclear proteins are bound to transcripts during the nuclear processing  are able to move through them.Mr . Although there is considerable sequence variation between species, the overall architecture of nuclear pores is generally conserved; albeit budding yeast nuclear pores are somewhat smaller than their metazoan counterparts . The transport barrier generated by these densely packed regions of FG-nups in the transport channel can be overcome by a range of nuclear transport factors (“carriers”) that mediate the import and export of specific macromolecular cargoes . The carrier:RanGTP complex then returns to the cytoplasm where the Ran GTPase-activating protein (RanGAP) catalyzes GTP hydrolysis, generating RanGDP, which dissociates from the karyopherin, freeing it for a further import cycle. The RanGDP then returns to the nucleus . The net result of these cycles is that the energy derived from GTP hydrolysis is used to impose directionality on transport by rectifying the thermal diffusion (Brownian motion) of the components of the transport machinery by facilitating assembly of the cargo:carrier complex in the donor compartment and its disassembly in the target compartment and so is an example of a Brownian ratchet mechanism . For example, in nuclear protein import  where techanism . This meechanism , and Nupechanism .C), although here the carriers are not members of the karyopherin-β family, and the energy used to rectify movement through the assembly and disassembly of the cargo:carrier complexes is provided by ATP hydrolysis on DEAD-box helicases , the overall mechanism of nuclear export of most mRNA is conserved within eukaryotes and employs an analogous thermal ratchet mechanism C, althouelicases –20. Tranlicases –, which eA) and the homologous metazoan NXF1:NXT1  complex function as general mRNP export factors to mediate the movement of mature mRNPs through nuclear pores to the cytoplasm , and finally a C-terminal UBA-fold domain . NXF1 haYra1 and Sub2, working in conjunction with the TREX complex, are necessary to generate mature, export-competent transcripts in the nucleus of budding yeast , 32, 38.The TREX complex, which is conserved between yeast and metazoans, contributes to the integration of the nuclear steps of the gene expression pathway and nuclear export . The yeaA hypothesis for the function of these DEAD-box helicases in rectifying the Brownian motion of the Mex67:Mtr2:mRNA complex has envisaged two reciprocal remodeling steps , perhapsB). Each motif in the CTE RNA is predicted to form a distinctive L-shaped stem loop, and structural studies have indicated how this motif binds primarily to the RRM, LRR, and NTF2-like domains as well as suggested how NXF1:NXT1 could dimerize to form a platform with its binding sites arranged to bind the two motifs simultaneously and so facilitate rapid nuclear export of the viral RNA  may give some clues to the RNA secondary structure elements that have greater affinity for NXF1:NXT1. The CTE that is found in some unspliced RNAs from simple retroviruses enables them to bypass the normal pathway for generating mature mRNPs and use the NXF1:NXT1 pathway more efficiently than host transcripts for nuclear export. The ∼130-nt CTE RNA has a 2-fold symmetric motif that enables it to bind to NXF1:NXT1 without a requirement for adapter proteins such as ALYREF B. Each miral RNA , 50. It in vivo  and the detailed mechanisms by which length is controlled vary, similar large multisubunit complexes mediate cleavage in the 3′-UTR and subsequent generation of a poly(A) tail in eukaryote transcripts before they are exported to the cytoplasm  that is organized into three structural modules that mediate key functions: a nuclease module that cleaves the transcript; a polymerase module in which Cft1 binds four other components, including poly(A) polymerase (Pap1) and Fip1; and a phosphatase/APT module that regulates 3′ end processing (Although the lengths of poly(A) tails  chain is prevented from leaving the enzyme by processivity factors, such as PABPN1 in mammals  polymerase (Pap1) through multiple interactions  RNA that seem to contain about 200–300 nt and so would be attractive candidates for the oligomerization that results in dissociation of poly(A) polymerase from CPSF  polymerase that binds the growing end of the poly(A) tail. Poly(A) polymerase is composed of three domains that encircle the active site , and in  mammals  or CF1 a mammals , 71, tharactions  and is tractions , 73, 74 ractions . The proractions  and possractions , after wractions  and ZC3Hractions  in higherevisiae . In highrevisiae , 79, andrevisiae , 67. In rom CPSF .S. cerevisiae is mediated by Nab2 (A), which contains seven Zn fingers (B). In vitro, binding poly(A) RNA induces Nab2 Zn fingers 5–7 to dimerize (C), and this feature could provide a mechanism  Although attachment of nuclear export factors is a prerequisite for movement through nuclear pores to the cytoplasm, it remains unclear how many of these factors are attached to an individual transcript. In higher eukaryotes, the EJC and 5′ cap generate attachment mediated through TREX, whereas completion of polyadenylation, at least in budding yeast, is thought to generate an analogous attachment. It appears that the 5′ and 3′ ends of the transcript are often quite close , but it In vitro, PABPN forms globular aggregates on poly(A) RNA (in vivo and whether there are one or two dimers per tail remain to be established.(ii) The termination of polyadenylation appears to be mediated by dissociation of poly(A) polymerase from the cleavage and polyadenylation machinery as a result of steric factors associated with the binding of PABPN1 in higher eukaryotes and Nab2 in budding yeast. y(A) RNA  that arey(A) RNA , but they(A) RNA  and proby(A) RNA , but the(iii) Polyadenylation is terminated by the dissociation of poly(A) polymerase from cleavage and polyadenylation machinery, but how this machinery detaches from the 3′-UTR has not been established, although it appears that the generation of export-competent mRNPs by Mex67 binding contributes to this step . It may (iv) In at least budding yeast, the termination of polyadenylation signals a TREX/Sub2-mediated remodeling of the transcript that results in Yra1 dissociation and Mex67:Mtr2 attachment . Pcf11 a(v) In budding yeast, there are indications of Mud2 functioning to coordinate splicing and polyadenylation, but the precise mechanism by which this is mediated and the role of other factors that show genetic interactions between these functions are unclear. Similarly, intron-containing transcripts appear to be retained in the nucleus, either at the nuclear basket in budding yeast or in nuclear speckles in higher eukaryotes, and although several components of the machinery involved have been identified, the precise mechanism by which this is mediated remains obscure.In summary, many of the components of the gene expression pathway that are involved in the nuclear processing and export of transcripts have been identified, but the precise mechanisms by which these steps are coordinated remain to be established. Although the thermal ratchet-based mechanism by which mRNPs are exported to the cytoplasm through nuclear pores has been established in considerable detail, many aspects of the generation of export-competent mRNPs in the nucleus remain unclear. Key checkpoints in the pathway leading to the generation of export-competent mRNPs, which entail establishing that both splicing and polyadenylation have been completed, need to be activated in a timely manner. Providing a comprehensive description of this complex series of integrated processes represents an important challenge and a fruitful area for future studies in this area.


Self-rated health (SRH) allows for comparison and identification of the health status of various populations. The aim of this study was to conduct a systematic review of the literature to expand the understanding of SRH after stroke.This systematic review was registered with PROSPERO (CRD42017056194) and conducted according to PRISMA guidelines. Studies published until December 2018 that evaluated the SRH of adults with stroke were included.Of the 2132 identified studies, 51 were included. Only four studies had experimental designs (7.8%). In 60.7% of the studies, SRH was assessed by variations on direct questions . Analog visual scales and quality of life instruments were also used to evaluate SRH, but there is no consensus regarding whether they are appropriate for this purpose. The results of cross-sectional and longitudinal studies revealed significant associations between poor SRH and stroke as well as between SRH, function, and disability. The power of SRH to predict stroke mortality is still uncertain. Two interventions  effectively improved SRH.Direct questions are the most common method of evaluating SRH after stroke. Studies reported significant associations between the SRH of individuals with stroke and several relevant health outcomes. However, few experimental studies have evaluated SRH after stroke. Interventions involving health education and family involvement had a significant impact on SRH. Self-rated health (SRH) is a simple measure of how individuals evaluate their own health status , 2 recomHealth status, quality of life (QoL), and health-related quality of life (HRQoL) are often used interchangeably . AccordiSRH has been considered a valuable outcome in studies with various objectives and populations. It has been demonstrated to have a significant association with the risk of diseases, such as depression  and typeStroke is the leading cause of serious long-term disability, and it accounts for most of the global burden of disease . GloballSRH has been associated with demographic, psychological, physical, and social factors in stroke patients  as well Due to the importance of SRH evaluations, the general aim of this study was to conduct a systematic literature review to expand the understanding of SRH after stroke. The specific aims were to (a) describe how SRH has been assessed and used, (b) synthesize previously reported results, and (c) verify the effects of interventions on the SRH of individuals who suffered from a stroke.This systematic review followed the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) guidelines , 24, andThis review included studies that assessed SRH in individuals ≥18 years of age who suffered from a stroke. The WHO’s definition of a stroke was adopted . Of the Searches were conducted in the following electronic databases: Medical Literature Analysis and Retrieval System Online (MEDLINE), Physiotherapy Evidence Database (PEDro), Latin American and Caribbean Health Sciences Literature (LILACS), and Scientific Electronic Library Online (SCIELO). An initial search strategy was created for MEDLINE and then was adapted to the other databases. The search strategy for stroke published in a recent systematic review of the Cochrane Database of Systematic Reviews  was elabAll studies found in the electronic databases were screened based on their titles and abstracts. Those that clearly did not meet the eligibility criteria were excluded. Then, the full texts of the remaining studies were analyzed to determine whether they met the eligibility criteria. A manual search in the references section was also performed in the included studies. The methodological quality of the clinical trials was evaluated using the PEDro scale , and then = 25) included subjects from European countries [n = 14) included subjects from North America [n = 30, 58.8%) [Of the 2132 identified studies, 51 were included in this review  , 63–69. , 58.8%) , 62, 65 , 58.8%)  was a qun = 32, 62.7%) [n = 12, 23.5%; Table The sample size of the included studies ranged from 19  to 104,8, 62.7%) , 70, 71,, 62.7%) , 70, 71 , 62.7%) , 67, 68,, 62.7%)  to 77 ± , 62.7%) . Stroke n = 37) [n = 21, 67.7%) [n = 15, 48.4%) [Regarding the SRH assessment, 72.5% of the studies n =  [37, 39,n = 37) , 73–77 u, 67.7%) , 76, 77 , 48.4%) , 73, 75., 48.4%) –56, 66 o, 48.4%) , 73, 75., 48.4%) , 62, 65 , 48.4%) , 44; the, 48.4%) , 61, 67;, 48.4%) , 59, 61.In 11 of the 21 cross-sectional studies (52.4%) , 75, 77,In the six longitudinal observational studies 23.1%) , 40, 60,.1% , 40,According to the PEDro scale, scores of the methodological quality of three experimental longitudinal trials , 62, 65 The quasi-experimental study , which iThe present study performed a systematic review of the literature on SRH after stroke. Most of the studies employed general and comparative direct questions to assess SRH. The cross-sectional and longitudinal observational studies revealed significant associations between poor SRH, stroke, and other important health outcomes. In addition, SRH was reported to predict the occurrence of stroke. However, the association between SRH and stroke mortality remains unclear. To date, few studies have evaluated the effects of interventions on SRH. However, those that do exist found two types of interventions that effectively improve the SRH of individuals with stroke.Very Good, Good, Fair, Bad, and Very Bad) and the European version . Although some differences were found, both versions were highly correlated after the items were resized to allow for comparison [SRH was mostly evaluated by direct questions, although there was great variability in the structure of these questions and the response items. Jürges et al.  reviewedmparison . Comparamparison .Studies investigating elderly people compared general and comparative direct questions but reported different results , 81. OneThe total scores of QoL/HRQoL instruments, such as the SF-36 and the SF-12, were used to evaluate SRH , 62, 65.The most common analog visual scale for SRH assessment is the EQ5D . This scIn most studies, poor SRH was associated with stroke. Since SRH is a measure and indicator of health status , this fiSome studies did not find any association between poor SRH and stroke. Tuomilehto et al.  observedMany studies have demonstrated the predictive power of SRH for mortality and morbidities among various populations , 36, 69.Only two of the four experimental longitudinal studies found improvements in SRH , 67. ThiSRH was used as an outcome less often in experimental longitudinal studies than in observational studies. Future experimental studies should examine SRH evaluation due to its importance and informative capacity for individuals’ health . In addiDirect questions were the most commonly mentioned and recommended way to measure SRH in subjects with stroke. It is unclear whether a certain type of question is superior to others, but general SRH is most commonly used. Cross-sectional and longitudinal studies have shown significant associations between SRH and several relevant health outcomes, while few experimental studies have evaluated the efficacy of interventions for improving SRH after stroke.


One of the most intriguing questions in biological science is how life originated on Earth. A large number of hypotheses have been proposed to explain it, each putting an emphasis on different events leading to functional translation and self-sustained system. Here, we propose a set of interactions that could have taken place in the prebiotic environment. According to our hypothesis, hybridization-induced proximity of short aminoacylated RNAs led to the synthesis of peptides of random sequence. We postulate that among these emerged a type of peptide(s) capable of stimulating the interaction between specific RNAs and specific amino acids, which we call “bridge peptide” (BP). We conclude that translation should have emerged at the same time when the standard genetic code begun to evolve due to the stabilizing effect on RNA-peptide complexes with the help of BPs. Ribosomes, ribozymes, and the enzyme-directed RNA replication could co-evolve within the same period, as logical outcome of RNA-peptide world without the need of RNA only self-sustained step. Life on Earth appeared 3.7–4.1 b. y. ago, shortly after the planet formed ,3,4. AnyMany hypotheses adopt a specific process—such as RNA replication, metabolism or membrane formation—as the organizing force of early events leading to the origin of life . An overAn attempt to explain this dilemma is the RNA world hypothesis ,9,10,11,Another argument used to support the RNA world hypothesis is the existence of different aminoacyl-tRNA synthetases (aaRS) for the same tRNA (Cys) found in methanogenic archaea and some bacteria ,26. AccoDespite the general agreement among supporters of RNA world hypothesis that a self-sustained ribozyme should have existed independently before the RNA/protein world came into being, there are a number of caveats with the hypothesis pointed so far ,28,29,30Irrespective of whether an RNA-only self-sustained system capable of Darwinian selection is possible, the key question in the origin of life as we know it is how translation of genetic information came into being? Several mechanisms establishing correspondence between codons and their cognate amino acids have been suggested, either direct or via RNA adaptors.Most of these are based on stereochemical compatibility (“code”) ,42,43,44On the topic of ribosome formation, advocates of the RNA world hypothesis are divided as well: Some believe the peptidyl transferase center first appeared in the large ribosomal subunit (LSU) ,24,37,46All hypotheses for the origin of life incorporate peptides as stabilizing factor, the differences being in the role they played in code formation. All variants of RNA world hypothesis by definition exclude the peptides from the initial events and place the involvement of peptides after code formation, with a variety of explanations of polymer transition and ribosome formation. In contrast, the hypotheses that reject the RNA world postulate that the peptides should have been part of the very first steps in the establishment of the genetic code ,48,49,50The two sets of theories also generally differ on the process that initiated Darwinian selection. The non-RNA world hypotheses theories put forward the emergence of proto-translation system, while the RNA world ones assume RNA replication as the trigger of selection. However, the gene set of the LUCA is thought to include largely highly conservative genes involved in translation ,52, giviRecently, Williams and colleagues rejected the RNA world and proposed a hypothesis in which the PTC in combination with peptides was central to the origin of life. The PTC originated via stabilizing interactions between peptides and RNA and subsequently evolved into the ribosome . In theiIn the post-LUCA time, there is only one type of system capable to recognize and couple specific amino acids to specific tRNAs: Amino-acyl transferases (aaRS class I and aaRS class II). The genetic code is embedded into the aaRS structure putting these proteins in the unique position as the only ones to “know” the genetic code. Even so, aaRS are mostly dismissed from the initial processes of codon formation due to variety of reasons, some of which were described above. In addition, it was shown that aaRS class I share protein HUP class domain with other proteins such as USPA, ETFP, photolyase, and PP-ATPase ,55. PhylContrary to this view, Schimmel was the first to propose that aaRSs and the genetic code must have coevolved “getting fitted” to each other ,57. ReceWe do not see controversy in the fact that the aaRS class I could evolve at a later stage before LUCA time and to share HUP class domain with another nucleotide-binding proteins. Additionally, based on the fact that today aaRSs are the only enzymes capable to aminoacylate the correct tRNAs with the correct amino acids we reason that some proto-forms of aaRSs might have played a role in codon formation. We postulate that the evolution of aaRS begun as a very short peptide that facilitated amino-acylation of RNA with low  specificity—we call it “bridge peptide” or BP. The BP was synthesized initially by chance as a result of the physical proximity of hybridized short random amino-acylated RNAs. The bridge peptide facilitated the emergence of a self-sustained RNA-peptide complex supporting a primitive translation. This concept offers an explanation for the development of the genetic code, the ribosome, the RNA relics , and RNA replication with minimal assumptions.Here, we propose a hypothesis that could have led to the emergence of RNA-encoded peptide synthesis. The events described may not have been strictly sequential.Prebiotic synthesis: As a result of chemical prebiotic expansion activated ribonucleotides ,61, (revAt temperatures between 1 °C and to denaturation (about 55 °C) temperature, short random RNA oligos would get stabilized via intra and intermolecular hybridization based on Watson-Crick base pairing, forming complexes of various 3D shape and size. Larger hybridized regions would confer greater stability and would be selected for. Highly self-complementary RNAs would be unlikely to exist, forcing intermolecular hybridization of short sequences and the emergence of complexes of several RNA oligos. The formation of RNA complexes also assumes a thermal cycle that would drive the process by sequential denaturation (~55–100 °C) and re-annealing (<55 °C) phases. Frequent repetition of the thermal cycle and stability selection would favor accumulation of complexes with higher degree of complementarity and higher GC content.Non-enzymatic aminoacylation between 2′ or 3′ positions of ribose and activated amino acids could occur ,82,83. IAminoacylated RNAs would be involved in complex formation (as in step 1.), bringing some of the aminoacylated RNA 3′-ends in close proximity. This would promote peptide bond formation between two adjacent amino acids , most liAminoacylation of RNAs would be governed by the availability of amino acids. Different experiments simulating prebiotic Earth conditions have proposed the molar ratios of amino acids that could have existed, reviewed in References ,66. WhilThe most “useful” property of HDPs would be to increase the stability of RNAs binding them. Under primordial conditions, in the absence of specific selection, increased time of existence (as a consequence of greater stability) is a measure of fitness of the complexes. In line with this thinking, several studies have suggested that the major role of early peptides was not catalytic, but to stabilize RNA ,91,92. TDue to the entirely probabilistic nature of the “chemical code” only very few of the HDPs will possess this property. We call these HDPs RNA-binding peptides (RBPs) a. As a rAs the BP is a short peptide, it would not possess activity specific for longer folded ones. As a result, the function(s) would directly reflect the properties of the incorporated amino acids. One possible mechanism of BP action is to link amino acids with opposite charge. The positively charged end would contact RNA and the negatively charged would attract amino acids of positive charge (their incorporation would provide the greatest RNA binding ability of RBP and greatest stability benefit to the RNA complex). The rest of the residues in the BP would facilitate the requirements like space between the charged amino acids or shape for bridging the RNA and the amino acid with some degree of specificity.Most likely the specificity of interaction between BP and its amino acid might have been very low. Even so, this preference would make the probability of insertion of the cognate amino acid into newly formed hybridization-dependent peptides higher than random. This property (to bind simultaneously a specific RNA and attract specific amino acid or group of amino acids) would enable for the first time the sequence of a template RNA to determinate the sequence of a newly formed peptide . We callWhile both BPs (and RBPs) could “work” within different RNA complexes, the ones that matter, in view of selection, would be those that stabilize their native complex. Selection forces would favor BPs that put “useful” amino acids at “successful” positions augmenting the fitness of RBPs. This would result in a positive feedback loop that would increase the chance of synthesis of peptides augmenting the stability of the RNA-peptide complexes. The contribution of BPs towards increased stability of their native complexes would be a consequence of the higher complexity of the synthesized HDPs within these complexes .At this point though, only non-enzymatic RNA replication would exist with the support of wet/dry natural cycles. Even if BPs would increase the stability of the RBP/BP producing complexes, they would be degraded eventually in the absence of enzymatic replication of RNA. The RNAs of the RNA-peptide complexes would change constantly. The supply of random RNAs (able to recombine with the RNA(s) already present in the RNA-peptide complexes) would be a permanent source of variability providing new properties to the HDPs—importantly to the RBPs and BPs subsets of these. One type of peptide would have a revolutionary effect on stability of RNA-peptide complexes and would be established relatively early—the RNA replication peptide (RRP). The consequence of having RRP would be the reproduction of the BP-stabilized RNA-peptide complexes. RRP most likely would derive from a BP or RBP and would facilitate the formation of phosphodiester bonds at temperatures at which hydrogen bonds could exist. The catalytic center of the nucleotidyltransferases and all modern polymerases is composed of two metal ions (like Mg++) positioned at a specific distance in a way that the first Mg++ deprotonates the 3′-OH of the RNA primer and attacks the alpha-phosphate of the incoming NTP. The second Mg++ stabilizes the NTP moiety ,94. We iWe presume that following this initial stage all components of the translation system would co-evolve in a stepwise way. For example, from certain point on, RNAs being aminoacylated and involved into peptidyl transfer should specialize to serve as tRNAs only. That would be possible if the longer peptides, evolved forms of BP, could recognize a specific 3D structure of the proto-tRNAs. In the predominantly GC sequences found in the RNA-peptide complexes, CCA-3′ is a particularly good recognition pattern. From that moment on CCA-3′ would have likely become the signature of the aminoacylation site of all tRNAs.Specialization of ribosomal Large Subunit—LSU will start with evolution of peptidyl transferase center (PTC). This event by itself would have been enormously beneficial for stability, since the peptidyl transfer had so far occurred “by chance”—due to close proximity of two adjacent amino acids brought together by hybridized aminoacylated RNAs.The evolution of peptides to proteins would occur from small motif to domains and finally—folded proteins. The functional repertoire would evolve as well from the very limited functional ability of short peptides to longer folded peptides and finally to proteins forming complex catalytic centers with almost unlimited possible functions. Therefore, the peptide/protein evolution offers a reasonable explanation why the ribozymes have been selected in the first place. The ribozymes, including the ribosome would have been selected during the RNA-peptide world where sophisticated folded proteins were still non-existent . An impoAccording to our hypothesis, the BP would influence the formation of the first proto-codon usage in the proto-translation system. Simultaneously, with the process of adding new codes, the role played by amino acid abundance  would decrease. The increase in complexity and the takeover of coded incorporation for all 20 canonical and some non-canonical amino acids should have been a gradual process of co-evolution of tRNAs, proto-forms of aaRS, the ribosome and peptide functions. The standard 3 base codon as we know it should be established as a result of the most advantageous fit of tRNA/ribosome structure with the functional requirement of amino acids.Each step of addition of new tRNA/amino acid combination should have been tolerable in its intermediate form—for example, lysine could have started to aminoacylate formerly arginine-specific tRNA, since both have similar charge and structure and initially may have covered similar functions. We imagine the same could have happened with tRNAs having different anti-codons but encoding the same amino acid. This would lead to the same amino acid being positioned more frequently in different peptide positions. These tolerable intermediate changes would have been possible due to the relative simplicity of the early complexes. With the acquisition of catalytic functions by the polypeptides, changes to the coding system would become more difficult and eventually impossible. However, in the meantime, the co-evolution of the protein coding apparatus would have led to the efficient translation system of LUCA with “well-fitted” tRNAs, aaRS, ribosomes and almost 600 genes working in synchrony. A similar mathematical concept has already been developed by Reijer Lenstra through codon progressive symmetry breaking .We have developed an RNA-peptide hypothesis for the origin of life with the principal assumption that if a structure-function is beneficial and selected during the origin of life, it could not be replaced later. This concept, called “the rule of continuity”, has been suggested before ,52,97, bThe application of the rule of continuity to translation, suggests that aaRSs (or their proto-forms) should have been important from the very inception of the process. Yet, phylogenetic analyses of modern aaRSs bring up the “protein evolution paradox”, highlighting the fact that by the time the ancestors of the aaRSs gave rise to specific proteins, extensive evolution of their domains had already produced a substantial diversity of other enzymatic and nucleotide-binding folds. This was argued to require high-fidelity translation and imply that specificity of amino acid–codon correspondence was initially determined by RNA molecules , followe“…takeover by aaRSs of the key role in the pairing of amino acids with the cognate anticodons ”.The transition of the chemical nature of the molecules determining the genetic code constitutes a major breach of the rule of continuity. Our hypothesis avoids this by postulating the existence of a proto-form of aaRS (bridge peptide and its evolved forms) involved in codon formation by facilitating proximity between a specific RNA and specific amino acid. In addition, BP is capable to stabilize the RNA-BP complex forming a feedback loop  where thWe explain the beginning of translation as a process of mutual cooperation between RNA and peptides as suggested by Williams and colleagues ,28,52, w++ or Fe++ in appropriate spatial proximity. Such an RNA replication peptide would provide a huge leap in stability, as part of an already established RNA-coded peptide complex. In addition, it seems likely that an efficient RNA replication ribozyme during early evolution would diminish RNA reactivity by forming complementary chains whose hybridization would eliminate any significant 3D structures. While, ways to form single stranded RNA could be envisaged—i.e., by toehold-based strand displacement [The rule of continuity is also at odds with the lack of any remnants of “RNA world” replication machinery, preserved by evolution, although ribozymes capable of RNA replication have been selected in the lab ,18,19,20lacement , it is uPenny and coauthors  have for“1. Without heritable templates, it is unlikely that multiple copies of a favorable peptide can be produced accurately and repeatedly.2. Peptides produced in a pre-genetic phase cannot improve for lack of preferential replication of favored forms, while genetically encoded RNA enzymes would predominate being improved via Darwinian evolution. 3. Complexity of modern translation is the result of an optimization process, impossible without natural selection of the early ribosomal machinery, on the code itself, and on early genes.”In our hypothesis, heredity begun because of BP and will exists as a result of increasing stability of complexes. The ability of the bridge peptides to bring specific amino acids into the same position would improve the odds to reproduce similar HDPs and selection would lead to more copies of the more favorable ones. The accuracy of bridging would also improve by selection  allowingSeveral hypotheses have been put forward that explore the advantages of RNA-peptide cooperation during the initial stages of the origin of life. Regarding the difficult-to-explain RNA replicating ribozyme George Fox in his “abbreviated RNA world” hypothesis suggested: “If peptide synthesis arises quickly, then there will neither be time nor need for extensive catalysis of biochemical reactions by RNA. If reasonable, the rapid appearance of a translation system may even eliminate the need to validate the RNA world by demonstrating the self-replicating RNA system that has proven experimentally difficult to achieve. ”Other authors, mathematically modeled the likelihood of emergence of a self-assembled coding system based on one heteropolymer (RNA Coding World) or two different heteropolymers (Protein Coding World), concluding the second is far more plausible than the “RNA world” scenario .While formation of the non-coded HDPs in our hypothesis could be considered a ribozyme function, the lack of positive feedback for selection means that it is not carried out by a self-sustained entity. Similar to Williams and team ,28, our Thus, it replaces an RNA-only self-sustained stage (RNA-world), with an RNA stage capable to produce HDPs . A very This applies for ribonucleotides (Nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD) capable of electron exchange for oxidation-reduction reactions. Theoretically, these reactions could be carried out by folded proteins, however these were not available and the selection went in that direction.Williams and Petrov developed a concept ,102 in wE. coli 16S and 23S RNA contain the entire set of transfer RNAs for all twenty amino acids as well as sequences encoding key fragments of ribosomal proteins, polymerases, ligases, etc. [We assume that immediately after the initial stabilization of the RNA/RBP/BP complex, co-evolution of the codon/A/P sites, PTC, tRNA, and the first peptides involved in RNA replication, ribosome stabilization, and more will occur . Therefoes, etc. . AccordiWithin this framework we could envision the evolutionary fate of aaRSs, based on the urzyme hypothesis ,30. The Caetano-Anolles and Caetano-Anolles published their variant of the origin of translation by including peptides named elementary functional loops (EFL) . EFLs weIn their latest review Koonin and Novozhilov described the biggest challenges for any theory explaining the origin of the Standard genetic code (SGC) and translation: “Evolution of the code is intimately linked to the origin and evolution of the translation apparatus itself... Evolution of translation involves an intrinsic and formidable paradox: A high-fidelity translation system requires a number of functional proteins, but the maintenance of such proteins is impossible without a reliable translation system.” . EssentiOur model is completely opposed to such a concept. We view code and translation as impossible to separate. This notion is usually countered by the argument that evolution had no foresight to create life. However, we believe foresight is unnecessary. The sole driving force of evolution from HDPs to coded translational system in our hypothesis is the selection of more stable forms. The one capable of coded translation would also be the most stable. Stability selection is the reason why the amino acids being incorporated by bridging will be those that contribute most to the overall stability of the complex, not the most abundant. Precisely because the nature has no foresight, it is unlikely that the relative abundance of the amino acids during the prebiotic synthesis would fit well to the “needs” of newly formed RNA-peptide complexes. For that reason, contrary to Higgs , in our Our hypothesis also avoids the apparent paradox of the need of a high fidelity translation system for protein evolution before it had evolved by postulating the gradual interrelated increases in complexity and fidelity of the translation apparatus composed of co-evolving components. It thus suggests a path for the Darwinian evolution of a self-sustained RNA-peptide entity.In summary, our hypothesis of the origin of life differentiates the following initial three major periods in its timeline :1. Prebiotic period during which very short 4–20 nt of the RNA and activated amino acids were synthesized.2. “RNA stage” in which RNA capable of recombination and importantly performing a low efficiency peptidyl transfers resulted in non-coded short hybridization-dependent peptides.3. “RNA-peptide world” beginning when the first amino acid was incorporated by bridging . This in


N7-methylguanosine (m7G), which forms the “cap” on the first 5′ nucleotide. Identification of the 5′ nucleotide on mRNA is necessary for determination of the Transcription Start Site (TSS). We explored the effect of various reaction conditions on the activity of the yeast scavenger mRNA decapping enzyme DcpS and examined decapping of 30 chemically distinct cap structures varying the state of methylation, sugar, phosphate linkage, and base composition on 25mer RNA oligonucleotides. Contrary to the generally accepted belief that DcpS enzymes only decap short oligonucleotides, we found that the yeast scavenger decapping enzyme decaps RNA transcripts as long as 1400 nucleotides. Further, we validated the application of yDcpS for enriching capped RNA using a strategy of specifically tagging the 5′ end of capped RNA by first decapping and then recapping it with an affinity-tagged guanosine nucleotide.Eukaryotic mRNAs are modified at their 5′ end early during transcription by the addition of  The capping occurs at an early stage of transcription and is involved in splicing, stabilizing and directing the transcript to the cytoplasm where it is translated. In higher eukaryotes, the canonical m7G cap can be further modified by 2′-O-methylation at nucleotide positions +1 (Cap 1) and +2 (Cap 2) of the RNA. Both capping and decapping of mRNA are critical components of gene expression. The enzymes responsible for the decapping have been described from two distinct protein families, the Nudix pyrophosphohydrolases1 and the HIT family of pyrophosphatases3. The distinguishing characteristic of catalysis by the two families of decapping enzyme is the resulting products of the reactions  and a 7-methyl-guanosine diphosphate (m7GDP). A few exceptions have been reported where nudix decapping activities result in mixed diphosphate and monophosphate 5′ RNA ends1. On the other hand, HIT decapping enzymes, including the so-called scavenger decapping enzyme DcpS, hydrolyze the phosphodiester bond between the gamma and beta phosphates to leave a 5′-diphosphate-RNA (ppRNA) and a 7-methyl-guanosine monophosphate5 (m7GMP) and are independent of divalent metal ions6. An exception to the HIT family is the FHIT subfamily some members of which were found to leave mixed 7mGDP and 7mGMP8. Recently an additional type of decapping enzyme was described from trypanosomes. TbALPH1 an ApaH-like phosphatase removes 7mGMP from the cap structure to leave a diphosphate 5′ end9.The 5′ ends of eukaryotic messenger RNA (mRNA) carry a 5′ to 5′ triphosphate linked 7-methyl-guanosine , as well as the identity  and methylation status  of the cap nucleotide, and the length of the internucleotidic phosphate bridge . Our results indicate that DcpS can decap most guanosine caps; in contrast, it shows no activity towards adenosine, cytidine or uridine capped RNAs.The yeast scavenger decapping enzyme is encoded by the 12 to enrich primary prokaryotic RNA transcripts by capping their 5′ triphosphate with 3′-desthiobiotin-GTP (DTB-GTP). In an effort to extend this method to eukaryotic mRNA, we demonstrate here that yDcpS can decap the 5′ end of m7G-capped RNA transcripts of a length from 90 to 1400 nucleotides without appreciable length bias. Further, the yDcpS-treated transcripts can be recapped with DTB-GTP and recovered after binding to, and eluting from, streptavidin beads.In a previous work, we developed a method termed Cappable-seq.et al.4 and Cohen et al.13 presented data indicating that human DcpS (hDcpS) does not decap RNAs longer than approximately ten nucleotides. Cohen et al. showed that the nematode DcpS was even more limited in its ability to decap oligonucleotides longer than 3 nucleotides13. This precept of acting solely on capped short oligonucleotides has been attributed to all homologous HIT enzymes14, despite Salehi et al.15 identifying a HIT protein in Schizosaccharomyces pombe, Nhm1, which was determined to decap long mRNA. Nhm1 has about 70% amino acid sequence similarity to the yeast and human DcpS enzymes. As these two disparate DcpS activities, one decapping only short RNA13 the other decapping mRNA15 are conflicting, we decided to characterize yDcpS with respect to its ability to decap RNA of different lengths. We used recombinant yDcpS expressed in E. coli and purified to homogeneity as described in Methods.Liu 7GpppA, as the characterization of DcpS typically involves pyrophosphorolysis of cap analogs which are dinucleotides of NpppN structure17. Cap analogs can be considered as a capped RNA of one nucleotide in length. Both Malys et al.18 and Liu et al.19 demonstrated the pyrophosphorolysis of m7GpppG dinucleotide with a rate constant of 0.012/sec for yDcpS and 0.09/sec for hDcpS, respectively. We compared the relative activity of hDcpS and yDcpS towards the m7GpppA dinucleotide. We determined the decapping rate at 50 µM m7GpppA dinucleotide by LC/MS analysis , the ionic strength was greater (100 mM KOAc) and the pH was higher (pH 7.0). Thus the catalysis of the dinucleotide observed with our preparation of yDcpS is consistent with published literature. The human DcpS was shown by Liu et al.19 to display negative cooperativity as indicated by a 10-fold higher rate at lower dinucleotide concentration (10 nM). The comparison of rates between human and yeast DcpS in our study was performed at much higher dinucleotide concentration (50 µM) which was well beyond the dinucleotide concentration necessary to demonstrate a 10 fold reduced rate.We first looked at catalysis of the dinucleotide cap analog msis Fig. . The resin vitro, was used as a substrate. As we have previously observed that addition of extraneous RNA acts as a competitive inhibitor for the reaction, we added E. coli RNA to the reaction shown in Fig. To determine whether yDcpS can decap an RNA longer than 15 nucleotides, a 25mer Cap 1 RNA synthesized and capped In vitro decapping of DcpS was assessed at various ionic strength and pH conditions by using a synthetic 25mer Cap 0 RNA 3′ modified with a 6-carboxyfluorescein (3′-FAM) label. Substrate and product of the decapping reaction were resolved and quantified by capillary electrophoresis (CE)  Fig. . As showTo determine whether the substrate secondary structure has an impact on the yDcpS activity, complementary synthetic RNA oligos were annealed to the 3′-FAM-labeled 25mer Cap 0 RNA to create a blunt end, a 10-nucleotide 5′ recessed cap, or a 5-nucleotide 5′ extended cap end. The extent of decapping was compared to the single-stranded capped 25mer. All 3 double-stranded substrates were decapped, with the 5′ extended cap substrate being decapped as efficiently as the single-stranded control, while the 5′ recessed and blunt ended caps were more resistant to decapping. These results indicate that while yDcpS prefers unstructured 5′ ends, structured ends can also be decapped at higher enzyme concentration  and Cap 2 (m7GpppNmNm), whereas Cap 0 (GpppN) has not been identified24. Additionally, a number of still uncharacterized cap structures have been reported to occur on short RNAs25. Hence, we sought to determine the susceptibility of 30 distinct 25mer RNAs featuring different cap structure permutations to decapping with yDcpS  were incubated for 60 minutes at 37 °C with increasing concentrations of yDcpS varying by a factor of three. The reactions were then analyzed by PAGE and the relative extent of decapping determined after staining and imaging the gels. A typical set of gel images was compiled in Fig. NAs Fig. . As for 7G from the cap in the form of m7GMP leaving a 5′-diphosphate on the 5′ terminus of the RNA4. 5′-diphosphate RNA is a known substrate for in vitro capping using the Vaccinia virus Capping Enzyme (VCE)27. We took advantage of these two reactions to tag a m7G-capped 25mer RNA with an affinity group, by first decapping the RNA with yDcpS, and then recapping it by treatment with VCE and DTB-GTP12. These reactions were performed in the presence of excess total E. coli RNA in order to simulate a complex mixture of RNA as would be found in total eukaryotic RNA. As shown by gel electrophoresis for a Cap 1 RNA . The produced RNA transcripts were capped with GTP by VCE to yield m7G-capped RNAs. The mixture of capped transcripts was then decapped by incubation with yDcpS. As a control, an aliquot of the m7G-capped transcripts was incubated in the absence of yDcpS. Both sets of transcripts were subjected to capping with DTB-GTP by VCE. The recapped products were exposed to streptavidin beads, and after washing steps to remove any unbound material, recovered by elution with biotin. As shown in Fig. 12.After demonstrating the efficient decapping and recapping of m7GpppN, m7GpppNm, m7Gpppm6A, or m7Gpppm6Am) does not significantly affect their susceptibility to decapping by this enzyme. We also examined non-canonical m7G caps where the ribose moiety was substituted with arabinose or deoxyribose. Although these caps have not been shown to exist in nature, they are also decapped by yDcpS.In characterizing yDcpS, we have shown that the methylation status of various canonical guanosine caps , while the human DcpS did not. We found that yDcpS, resembling hDcpS, did not decap 2,2,7-trimethylated guanosine. The in vitro characterization of yDcpS presented here provides evidence that yDcpS should be a useful reagent for generating recappable 5′ ends from capped RNAs. We demonstrate that yDcpS decaps m7G-capped RNAs from as short as 25 to at least 1400 nucleotides, leaving a 5′-diphosphate end. The implications of these in vitro findings suggest that it is possible that the in vivo function of the scavenger decapping enzymes may be more complex.Under the conditions described, yDcpS decaps the majority of the guanosine-based caps, regardless of the nucleotide at position +1. The enzyme prefers m12 to include eukaryotic mRNAs. Cappable-seq has been used to determine the transcription start sites of primary transcripts of prokaryotes at single base resolution via the attachment of a biotinylated GTP analogue to the 5′-triphosphate end of primary RNA transcripts by action of the Vaccinia capping enzyme (VCE). The extension of this method to canonical eukaryotic mRNAs requires the removal of the m7G moiety to generate a recappable end (such as a tri- or diphosphate 5′ end), which we now show is attainable with the use of yDcpS. Therefore, we anticipate being able to enrich eukaryotic mRNA by combining yDcpS-mediated decapping followed by recapping with affinity tagged GTP and generating libraries for high-throughput sequencing. Progress towards this goal is underway in our laboratory.The process of decapping and recapping with an affinity-tagged nucleotide would be advantageous for identifying and enriching 5′ capped termini of mRNAs in eukaryotes. This would expand the utility of the prokaryotic Cappable-seq method7GpppA dinucleotide cap analog m7G(5′)ppp(5′)A was obtained from New England Biolabs, Ipswich, MA (NEB). The 5′-[m7Gppp]GUAGAACUUCGUCGAGUACGCUCAA[FAM]-3 was purchased from Bio-Synthesis, Inc. The complementary oligos used for secondary structure experiments for blunt end (UUGAGCGUACUCGACGAAGUUCUAC), 5′ cap extension (UUGAGCGUACUCGACGAAGU), and 5′ cap recession (UUGAGCGUACUCGACGAAGUUCUACAAUGACCAUC) were purchased from Integrated DNA Technologies (IDT). The 5′-triphosphate 25mer RNAs  utilized for the generation of m7GpppN, m7GpppNm, m7dGpppN, GpppN, and m7AraGpppN were individually synthesized as previously described28. The 5′-monophosphate 25mer RNAs  utilized for the generation of m7Gpppm6A, m7Gpppm6Am, GppppA, GppA, and NpppG  were synthesized by standard phosphoramidite chemistry on an ABI 394 DNA/RNA synthesizer. For the synthesis of 5′-p-m6AUAGAACUUCGUCGAGUACGCUCAA-3′, a N6-Methyl-A-CE phosphoramidite from Glen Research was utilized. Detailed protocols for the enzymatic or chemical generation of the 5′-capped 25mer RNAs utilized in this study are described in the Supplementary Information section.The mSaccharomyces cerevisiae yDcpS (YLR270W) was codon optimized for E. coli expression and designed with an amino terminal His tag to be expressed from a T7 promoter in plasmid pET28a  by chromatography over HisTrap, SP, and Q resins. The protein’s size as determined by SDS-PAGE is consistent with its predicted molecular weight  was purchased from Enzymax LLC, Lexington, KY. The protein concentration of DcpS was determined by OD7GpppA dinucleotide cap analog were terminated by 70 °C heat inactivation.Unless otherwise noted, the decapping was carried out at 37 °C in the decapping buffer 10 mM Bis-Tris pH 6.5, 1 mM EDTA. With the exception of the reactions containing FAM-labeled RNAs (see below), all reactions using capped 25mer RNA substrates were terminated by the addition of 1 volume of 2X RNA loading dye (NEB). The reactions using the mIn vitro decapping reactions were carried out in a 20 µL reaction containing 10 mM Bis-Tris pH 6.5, 1 mM EDTA, 500 nM substrate RNA , and 60 nM yDcpS at 37 °C for 60 minutes, unless otherwise indicated (Table S. cerevisiae Cet129. The 5′-monophosphate standard was generated by treating the 5′-triphosphate/3′-FAM-labeled 25mer with Apyrase (NEB). The 5′-hydroxyl standard was generated by treating the 5′-triphosphate/3′-FAM-labeled 25mer with calf intestinal phosphatase (NEB). The cap 0 standard was generated by treating the 5′-triphosphate/3′-FAM-labeled 25mer with Vaccinia virus capping enzyme  in the presence of GTP and S-adenosylmethionine (SAM). The RNA standards were purified using RNA Clean and Concentrator kit (Zymo Research). The identity of the standards was verified by intact mass spectrometry. The 25mer Cap 1 RNA was generated by treating the chemically synthesized 3′-FAM-labeled Cap 0 25mer RNA with mRNA Cap 2′-O-Methyltransferase (NEB) in the presence of SAM. The Cap 1 25mer RNA was purified using Monarch RNA Cleanup kit . Complete methylation was verified by digestion of RNA with the Nucleoside Digestion Mix  and LC-MS analysis.The reaction conditions for decapping consisted of 100 nM 5′-capped 25mer RNA and 0, 0.3, 0.9, 2.7, 8 or 25 µM DcpS in a 10 µL reaction volume in 10 mM Bis-Tris pH 6.5 and 1 mM EDTA. The reactions were incubated for 60 minutes at 37 °C and terminated by the addition 2 µL of Proteinase K (NEB) which had been supplemented with 0.1 reaction volume of 1 M Tris-HCl pH 8.0, and further incubated for 5 minutes at room temperature. To each 12 µL reaction was added 12 µL of RNA loading dye (2X) (NEB). This mixture was heat denatured at 80 °C for 3 minutes, and an aliquot of 5 µL was loaded onto a 15% polyacrylamide TBE-Urea gel (Invitrogen). The gel was run at 180 volts for 75 minutes. The RNA was visualized with SYBR Gold stain.E. coli RNA, was incubated for 2 hours at 37 °C. Before addition of the yDcpS, a 2 µL sample of the reaction was removed. The yDcpS reaction was terminated by addition of 4 µL of 1 M Tris HCl pH 8.0 and 2 µL of Proteinase K. The reaction was further incubated for 30 minutes at 37 °C and then heated to 94 °C for 3 minutes. The RNA was purified by binding to Ampure XP beads (Beckman Coulter) by addition of 2 volumes of beads, and to that final volume additional 1.5 volumes of ethanol. After washing the beads with 80% ethanol, the RNA was eluted in 30 µL of 1 mM Tris pH 7.5 and 0.1 mM EDTA. A 20 µL reaction containing 1X VCE buffer (NEB), 10 µL of the eluted RNA, 0.1 mM SAM, 0.5 mM DTB-GTP (NEB) was split into two 9.5 µL reactions: in one tube was added 1 µL of VCE and in the other tube was added 1 µL of water. The reactions were incubated at 37 °C for 30 minutes. Samples were electrophoresed on a 15% TBE-Urea gel (Invitrogen) and stained with SYBR Gold  transcription of different DNA templates generated from an FLuc plasmid, each digested with one of the following restriction endonucleases: NlaIV, HpyCH4V, Hpy166II, PsiI, AflIII, EcorI, BspQI, BcoDI, or EcoRV in separate reactions. The resulting RNA transcripts comprised 90, 171, 240, 419, 587, 681, 896, 1117, and 1432 nucleotides, respectively. The individual transcripts were combined in approximate equal amounts. The transcript mixture was subsequently capped by standard VCE capping reaction with GTP and SAM.mRNA was loaded onto 6% or 15% PAGE Urea gels and electrophoresed for 75–85 minutes at 180 V. The gels were stained with SYBR Gold and imaged with an AlphaImager HP. The images were imported into Photoshop, inverted and levels were auto-adjusted.Supplementary Information