Modifications of m6A are dynamic and reversible in mammalian cells, which have been proposed as another layer of epigenetic regulation similar to DNA and histone modifications. m6A RNA methylation is involved in all stages in the life cycle of RNA, ranging from RNA processing, through nuclear export, translation modulation to RNA degradation, which suggests its potential of influencing a plurality of aspects of RNA metabolism. All of the recent studies have pointed to a complicated regulation network of m6A modification in different tissues, cell lines, and space–time models. m6A methylation has been found to have an impact on tumor initiation and progression through various mechanisms. Furthermore, m6A RNA methylation has provided new opportunities for early stage diagnosis and treatment of cancers.First identified in 1974, m6A RNA methylation, its multiple functions and potential significance in cancer.Herein, we review the chemical basis of m Termed as “epitranscriptome,” RNA harbors the potential of being dynamically and reversibly regulated by the addition and removal of distinct chemical moieties, which extends the RNA repertoire and alters its chemistry in various ways .N6-methyladenosine (m6A) RNA methylation is one of the most prevalent modifications, accounting for about 50% of total methylated ribonucleotides and 0.1–0.4% of all adenosines in total cellular RNAs [6A sites are found within the consensus sequence Rm6ACH , which is consistent with the enriched binding motifs observed in studies of methyltransferase-like 14 (METTL14), methyltransferase-like 3 (METTL3), and Wilms tumor 1-associated protein (WTAP)  [Among more than 100 types of post-transcriptional modifications identified in RNAs so far –4, N6-mectively) . There hctively) .6A RNA methylation are dynamic and reversible in mammalian cells, which have been proposed as another layer of epigenetic regulation similar to DNA and histone methylation. The biological function of m6A RNA methylation is highly variable depending on context and little is known about the underlying mechanisms; however, emerging evidence has suggested that m6A modification plays a pivotal role in pre-mRNA splicing, 3′-end processing, nuclear export, translation regulation, mRNA decay, and miRNA processing [6A RNA methylation has implied its important and diverse biological functions in mammals including tissue development, circadian rhythm, DNA damage response, sex determination, and tumorigenesis [Histone methylation in localized promoter regions is one kind of histone codes for chromatin packing and transcription. Generally, methylation of H3K4, H3K36, and H3K79 is linked to gene expression activation, whereas H3K9me2, H3K9me3, H3K27me3, and H4K20 are associated with gene repression . Modificocessing . Growingigenesis .6A RNA methylation participates in tumorigenesis, invasion, metastasis, and drug resistance and how to use m6A modification as new diagnostic biomarkers and therapeutic targets.In this review, we will discuss how m6A methylase complex is consisted of at least five “writer” proteins where METTL3 acts as the catalytic core. METTL14 serves as structural support for METTL3, while WTAP stabilizes the core complex. RNA-binding motif protein 15 (RBM15) helps to recruit the complex to its target sites. The molecular function of KIAA1429 is still elusive [6A modification [6A methyltransferases and demethylases probably determines the dynamic regulation of m6A modification.The mfication , 17. The6A marks on mRNA. They display a 10- to 50-fold higher affinity for m6A-methylated mRNA than for unmethylated mRNA [6A sites and produce different effects on gene expression. For example, YTHDF2, the first identified m6A reader, increases turnover of m6A-modified mRNA by promoting co-localization with decay factors [6A sites near the stop codon, which then interacts with the translation initiation factor eIF3, causing a stimulatory effect on translation in mammals [6A RNA methylation comes into play during stress responses where preferential m6A methylation of the 5′-UTR of stress response genes leads to cap-independent translation, which involves nuclear relocalization of YTHDF2 to prevent m6A removal by FTO [YT521-B homology (YTH) domain-containing proteins including YTHDF1-3, YTHDC1, and YTHDC2 have recently been identified as “readers” of mted mRNA –22. When factors , 24. Con mammals . Anotherl by FTO , 27. YTHl by FTO , 29. YTHl by FTO .6A sites, indicating its important role in promoting pri-miRNA processing [As to the processing of pri-miRNAs, hnRNPA2B1 has the capability of recruiting DGCR8 to RNA by targeting mocessing , 32.6A RNA methylation has been shown to modify RNA structure, as a direct effect, in a way that improves accessibility for RNA-binding proteins such as hnRNPC, which in turn mediates an indirect effect. This phenomenon is termed as “m6A-switch” [A special combination of direct and indirect mediation has also been observed and studied. m-switch” .To sum up, the emerging new data and discoveries have revealed a complex picture concerning epitranscriptome Fig. .Fig. 1Ch6A modification has been reinvigorated due to the advent of potent analytical methods, which harness the effects of m6A RNA methylation on RNA structure and metabolism, in combination with novel, high-throughput methods.The research related with m6A modification, while they are not suitable for widespread identification and localization of modified sites [Several methods, including dot-blot and high-performance liquid chromatography coupled to triple–quadrupole mass spectrometry (LC–MS/MS) can be utilized to yield important quantitative information about existence and abundance of med sites .6A-seq or MeRIP-seq has received considerable attention recently due to its accuracy and reproducibility. This novel method is based on the high specificity of one antibody raised against m6A in combination of high-throughput sequencing, making the mapping of m6A in the mammalian transcriptome possible [Thus, an emerging method termed mpossible , 35.6A-seq localizes m6A residues to 100- to 200-nt-long regions of transcripts and is incapable of single-nucleotide-resolution detection of methylation sites. Contrary to other base modifications, such as 5-methylcytosine (5mC), which can make use of chemical conversion of cytosine as a mapping approach at single-nucleotide resolution in RNA [6A RNA methylation has no such chemical conversion. Additionally, m6A does not introduce errors during reverse transcription that would allow direct mapping of its position [6A individual-nucleotide-resolution crosslinking and immunoprecipitation (miCLIP) has marked a major step forward in the field, which augments m6A-seq with UV-induced crosslinking of the antibody to the immunoprecipitated RNA fragments [6A residues throughout the transcriptome at single-nucleotide resolution.However, the immunoprecipitation-based approach mn in RNA , m6A RNAposition . A novelragments . Reverse6A RNA methylation as a complementary approach [Moreover, it is currently possible to directly test the influence of altering any modification site in many organisms with the use of CRISPR-based genome engineering. CRISPR multiplexing strategies could potentially permit interrogation of many sites in parallel and hasten functional discoveries and would be valuable for the research of mapproach .6A RNA methylation has been found to have diverse biological regulatory functions in cancer initiation and progression and dysregulated expression of m6A RNA methylation is closely associated with various kinds of cancers. Herein, we mainly discuss four aspects of these functions exerted by m6A RNA methylation in cancer , have aroused interests of researchers . CSCs, tHematopoietic stem cells (HSCs) have well-defined developmental trajectories, and it is plausible to monitor and quantify their differentiation, providing an ideal model system in which to explore differentiation states. Abnormal or blocked differentiation is a common feature of myeloid hematological malignancies.6A levels of its target genes including myelocytomatosis (MYC), B cell lymphoma 2 (BCL2), and phosphatase and tensin homolog (PTEN) genes in the human acute myeloid leukemia (AML) MOLM-13 cell line, thereby promoting the translation of these mRNAs. These data suggest that AML cells regulate their translational state through m6A RNA methylation of specific transcripts to retain pluripotency properties and inhibit cell differentiation. Depletion of METTL3 in human myeloid leukemia cell lines induces cell differentiation and apoptosis and delays leukemia progression in recipient mice in vivo [According to the recent research conducted by Ly P Vu and colleagues, leukemia cells show an elevated abundance of METTL3 as compared to normal hematopoietic cells. Utilizing miCLIP coupled with ribosome profiling, it is elucidated that METTL3 augments m in vivo .6A modification, while the protein itself is negatively regulated by spleen focus-forming virus proviral integration oncogene (SPI1), collectively forming a SPI1/METTL14/MYB, MYC, et al. signaling axis in myelopoiesis and leukemogenesis. This highlights the critical roles of METTL14 and m6A modification in normal and malignant hematopoiesis [Consistent with METTL3, METTL14 is also highly expressed in AML cells carrying t(11q23), t, or t and is downregulated during myeloid differentiation. Mechanistically, METTL14 exerts its oncogenic role by regulating its mRNA targets  and MYC) through mopoiesis .6A writer complex, is linked to myeloid leukemia as well where AML initiation is mediated by a chromosomal translocation t  of RBM15  with the myelin and lymphocyte (MAL) gene [6A RNA methylation in myeloid leukemia that WTAP expression was elevated in cells derived from 32% of patients with AML and knockdown of WTAP resulted in reduced proliferation, increased differentiation, and increased apoptosis in a leukemia cell line [RBM15, another component of the mAL) gene . Bansal ell line .6A RNA methylation expression can result in anti-proliferation effects in some circumstances. Su and colleagues have elaborated that R-2-hydroxyglutarate (R-2HG) exhibits broad and variable anti-proliferation effects in leukemia and glioma since it increases global m6A RNA modification in the sensitive cells via suppressing FTO. The R-2HG/FTO/m6A axis regulates MYC and CCAAT/enhancer-binding protein alpha (CEBPA) gene expression and downstream pathways [However, upregulated mpathways .6A RNA methylation levels may exert oncogenic functions in some certain types of AML. For example, FTO, an m6A demethylase, is highly expressed in AML with t(11q23)/MLL rearrangements, t/PML-RARA, FLT3-ITD, and/or NPM1 mutations and functions as an oncogene that promotes leukemic oncogene-mediated cell transformation and inhibits all-trans-retinoic acid (ATRA)-mediated leukemia cell differentiation. The oncogenic role of FTO is exerted through regulating expression of its target genes such as a suppressor of cytokine signaling box-2 (ASB2) and the retinoic acid receptor alpha (RARA) by reducing m6A RNA methylation levels in these mRNA transcripts. RNA stability assays have proposed that FTO-induced repression of ASB2 and RARA expression is at least in part due to the decreased stability of ASB2 and RARA mRNA transcripts upon FTO-mediated decrease of m6A RNA methylation levels. However, the reader(s) that targets m6A modification and impairs mRNA stability remains to be identified, which indicates an additional reading process controlling the stability of FTO target transcripts. Studies have [On the other hand, decreased mies have –51 demonies have .6A demethylation and stabilization of NANOG mRNA. Intratumoral hypoxia is a critical feature of the tumor microenvironment caused by dysregulated cell proliferation in combination with abnormal blood vessel formation and function, which drives cancer progression [6A RNA methylation level and promotion of breast cancer stem cell (BCSC) phenotype [6A methylation of mRNAs encoding NANOG and Kruppel-like factor 4 (KLF4), which is another pluripotency factor that mediates BCSC speciation [In breast cancer, Zhang and colleagues proposed that the exposure of breast cancer cells to hypoxia could stimulate hypoxia-inducible factor (HIF)-1α- and HIF-2α-dependent expression of ALKBH5, which induced mgression –55. NANOhenotype . The reseciation .6A level is critical for maintaining glioblastoma stem-like cell (GSC) growth, self-renewal, and tumor development as downregulation of METTL3 or METTL14 expression reduces mRNA m6A levels of theirs target gene A disintegrin and metallopeptidase domain 19 (ADAM19) and promotes ADAM19 expression. ADAM19 is a metalloproteinase disintegrin gene that exhibits elevated expression in glioblastoma cells and promotes glioblastoma cell growth and invasiveness [When it comes to glioblastoma (GBM), a study carried out by Cui et al. demonstrates that reduced mRNA msiveness –60.6A demethylase ALKBH5 is highly expressed in GSCs and demethylates forkhead box protein M1 (FOXM1) nascent transcripts [6A modification and exert stabilizing effects on its bound RNAs [6A level mediated by ALKBH5 in GBM helps to promote tumor progression.Similarly, Zhang and colleagues have identified that mnscripts . The nucnscripts , 63, hasund RNAs . Recruitund RNAs  and is ound RNAs –67. Take6A modification, forming a positive feedback loop of HBXIP/let-7g/METTL3/HBXIP, which leads to accelerated cell proliferation in breast cancer [m6A RNA methylation has been found involved in cancer cell proliferation in many kinds of cancers. There remains a coherent natural link between the oncoprotein hepatitis B virus X-interacting protein (HBXIP) and METTL3 in the development of breast cancer (BC). Mechanistically, HBXIP upregulates METTL3 in breast cancer cells by inhibiting miRNA let-7g, which downregulates the expression of METTL3 by targeting its 3′UTR. METTL3 then promotes the expression of HBXIP gene through mt cancer .6A modification and downregulates SOCS2 mRNA expression by degrading SOCS2 mRNA transcripts through m6A “reader” protein YTHDF2-dependent degradation pathway, suggesting a new dimension of epigenetic alteration in liver carcinogenesis [In human hepatocellular carcinoma (HCC), METTL3 has been elucidated to be frequently upregulated and contributes to HCC progression through a distinct mechanism. SOCS2, one of the members of suppressor of cytokine signaling (SOCS) family, functions as a tumor suppressor gene by negatively regulating the JAK/STAT pathway . METTL3 ogenesis .6A RNA methylation can act on cancer cell migration and tumor metastasis has now blossomed into a full-fledged field of research.The topic that m6A modifications is observed and METTL14 is addressed to be the main factor involved in aberrant m6A modification [6A modification can mark pri-miRNAs for processing by recognizing DiGeorge critical region 8 (DGCR8) in a manner dependent on METTL3/m6A, highlighting the important role of m6A modification in RNA processing, including mRNAs and pri-miRNAs [6A-dependent manner and thus METTL14 depletion in HCC results in pri-miR126 processing arrest and reduces the expression of mature miR126 who has been identified as a metastasis suppressor, leading to advanced metastasis capability.In HCC, especially in metastatic HCC, a decreased tendency of mfication . It has i-miRNAs . Similar6A modification on miRNAs promotes cancer metastasis [Besides the genetic background of cancer cells, alteration in microenvironment has emerged as a vital layer of regulating cancer metastasis. In colorectal carcinoma (CRC), serine proteases are important component in microenvironment and can selectively activate protease-activated receptor 2 (PAR2) through proteolysis of the receptor . The restastasis .6A RNA methylation sites in YAP mRNA transcript with one site in coding DNA sequences (CDS) and the other site in exon [6A sites of YAP mRNA to decrease the stability of mRNA, while the direct link between YTHDF2 and YAP remains to be clarified.In human pancreatic cancer (PC), the research conducted by Chen et al. showed that YTHDF2 was upregulated at both mRNA and protein levels and orchestrated proliferation and epithelial–mesenchymal transition (EMT) dichotomy . Two of  in exon . Therefo6A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathway. This prompts the very first attempt at studying m6A RNA methylation as key regulators of T cell differentiation, suggesting their involvement in tumor–immune system communication and importance in tumor growth and spread [As elucidated by Li et al., md spread .6A modification are found incapable of activating TLR3, and those with 5mC and/or m6A do not activate TLR7 or TLR8, leading to non-recognition of pathogens carrying these RNA modifications by TLR receptors . The methylation in m6A interferes with Watson–Crick base pairing; thus, its presence destabilizes RNA duplexes, which may explain why RNAs containing m6A modification are incapable of stimulating TLR3 [In other circumstances, toll-like receptors (TLRs) establish the first line of defense against besieging pathogens as the most conserved molecules of the innate immune system, which recognize pathogen-associated molecular patterns to facilitate an immune response. RNAs with ming TLR3 . Dendriting TLR3 –83.6A RNA methylation in circulating tumor cells (CTCs) compared with whole blood cells for the first time, constituting the first step for the investigation of RNA methylation in CTCs from lung cancer patients. This research outcome may facilitate uncovering the metastasis mechanism of cancers in the future. It is worth noting that early detection and characterization of upregulated m6A expression in CTCs can make contributions to monitoring and preventing the development of overt metastatic diseases [Huang et al. have demonstrated a significant increase of mdiseases .6A RNA methylation represents brand new therapeutic targets. All the subtypes of AMLs with high levels of endogenous FTO expression, such as those carrying t(11q23), t, NPM1 mutations, and/or FLT3-ITD, are more sensitive to ATRA treatment than the other AML subtypes [Moreover, msubtypes . It is p6A-containing nucleic acid and treatment of HeLa cells with the ethyl ester form of MA (MA2) led to elevated levels of m6A modification in mRNA [6A methylation could be a promising target for anti-glioblastoma therapy [Meclofenamic acid (MA) is a US Food and Drug Administration (FDA)-approved non-steroidal anti-inflammatory drug primarily known for its inhibition of prostaglandins synthesis as well as for its more specific inhibition of cyclooxygenase enzymes and lipoxygenases . Intrigu in mRNA . The ide therapy .6A regulatory complex themselves can also function as novel options for cancer treatment. For example, researchers have endeavored to design a peptide mimicking the critical METTL14 domain and investigate whether it has a potential therapeutic effect in HCC [The cellular components of mt in HCC .6A RNA methylation and clinical features including predict treatment-free survival (TFS) and overall survival (OS) in cancer still warrants further research.However, the correlation between m6A RNA methylation research has contributed to revealing underlying mechanisms of cancer initiation and progression. However, there remain a large number of challenges.As described above, the rapid development of m6A modification. Cellular components of m6A methylation complex are well established to be involved in cancer carcinogenesis and may function as tumor promoters or suppressors [6A reader proteins downstream of nuclear METTL3, since both wild-type and catalytically inactive METTL3 promote translation when tethered to a reporter mRNA [The first issue we should pay attention to is that whether the multiple functions proposed in cancer is actually dependent on mpressors , 88–93. ter mRNA .6A methylation process has been shown to suppress cell growth in cancer [6A RNA methylation caused by SAM and its cancer growth-inhibitory effect remains to be established.Additionally, the elevated level of the S-adenosyl methionine (SAM) donor of the methyl group in the mn cancer –98. Howe6A RNA methylation is introduced. Despite the strong consensus, only a small fraction of RACH sites is detectably methylated in vivo, arguing that the sequence motif is not sufficient to determine the distribution of m6A [6A modification via a sequence pairing mechanism, through which miRNA sequences alter m6A modification levels via modulating the binding of METTL3 to mRNAs containing miRNA targeting sites. These results reveal how miRNAs involves in regulating the formation of m6A and partially explain the site selection mechanism of m6A [6A methylation so that the current view of methylation regulation can be completed.Secondly, we need to identify through which mechanism mn of m6A . It has m of m6A . Similarm of m6A . More thm of m6A . It is w6A is regulated refers to that METTL3 can associate with chromatin and localize to the transcriptional start sites of active genes. Thereby, METTL3 induces m6A modification within the coding region of the associated mRNA transcript and enhances its translation by relieving ribosome stalling. One example is that METTL3 modulates nn n zSP1, an oncogene in several cancers which regulates c-MYC expression in this way [Another mechanism by which the emplacement of mthis way , identifthis way .6A recruitment modulation. ALKBH5 belongs to the AlkB family of non-heme Fe(II)/a-ketoglutarate-dependent dioxygenases, whose activity is iron dependent [Iron metabolism also participates in mependent . Gene exependent , 66.6A RNA methylation and transcript-specific fates in different cell types, it remains to be determined whether m6A is differentially recognized by distinct YTH domain proteins or whether m6A RNA methylation is used in a cell type-specific manner to either direct translational enhancement or mRNA decay.The third issue is that although there exist emerging links between m6A abundance mainly on the 3′-terminal exon, while FTO promotes stability of MYC mRNA through inhibition of YTHDF2-mediated RNA decay which can be attributed to decreased m6A abundance on the 5′-terminal and internal exons of MYC mRNA [6A modifications on different regions of the same mRNA transcript  may result in distinct fates likely due to recognition by different readers. It also leads to very open and constructive discussion that YTH domain family proteins may depend on sequence context to control their selectivity towards m6A sites and may be dynamically regulated.Interestingly, METTL14 promotes stability of MYC mRNA through affecting mMYC mRNA . This ph6A RNA methylation and its impacts on downstream mRNA processing events and cancer progression.However, much remains to be learned before we can understand the dynamic regulation of the co-transcriptional installation of m6A RNA methylation has a deep root in modulating gene expression and is implicated in affecting many cellular processes and diseases, including cancers. Over the past few years, empowered by the availability of highly specific antibodies and the accessibility of high-throughput sequencing technologies, it has become feasible to identify the chemical basis and multiple functions of m6A RNA methylation. There remain many other kinds of RNA post-transcriptional modifications which may share similar properties with m6A RNA methylation, shedding light on the advancement of epitranscriptome.As the most prevalent modifications of mRNA, m


Corresponding to life’s two distinct aspects: Darwinian evolution and self-sustainment, the origin of life should also split into two issues: the origin of Darwinian evolution and the arising of self-sustainment. Because the “self-sustainment” we concern about life should be the self-sustainment of a relevant system that is “defined” by its genetic information, the self-sustainment could not have arisen before the origin of Darwinian evolution, which was just marked by the emergence of genetic information. The logic behind the idea of the RNA world is not as tenable as it has been believed. That is, genetic molecules and functional molecules, even though not being the same material, could have emerged together in the beginning and launched the evolution—provided that the genetic molecules can “simply” code the functional molecules. However, due to these or those reasons, alternative scenarios are generally much less convincing than the RNA world. In particular, when considering the accumulating experimental evidence that is supporting a de novo origin of the RNA world, it seems now quite reasonable to believe that such a world may have just stood at the very beginning of life on the Earth. Therewith, we acquire a concrete scenario for our attempts to appreciate those fundamental issues that are involved in the origin of life. In the light of those possible scenes included in this scenario, Darwinian evolution may have originated at the molecular level, realized upon a functional RNA. When two or more functional RNAs emerged, for their efficient cooperation, there should have been a selective pressure for the emergence of protocells. But it was not until the appearance of the “unitary-protocell”, which had all of its RNA genes linked into a chromosome, that Darwinian evolution made its full step towards the cellular level—no longer severely constrained by the low-grade evolution at the molecular level. Self-sustainment did not make sense before protocells emerged. The selection pressure that was favoring the exploration of more and more fundamental raw materials resulted in an evolutionary tendency of life to become more and more self-sustained. New functions for the entities to adapt to environments, including those that are involved in the self-sustainment per se, would bring new burdens to the self-sustainment—the advantage of these functions must overweigh the corresponding disadvantage. To avoid discussing this issue in an ambiguous context, we ought to clarify what on earth is “life”. Unfortunately, in respect of the latter question, few of us can offer a convincing answer ,2. IndeeRecently, I made an assertion about the reason underlying this situation . That isSo, we know that the origin of life should also imply two distinct issues: the origin of the life form—or say, the origin of Darwinian evolution, and the origin of living entities—or say, the origin of the self-sustainment. Then comes a question of “order”: which originated at the very beginning? In fact, there has long been a disputation concerning “replication first or metabolism first” during the emergence of life ,5,6,7. TTo answer the “order question”, we should examine the relevant concepts more carefully. Actually, as it was noted, “self-sustainment” has a rather blurry meaning . What doThat is to say, even if the so-called self-sustaining autocatalytic cycle  emerged first, the self-sustaining living system emerging after the appearance of genetic materials would have no essential relationship with the original cycle, due to the shifting of the connotation of “self-”. The autocatalytic cycle, if ever existed, seemed to have only made its sense as some environment, providing the necessary energy and raw materials, for the emergence of those early genetic molecules (similar ideas have been expressed previously ). IndeedTo conclude, as to the life’s feature “self-sustainment”, what we really concern about should be the self-sustainment of the system “defined” by the genetic information. Therefore, the problem of the origin of the life can be summarized as: first, the origin of Darwinian evolution, as labeled by the emergence of the genetic material, and next, the arising of the self-sustainment guided by the genes carried on the genetic material. At least, the self-sustainment should not have arisen before the origin of Darwinian evolution, if the possibility for simultaneous advent of the two features could not be ruled out. In terms of the splitting definition of life (mentioned above) , that isAs long ago as the 1960s, it was speculated that there might have been an early stage of life in which RNA played both the roles of DNA and proteins ,12,13. IThereafter, evidence accumulated quickly in favor of the hypothesis ,18,19. THowever, the proponents of the hypothesis almost immediately have to face a further problem: how did the so-called RNA world itself come into being? May it have appeared de novo? In those days, experimental work to simulate the prebiotic synthesis of nucleotides apparently had not transferred messages so optimistic. In fact, this area was even described as the prebiotic chemist’s “nightmare” . NonetheFirst of all, according to the conclusions that are made in Notably, there could be alternative scenarios adopting a similar solution. For example, in those years, the “nightmare” regarding nucleotides’ prebiotic synthesis was so awful that some people began to search out for other RNA-like polymers, which might also, possibly, act as both functional and genetic material, such as p-RNA (pyranosyl-RNA), PNA (peptide nucleic acid), TNA (threose nucleic acid) and GNA (glycol nucleic acid) ,22,24,27Perhaps somewhat unexpectedly, here it is underlined that there can still be other possible scenarios—adopting a different solution. That is, genetic material and the functional material it encoded, even if distinct from each other, could have emerged together in the very beginning and launched Darwinian evolution as well. This assertion is unexpected because it casts doubt on the logic that is followed by the original proposition of the idea of the RNA world. Indeed, why genetic material and functional material, if different from each other in molecule type, cannot emerge together, especially considering that the “prebiotic pool” may have been a rather complex “soup” ? For example, if DNA and RNA could both be synthesized in the prebiotic pool, DNA might act as genetic material and encode functional RNA molecules. It should be noted that DNA can simply instruct the synthesis of RNA via template-directed copying, just like the transcription shown in modern cells. The problem with this scenario, however, is that if DNA and RNA were simultaneously synthesized in a common prebiotic environment, the synthetic reactions may have interfered with each other . Thereby, mosaic nucleic acids (with a combination of nucleotide and deoxynucleotide residues) may have arisen. A recent interesting study showed that mosaic nucleic acids might also carry functions, albeit less efficient than RNA , which sAdditionally, one may also conceive: if RNA and proteins could both be synthesized in the prebiotic pool, RNA might act as genetic material and proteins may act as functional material. Indeed, there are now some proponents for the hypothesis of the “RNA/proteins (peptides)” world e.g., ,35), esp, esp35])Taken together, we can conclude that though we have not obtained sufficient evidence to be sure about the existence of an RNA world at the start point of life, and even the logic for the raising of the idea of the RNA world is not, as it is usually taken for granted, so obliged, this world is still most likely to have been the earliest scenario for the life on our planet—at least according to the knowledge that we have so far. Above we have conceptually inquired into the problem of the origin of life and have interpreted in depth the idea of the RNA world, as a central hypothesis in this field. It is then interesting to relate the two contents to each other and ask: what on earth does “the RNA world” mean to “the origin of life”?Previously, the process of the origin of life is only pondered and discussed as a blurry and abstract issue. If, as mentioned above, there was an RNA world standing at the start point of life’s history, the origin of this world per se, together with its early development, would have represented the very process of the origin of life. Then, we obtain a “concrete background” to think, discuss, and even debate about this issue. For example, based upon the analysis in Since RNA can both act as genetic molecules and functional molecules, it is natural to think that RNA may have evolved initially just in a naked way, at the molecular level. It is usually supposed that some RNA, catalyzing the template-directed copying of RNA, may have favored its own replication (often referred to as an “RNA replicase”) and spread in a prebiotic pool ,22,23. ITo avoid a diverging representation, here let us adhere to the assumption that the replicase emerged first. A key problem of the scene regarding the emergence of the replicase is the parasite problem; that is, since other RNA sequences might also exploit the ribozyme, how could the replicase “win the game”? Through theoretical work in this area, it is now well known that the spatial limitation may have played an important role for the replicase to overcome the parasite problem ,54,55,56Certainly, if the RNA molecules were encompassed within a lipid vesicle, forming a so-called protocell, then the parasite problem would be much less serious. For example, it was suggested—not surprisingly—that the simplest protocell may have been an RNA replicase trapped in a lipid vesicle . Parasit2+, which is a usual condition for RNA’s template-directed copying [2+’s surface with citric acid, obviously, the scene is not quite convincing as the authors admitted in the same paper [A logic justifying the idea of “a naked stage first” is: “the simpler, the more likely to emerge de novo”. However, if the prebiotic pool was a rather complex soup , for exa copying ,74. Thoume paper . Interesme paper ,76. Indeme paper ,48,49,50No matter how, let us come back to our mainline as it is assumed: an RNA replicase emerged first in a naked way. Indeed, on account of the belief about the importance of this functional RNA to the origin of the RNA world, there have been long-standing efforts to construct such a ribozyme in laboratory ,80,81,82Therefore, the search for shorter RNA replicase, even with some sacrifice of efficiency, would be an urgent task for supporting this “earliest scene” of the RNA world. Now that the tail-binding of the ribozyme onto the template can greatly improve the ribozyme’s efficiency, it would be natural to expect that a shorter version of the replicase (perhaps about 30–50 nt long), with the tail-binding retained, may have had an efficiency, albeit somewhat lower, yet enough to support its own thriving in the system. Importantly, the tail subsequence on the target template, which is recognized by the replicase through base-pairing can just serve as a so-called “tag”. As mentioned above, one of our theoretic studies on evolutionary dynamics (by computer simulation) has concluded that the introduction of a tag mechanism would greatly enhance the replicase’s ability to resist parasites . Maybe tLet us go on with our scenario. What may have occurred after the emergence of the RNA replicase? Indeed, even if a nucleotide synthetase ribozyme had not emerged before the emergence of the replicase, here it seems inevitable to have emerged subsequently. When the replicase spread in the pool, nucleotides would become scarce, and a ribozyme that could supply the building blocks would be strongly favored in the context of natural selection. The nucleotide synthetase ribozyme would enable the system to exploit more fundamental materials, namely precursors of nucleotides. It has been demonstrated by computer simulation from our group  and HiggThat is, the subsequent scene (before the emergence of the third ribozyme) is likely to have been the appearance of protocells. In addition, it is worth noting that, after the emergence of the nucleotide synthetase ribozyme, the appearance of protocells would have become more feasible (as mentioned above)—what need to permeate into the vesicle are, instead of nucleotides, only precursors of nucleotides. The two ribozymes, if by chance, were “engulfed” into some lipid vesicle, would then cooperate within a protocell. Initially, the protocell should have yet been unable to synthesize the components of its membrane. The membrane appears to have been no much more than an environmental factor. We would like to call it a “pseudo-protocell” . InteresNonetheless, at that time, there should have been a strong selection pressure for the emergence of a ribozyme to synthesize the lipid molecules. This ribozyme would provide the membrane components from the inside and allow or the protocell’s membrane to grow faster, thus achieving the potential to accommodate more contents. Then, the replication of the inner RNAs would be promoted when more raw materials could flow in. In contrast to the situation for pseudo-protocells, which is the content-triggered growth, here is a sort of membrane-triggered growth. This growth, of course, would also eventually lead to “cell division” due to the instability of the protocells, thus completing the circle of reproduction. Indeed, in one of our modeling studies, the emergence of such a ribozyme has been demonstrated to be favored in evolution  because Up to this stage, though lipid vesicles provided a good mechanism for the cooperation of different ribozymes, accompany with the emergence of more ribozymes, another problem would become increasingly apparent. That is, due to the random distribution of the ribozymes between offspring protocells during the “cell division”, there is a risk of “gene loss” ,90. ObviIn fact, quite a few scenes above concerning the origin and development of the RNA world have been described previously ,93. WhatAs mentioned above, the origin of life is, firstly, the origin of Darwinian evolution. Then, how may Darwinian evolution have got started? According to the scenario described above, the RNA world may have begun at the molecular level. From the prebiotic pool, nucleotides may have been synthesized abiotically, and linked together to form RNA—perhaps by mineral catalysis ,93. ThenNo matter how, then, a nucleotide synthetase ribozyme is likely to have emerged and become co-thriving with the RNA replicase in the naked stage. Indeed, Darwinian evolution in this scene may have taken some sense of a higher level—in regard to the locally distributed clusters of the two ribozymes . HoweverIt is only when the two ribozymes were encompassed within lipid membranes that Darwinian evolution at a higher level began to show clearly. The fate of the functional RNAs would be tightly tied up with the fate of the protocell they resided in. For pseudo-protocells, on account of the “osmotic pressure” effect, the more efficient replication of functional RNAs would directly result in a faster reproduction of the protocells containing them. Even a functional RNA having no direct influence on the synthesis of RNA may have been favored in the evolution as long as it favored the reproduction of the protocell. As a case in point, the protocells containing a ribozyme favoring the synthesis of membrane components would reproduce faster than those without the ribozyme ,89—so thThe emergence of the unitary-protocell, that is, a protocell with all its genes linked into a chromosome is significant for the Darwinian evolution at the cellular level. It has been pointed out that even when the protocells appeared, the competition at the molecular level went on. That is, at the stage of protocell, there is still a two level Darwinian evolution: the molecular level and the Darwinian level ,94,95. AThe arising of self-sustainment is another issue for the origin of life. If Darwinian evolution started at the molecular level, e.g., as described in the scenario we focused on—an RNA replicase emerging first in a naked way, there would have been no problem of self-sustainment at that time. There is no sense for a molecule to self-sustain. The key reason is that the covalent bonds within a molecule would prevent it from refreshing—a molecule either stays unchanged or is “ruined” on account of any change . If self-sustainment is a key feature for a living entity, the replicase molecule is not “alive” (like viruses in our modern living world ); nonethThen, when a nucleotide synthetase ribozyme ensued, what about the problem of self-sustainment? Indeed, the emergence of the nucleotide synthetase ribozyme would have rendered the whole system more self-sustaining, by exploiting more fundamental raw material. However, without a clear boundary, there would be no definite entities at a level above the level of molecules. Therefore, self-sustainment still made little sense at this stage.When the two ribozymes were encompassed into the lipid membrane and formed protocells (pseudo-protocells), things became different. The RNA molecules within the vesicle could be replenished if some of them were degraded, and the membrane components (lipid molecules) could also be replenished if some of them degraded or left the membrane. Here, it becomes clear that the key factor rendering “self-sustainment” meaningful is the establishment of entities at a level above that of molecules.For a protocell containing the two ribozymes, it should be noted that, while these functional RNA molecules may be replenished via replication, the lipid molecules on the membrane could only be replenished by recruiting new ones from environments. The self-sustainment of such protocells was apparently “incomplete”. That is just why we named them “pseudo-protocells” . When anHere, it is worth talking about the relation of replication, self-sustainment, and reproduction. It has been noted that there is a difference between replication and reproduction ,88. IndeFinally, the tendency to become more self-sustained or evolve other features for adapting to the environment would result in more “components of its own” for the living entity. Thus, the task for its self-sustainment would be “heavier”. The advantage brought about by these new components ought to cover the burden to synthesize them. In a scene mentioned above, for example, for the pseudo-protocells containing an RNA replicase ribozyme and a nucleotide synthetase ribozyme to incorporate a new ribozyme—the amphiphile synthetase ribozyme (which would give rise to the true-protocells), the advantage of synthesizing their own membrane components must outweigh the burden in respect to producing such a ribozyme. Likewise, the task per se should not be too heavy to synthesize those new “metabolic” ribozymes, with which the protocells could go further to exploit raw materials that are more fundamental than the nucleotide precursors and amphiphile precursors. Certainly, a most “typical” case in this point should be the one concerning the emergence of proteins in the RNA world. That is, the burden in respect to sustaining the translation machine (though rather complex) should have been trivial when considering the great advantage of the introduction of proteins, as a significantly more efficient functional material.Here, we have talked about the origin of Darwinian evolution and the arising of self-sustainment in a concrete background of the RNA world scenario. As it is implied in the text, though we say that the RNA world is most likely the earliest stage for life, this assertion is far from certain, especially considering that the idea of the RNA world is not obliged in logic and our knowledge on prebiotic environments and prebiotic chemistry remains quite limited. However, at least, we can benefit a lot from the way to talk about the origin of life—previously a rather abstract problem—based upon a concrete scenario. In particular, the RNA world scenario has a simple base of material, that is, the central molecules, genetic and functional, are identical in material. Such a “succinct” world takes our focus to the core issues of the problem: the origin of Darwinian evolution and the arising of self-sustainment. If the earliest scenario in the history of life is, in the future, shown likely to be a different one, either a different version of the RNA world scenario or even a non-RNA-world story, the discussion here may still be helpful for the new interpretation possibly involved because the principles concerning the implementation of Darwinian evolution and that of self-sustainment remains unchanged. To the most extreme end, even for another type of life (different from the “life as we know it” on the Earth)—if possibly existing, these analyses would also be useful as long as our concept of life defined in terms of “Darwinian evolution” and “self-sustainment” stands.


Takydromus tachydromoides and compared the polyadenylation profiles with those identified similarly for 23 tetrapod species, featuring sauropsid taxa (reptiles and birds).Genes encoded in vertebrate mitochondrial DNAs are transcribed as a polycistronic transcript for both strands, which is later processed into individual mRNAs, rRNAs and tRNAs, followed by modifications, such as polyadenylation at the 3′ end of mRNAs. Although mechanisms of the mitochondrial transcription and RNA processing have been extensively studied using some model organisms, structural variability of mitochondrial mRNAs across different groups of vertebrates is poorly understood. We conducted the high-throughput RNA sequencing to identify major polyadenylation sites for mitochondrial mRNAs in the Japanese grass lizard, Pelusios castaneus. In the last case of P. castaneus, as well as another example for NADH dehydrogenase subunit 1 mRNAs of some birds, the association between the polyadenylation site change and the gene overlap was highlighted. The variations in the polyadenylation profile were suggested to have arisen repeatedly in diverse sauropsid lineages. Some of them likely occurred in response to gene rearrangements in the mitochondrial DNA but the others not.As compared to the human, a major polyadenylation site for the NADH dehydrogenase subunit 5 mRNA of the grass lizard was located much closer to its stop codon, resulting in considerable truncation of the 3′ untranslated region for the mRNA. Among the other sauropsid taxa, several distinct polyadenylation profiles from the human counterpart were found for different mRNAs. They included various truncations of the 3′ untranslated region for NADH dehydrogenase subunit 5 mRNA in four taxa, bird-specific polyadenylation of the light-strand-transcribed NADH dehydrogenase subunit 6 mRNA, and the combination of the ATP synthase subunit 8/6 mRNA with a neighboring mRNA into a tricistronic mRNA in the side-necked turtle These results demonstrate structural variability of mitochondrial mRNAs in sauropsids. The efficient and comprehensive characterization of the mitochondrial mRNAs will contribute to broaden our understanding of their structural and functional evolution.The online version of this article (10.1186/s12864-017-4080-0) contains supplementary material, which is available to authorized users. They contain intronless genes for 2 rRNAs, 22 tRNAs, 13 respiratory proteins , while the ND6 and eight remaining tRNA genes are encoded by the light strand (L-strand). In the late twentieth century, gene expression of mtDNAs was intensively investigated using some model organisms . It was found that transcription of mitochondrial genes is initiated from within the MNCR in both directions , 6. PolyPolyadenylation of mRNA is an important process in gene expression. In eukaryotes, a polyA tail works for stabilization of mRNA, transportation from nucleus to cytosol, and promotion of translational initiation whereas the polyadenylation stimulates RNA degradation in bacteria and chloroplasts , 9. PolyAlthough some enzymes, such as the mitochondrial polyA polymerase and polynucleotide phosphorylase, have been shown to regulate the polyadenylation, regulatory mechanisms in the control of polyadenylation/deadenylation and stabilization/degradation of polyadenylated RNAs are not fully understood albeit with many modern investigations  technology to gain new insights into the structural diversity and evolution of mt-mRNAs. The high-throughput sequencing is a technique which can parallelize the sequencing process, producing tens of millions of sequences concurrently . To the Takydromus tachydromoides) used for experiments was collected at Chikusa-ku, Nagoya, Japan in 2012. In sacrificing the animal, we employed the decapitation as a physical euthanasic procedure based on the permission by the Animal Experiment Committee of Nagoya City University (Permit No. H21N-02). A mirVana miRNA Isolation Kit (Life Technologies) was used to extract total RNA from fresh liver of T. tachydromoides. The extracted RNA was quantitated with NanoVue Plus , and its intactness was confirmed with the Agilent 2100 BioAnalyer (Agilent Technologies). A TruSeq RNA Sample Preparation Kit (Illumina) was used to prepare the high-throughput sequencing library. Briefly, enrichment of polyA-containing RNA from the total RNA was conducted with the oligo-dT beads. After the polyA-containing RNA was cut randomly with cation and heat, 1st and 2nd strand cDNA synthesis was carried out with random hexamers. After the end repair, index adaptor sequences were ligated to both ends. The ligate was amplified by the bridge PCR and amplified products were purified with AMPure XP beads (Beckman Coulter) that removed short constructs with less than 100-bp inserts. The prepared samples were run for the paired-end 101-bp sequencing with the Illumina HiSeq™ 2000 Sequencer.The Japanese grass lizard  was used for the PCR. Amplified products were subjected to the ExoSAP-IT (Affymetrix) treatment and dye termination reaction with a Big Dye Terminator version 3.1 Cycle Sequencing Ready Reaction Kit (Life Technologies). Sequences obtained with the Applied Biosystems Genetic Analyzer 3500 Sequencer were edited and assembled using Sequencer ver. 4.8 (Gene Codes) to give rise to a nearly complete mtDNA sequence.A nearly complete mtDNA sequence of Kumazawa   alt editing  and refs editing . The map editing . Some unWe wrote a script named polyA_seq.pl  using BlastStation-Local 64 (TM Software) was conducted with the oligoA-containing reads as a database and the determined/deduced mtDNA sequence of the individual used for the RNA-Seq as a query. Reads that showed the matching in the E-value criterion were assembled with Sequencher 4.8 (Gene Codes) into contigs. After the oligoA parts were removed from the 3′ end of individual contigs, they were assembled to the mtDNA sequence. When a stretch of A follows the assembled sites in the mtDNA sequence, this contig represents the false positive of the polyadenylation signal . These falsely positive contigs were removed and the polyadenylation sites were identified based on the assembled sites of the remaining contigs and singletons. OligoA-containing reads constituting these remaining contigs and singletons are called polyA-containing reads hereafter. Polyadenylation sites were identified as the last base encoded in the mtDNA just before the stretch of A starts. However, when the range of a neighboring tRNA can clearly specify a processing site that leaves one or two adenosine residues at the 3′ end of the processed RNA, this information was used to specify the polyadenylation sites. In this study positions at which the polyA attachment was confirmed with 10 or more independent polyA-containing reads were considered as major polyadenylation sites. The proportions of mtDNA-derived reads and polyA-containing reads in total RNA-Seq reads were rather heterogeneous across the species (Table Confirmation of the polyadenylation sites by 3′ RACE experiments was conducted using a SMARTer™ RACE cDNA Amplification Kit (Clontech). After amplifying 3′ end portions of targeted genes using a specific forward primer for the gene and a common reverse primer provided in the kit, amplified products were recovered from an agarose gel using a MinElute Gel Extraction Kit (Qiagen) and ligated to a plasmid vector by a Mighty TA-cloning Kit (Takara). After cloning with NEB 10-beta competent cells (New England Biolabs), several colonies were subjected to the colony PCR and subsequent sequencing.http://rna.urmc.rochester.edu/RNAstructureWeb/) in the default conditions.Secondary structures of mRNA sequences around the polyadenylation sites (a range covering 100 bp before and after the polyadenylation site) were sought by the RNA Secondary Structure option of DNASIS-MAC version 3.5 (Hitachi) and RNAstructure  and shoThese mtDNA-derived reads were mapped to the mtDNA sequence of the RNA-sequenced individual with Bowtie 1.1.2. The mapped information was output to a sam file, which was then converted to a bam file sorted by position using SAMtools. The bam file was analyzed using the genomeCoverageBed software of the bedtools v2.26.0 utilities  to calcuA rationale behind this approximation is as follows. The reads mapped to coding regions of protein genes greatly outnumber those mapped to other regions including the MNCR and tRNA genes. This is because short RNAs of less than 100 nucleotides  and any RNAs without the polyA tail do not contribute to the RNA-Seq reads in the standard RNA-Seq condition. Thus, an mRNA should have contiguously high frequencies of mapped reads throughout its range and the 5′ end position of mRNAs may be approximated around a site where the slope of mapped read frequencies considerably  increases.Using these procedures, we approximated ranges of H-strand-transcribed mRNAs, which usually count to be ten because ATP8/ATP6 and ND4L/ND4 are two pairs of genes that make a dicistronic mRNA , 34. TheT. tachydromoides liver mRNAs . Because a 65-bp sequence repeated at least 10 times in the 5′ half of the MNCR, it was difficult to obtain the complete mtDNA sequence. We did not find any site that showed heteroplasmy in the determined 17,923-bp mtDNA sequence. The gene arrangement of the mtDNA  Table  were ideT. tachydromoides. The blastn search subsequently found 18,554 mtDNA-derived oligoA-containing reads  supported by 13 polyA-containing reads and another is within an antisense sequence of the tRNATyr gene (4 bp before the CO1 gene) supported by 10 polyA-containing reads. When 5′ ends of paired reads for these polyA-containing reads were examined, none of them extended their 5′ ends to the ND2 coding region  near a start codon for the CO1 gene (position 5285), stayed at high levels throughout its coding region, and rapidly decreased near the polyadenylation site (position 6898)  increased around base position 5290  larger than the starting position of the mt-mRNA coding regions  . In T. tite Fig. . Thus, adon Fig. . We weredon Fig. , as has T. tachydromoides, except that mtDNA sequence of the RNA-sequenced individual was not experimentally determined but deduced from the RNA-Seq data. In 11 species among them (including H. sapiens) polyadenylation sites for the ten mt-mRNAs were located in similar positions as found for the human  had the typical mtDNA gene arrangements but its polyadenylation sites for ND5 and CYTB mRNAs were found to be different from those in the human  supported by 200 polyA-containing reads. The second polyadenylation site was identified 78 bp downstream of the ND5 stop codon, which was supported by 109 polyA-containing reads  commonly have a rearranged QIM  tRNA gene order  having the typical mtDNA gene arrangements, the far polyadenylation site for the ND5 mRNA located next to the CYTB gene was entirely lost and new polyadenylation sites were created at closer positions to the ND5 stop codons  had a striking change in the range of ATP8/ATP6 and CO3 mRNAs. A polyadenylation site for the ATP8/ATP6 mRNA usually occurs immediately 3′ to the ATP6 gene for other species  of paired reads of the 614 polyA-containing reads had their 5′ ends in the CYTB gene (data not shown), supporting that A. mississippiensis CYTB mRNA has its primary polyadenylation site just before the tRNAPhe gene.In addition, there was a novel polyadenylation site supported by 614 polyA-containing reads for the ene Fig. , which hGlu are translocated to the proximity of the MNCR [G. gallus, U. epops, F. tinnunculus and A. monachus) occurred immediately after the ND5 stop codon in response to this gene rearrangement . However, the ND1 polyadenylation site occurred immediately before the tRNAMet gene in two other birds (U. epops and A. monachus) although gene arrangements around the IQM tRNA gene cluster were common among the four birds  with 123 supportive polyA-containing reads. The second polyadenylation site was identified immediately before the tRNAMet gene with 80 supportive polyA-containing reads.It is also noteworthy that polyadenylation sites for ND1 mRNAs varied among the four birds. The ND1 polyadenylation sites usually occur immediately before the tRNArds Fig. . Also, iNAs Fig. . One wasStudies on gene expression from vertebrate mtDNAs have been carried out using model organisms  by molecular biological characterization of transcripts of some target genes, as well as proteins interacting with the mtDNAs and transcripts   for the Second, standard RNA-Seq experiments do not retain information on the strand of RNAs from which the RNA-Seq library was constructed. However, directional RNA-Seq is not necessary for the purpose of identifying polyadenylation sites at the 3′ end of mt-mRNAs because coding strands for the mt-mRNAs are obvious based on the mtDNA sequences. When we analyzed directional RNA-Seq data for the human (SRR3151753), we could identify similar major polyadenylation sites  mtDNA sequence. Similar methods were reported by Nabholz et al.  and TianIn our study, RNAs used for the RNA-Seq mostly came from liver for reptilian taxa but those for non-reptilian taxa were derived from different organs including liver, brain, blood, retina and cochlea Table . AlthougIn this study, we examined the variability of sauropsid mt-mRNAs with respect to their ranges and polyadenylation sites in comparison with other tetrapods . As a result, different polyadenylation profiles from that for the human were found in 12 of 23 tetrapods. We estimated the timing for changes in the polyadenylation sites by the parsimony criterion based on well-established phylogenetic relationships Fig. .Fig. 4EvT. tachydromoides, E. macularius, P. castaneus, C. serpentina, and T. carolina) . Based on the phylogenetic relationships of Fig. The polyadenylation site for the human ND5 mRNA is located far away from the ND5 stop codon just before the CYTB gene, creating a long 3′ UTR for the ND5 mRNA Fig. . In thisa) Figs.  and 3. Ides Fig.  and E. mWith respect to the functional consequences of shortening the 3′ UTR of ND5 mRNAs, the long 3′ UTR for ND5 mRNAs was suggested to act like a long noncoding RNA that interacts with the L-strand-transcribed ND6 mRNA for an unknown mechanism of gene expression regulation , 48. MamT. tachydromoides . In this secondary structure, base pairings resembling the acceptor stem and the T stem of vertebrate mitochondrial tRNAs [de novo polyadenylation site, followed by the polyadenylation to the 3′ end by the mitochondrial polyA polymerase [In order to consider evolutionary processes for the changes of the ND5 polyadenylation sites, the RNA secondary structure was inferred near the new polyadenylation sites. We found a tRNA-like secondary structure for al tRNAs  were conal tRNAs , 53) comal tRNAs . Thus, tlymerase .Glu to the vicinity of the MNCR, resulting in the juxtaposition of genes for the ND5 and CYTB, as well as for the tRNAPro and ND6 [Pro probably serves as a punctuation signal on the precursor L-strand transcript for the subsequent polyadenylation at this site. Polyadenylation for the ND5 mRNA occurs immediately after the ND5 gene  in contrast with mammals that have a very long 3′ UTR for the ND5 mRNAs , the ND1 polyadenylation site shifted from the 5′ end of the tRNAIle gene to the 5′ end of the tRNAMet gene without involving a gene rearrangement in the IQM tRNA gene cluster  had two major polyadenylation sites at the 5′ ends of the tRNAIle gene (159 reads) and the tRNAMet gene (105 reads)  Fig. . This maP. castaneus, a polyadenylation site for the ATP8/ATP6 mRNA appears to have been lost Primers to determine the mtDNA sequence of Additional file 2:A Perl script named polyA_seq.pl used to find oligoA-containing reads. (TXT 1 kb)Additional file 3: Figure S1.Figure S2. Minor polyadenylation sites for T. tachydromoides mitochondrial RNAs. Figure S3. Confirmation of the new polyadenylation site for T. tachydromoides ND5 mRNA by 3′ RACE. Figure S4. 5′ end mapping of T. tachydromoides cDNA fragments with polyadenylation at their 3′ end inside mitochondrial tRNA genes. Figure S5. Major polyadenylation sites for 11 species in which the polyadenylation profile was similar to that of the human. Figure S6. Characterization of E. macularius mt-mRNAs using RNA-Seq reads. Figure S7. Characterization of P. castaneus mt-mRNAs using RNA-Seq reads. Figure S8. Characterization of A. mississippiensis mt-mRNAs using RNA-Seq reads. Figure S9. Characterization of Homo sapiens mt-mRNAs using directional RNA-Seq reads (SRR3151753). Figure S10. The secondary structure of the L-strand sequence around the de novo  polyadenylation site for the T. tachydromoides ND5 mRNA. (PDF 1437 kb)Read numbers obtained using the polyA_seq.pl program in different conditions.


Bamboo mosaic virus (BaMV), which belongs to the genus Potexvirus in the family Alphaflexiviridae, has a single-stranded positive-sense RNA genome that is approximately 6400 nucleotides (nts) in length. Positive-sense RNA viruses can use genomic RNA as a template for translation and replication after entering a suitable host cell. Furthermore, such viral RNA is recognized by capsid protein for packaging and by viral movement protein(s) or the movement protein complex for cell-to-cell and systemic movement. Hence, viral RNA must contain signals for different functions to complete the viral infection cycle. In this review, we examine various cis-acting elements in the genome of BaMV. The highly structured 3′ untranslated region (UTR) of the BaMV genomic RNA plays multiple roles in the BaMV infection cycle, including targeting chloroplasts for RNA replication, providing an initiation site for the synthesis of minus-strand RNA, signaling for polyadenylation, and directing viral long-distance movement. The nt at the extreme 3′ end and the structure of the 3′-terminus of minus-strand RNA are involved in the initiation of plus-strand genomic RNA synthesis. Both these regions have been mapped and reported to interact with the viral-encoded RNA-dependent RNA polymerase. Moreover, the sequences upstream of open reading frames (ORFs) 2, 3, and 5 are involved in regulating subgenomic RNA synthesis. The cis-acting elements that were identified in BaMV RNA are discussed and compared with those of other potexviruses. The genome contains five open reading frames (ORFs) . ORF1 encodes a replicase for viral RNA replication, ORF2 encodes a 28-kDa protein (a silencing suppressor) required for viral movement, ORF3 and ORF4 encode membrane-anchoring proteins required for virus movement, and ORF5 encodes a 25-kDa capsid protein for viral encapsidation, movement, and symptom development.cis-acting elements of BaMV RNA involved in viral RNA replication, intracellular trafficking, and movement have been extensively studied in the last two decades. This report comprehensively reviews these studies and discusses the common theme of the roles of these cis-acting elements that could be applied to other members of potexvirus including the Potato virus X (PVX), one of the top 10 plant viruses in molecular plant pathology  and host proteins in the replicase complex was studied, the involvement of elongation factor 1a (eEF1a) and chloroplast phosphoglycerate kinase (PGK) was revealed  in most viruses play a critical role in recognition by the replicase complex. Typically, the 3′-terminal nucleotides (nt) or penultimate nt of non-poly(A)-tailed RNA viruses is used as the initiation site for minus-strand RNA synthesis (cis-acting elements in the 3′ UTR are far from the extreme 3′ end of the initiation site if poly(A)-tailed viruses use a similar synthesis mechanism as described for non-poly(A)-tailed RNA viruses. Therefore, poly(A)-tailed RNA viruses might have a different strategy or use initiation sites that are close to the cis-acting elements. In vitro and in vivo studies of BaMV revealed that the extreme 5′ end of minus-strand RNA contains stretch of uridine residues ranging from 1 to 15 nt, usually about 7–10 uridines .During initiation of minus-strand RNA synthesis, ynthesis , 2009. Hynthesis . Thus, curidines . These rCis-acting elements for minus-strand RNA synthesis, as mentioned previously, are usually situated in the 3′ UTR and form secondary or tertiary structures, such as the stem–loops of Alfalfa mosaic virus  sequence adjoining the 3′ UTR, designated as the E domain . Furthermore, results derived from ultraviolet (UV)-crosslinking and foot-printing assays indicate that the polymerase and helicase-like domains of the replicase (ORF1 of BaMV) interact with the D and E domains and ABC domain of the 3′ UTR, respectively. The potexviral conserved hexamer motif (ACXUAA) involved in the accumulation of virus was discovered in Clover yellow mosaic virus , and was protected from RNase digestion during interaction with the polymerase .The stem–loops B and C of the ABC domain in the 3′ UTR play a lesser, but significant, role in RNA replication . Accumulin vitro transcription assay, short transcripts of 39, 77, and 173 nts in length, corresponding to the 3′ terminus of minus-strand RNAs, were used as templates to examine their ability to direct RNA synthesis. The 3′-terminal 77-nt RNA, designated Ba-77, was the most efficient RNA template . It harbors two complete stem–loops confirmed by enzymatic structural probing and is required for plus-strand RNA synthesis   and retaining the terminal UUUUC preserves up to 60% of the template activity of Ba-77  (At least three igure 3) .cis-acting element AAUAAA in the 3′ UTR plays a role in polyadenylation  polymerase is located mainly in the nucleus. Furthermore, the polyadenylation of mRNAs with poly(A) polymerase is independent of the recognition of the AAUAAA motif, whereas the polyadenylation of BaMV is associated with the AAUAAA motif  tail; therefore, the extreme 3′ end of plus-strand RNA immediately after synthesis accordingly has a short run of adenylates (most frequently 7–10). However, to maintain the approximately 250 adenylates at the extreme 3′ end of genomic RNA after synthesis, the nylation . InteresAA motif .cis-acting RNA elements to direct the viral-encoded RdRp to transcribe these RNAs (The genomes of many positive-sense RNA viruses are multicistronic organizations that produce subgenomic RNAs (sgRNAs) to serve as messengers, allowing the translation of downstream ORFs . A few sese RNAs .cis-acting elements required for sgRNA synthesis (cis-acting element of the SGP for synthesis of the 1-kb sgRNA covers the region between nt -91 to +52 (the transcription start site is designated as +1). Further analysis indicated that the SGP can be split into four elements: the core (nt -30 to +16), two upstream enhancers (nt -59 to -31 and -91 to -60), and a downstream enhancer (nt +17 to +52). The core sequence is the minimum region required for 1-kb sgRNA synthesis, which folds into two stem–loops, stem–loop (SL)1 and SL2, in minus strand . Maintaining the integrity of SL2 structure and the conserved octamer motif (3′-CAAUUCAA-5′) in the loop are essential for 1-kb sgRNA synthesis. Furthermore, the cis-acting elements of SGP for 2-kb sgRNA synthesis are located at nt -119 to +11 (the transcription start site of the 2-kb sgRNA is designated as +1). The minus-strand SGP sequence for 2-kb sgRNA synthesis was predicted to have similar stem–loops to those of the 1-kb SGP. The conserved octamer motif (3′-CAAUUCAU-5′) is also located in the loop of SL2  . Although BaMV SGPs were inserted into the satBaMV cassette without a BaMV minus-strand 3′-terminal sequence, the octamer motif could also interact with the 3′-terminal sequence of minus-strand satBaMV RNA  . The long-distance RNA–RNA interaction between of the 3′-terminal sequence and the conserved octamer motif of the SGPs observed in PVX and BaMV favors the internal initiation mode of sgRNA synthesis.Compared with the putative SGPs of ORF2 and ORF5 of potexviruses, the octamer motif is highly conserved . The longure 4B) . As in agure 4B) . Redundagure 4B) . Complemcis-acting elements for intracellular trafficking to organellar membranous target sites, minus-strand RNA synthesis, plus-strand genomic RNA synthesis, subgenomic RNA synthesis, viral movements, and viral encapsidation. In this review, we summarize studies of most of the cis-acting elements identified in the BaMV genome, except for those involved in viral movement and viral encapsidation. The signal for BaMV genomic RNA encapsidation is very likely in the 5′ UTR, similar to those identified in PVX (For accomplishing an efficient infection by a positive-sense RNA virus, the viral genome consists of various d in PVX , 2015. TI-HC wrote the section on the plus-strand RNA synthesis. Y-WH wrote the section of subgenomic RNA synthesis. C-HT wrote the rest part of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RBM10 is an RNA-binding protein that plays an essential role in development and is frequently mutated in the context of human disease. RBM10 recognizes a diverse set of RNA motifs in introns and exons and regulates alternative splicing. However, the molecular mechanisms underlying this seemingly relaxed sequence specificity are not understood and functional studies have focused on 3΄ intronic sites only. Here, we dissect the RNA code recognized by RBM10 and relate it to the splicing regulatory function of this protein. We show that a two-domain RRM1–ZnF unit recognizes a GGA-centered motif enriched in RBM10 exonic sites with high affinity and specificity and test that the interaction with these exonic sequences promotes exon skipping. Importantly, a second RRM domain (RRM2) of RBM10 recognizes a C-rich sequence, which explains its known interaction with the intronic 3΄ site of NUMB exon 9 contributing to regulation of the Notch pathway in cancer. Together, these findings explain RBM10's broad RNA specificity and suggest that RBM10 functions as a splicing regulator using two RNA-binding units with different specificities to promote exon skipping. RBM10 gene are frequently found in patients with the TARP syndrome , an X-linked inherited pathology associated with malformation of multiple organs and significant early-life mortality  and two zinc fingers (ZnFs) Figure . Three oIt is possible that RBM10 encodes a very low RNA-binding specificity and that this makes the protein a non-discriminant RNA binder. However, RRMs and ZnF are typically sequence-specific domains and we reasoned that the difficulties in identifying RBM10's sequence selectivity might stem from the distinct specificities of the different domains, which could be used to target varied sets of RNAs. We now provide a combination of biophysical, molecular and bioinformatics data to show that RBM10 possesses two distinct RNA recognition units with very different sequence preferences. We propose that this expands the repertoire of RBM10 splicing targets by facilitating its interactions with naturally dissimilar intronic and exonic sequences.http://www.origene.com). Primers were designed using the Crystallization Construct Designer Software  and used to amplify regions of the RBM10 gene while introducing flanking ligation independent cloning sites. The details of constructs used throughout the study are shown in Figure 6 fusion proteins. DNA sequencing confirmed all plasmid insert sequences prior to recombinant protein expression.The gene encoding human RBM10 (Uniprot P98175) was purchased from OriGene (6 tag or His6SUMO3 fusion proteins in Escherichia coli BL21(DE3) cells grown to an OD600 of 0.6 before the temperature was reduced to 22°C and protein expression was induced with Isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM. Cells were lysed and the fusion proteins purified from the soluble fraction of lysate by nickel-affinity chromatography. HRV 3C protease was used to cleave the His6 tag or His6SUMO3 tag by incubation overnight at 4°C then both the tags and protease were separated from the cleaved protein by nickel-affinity chromatography. The protein was purified further on a HiTrap Heparin HP 5ml column  to remove nucleic acid contamination and a final purification step of size exclusion chromatography was performed using an ÄKTA purifier system  with a Hiload S75 16/60 Superdex prep grade column .Proteins were expressed as His2 and concentrated in a Vivaspin MWCO 10 000. Protein concentration was determined from the absorbance at 280 nm.Fractions of pure protein were pooled and concentrated before being dialyzed into a final buffer of 10 mM phosphate pH 6.9, 50 mM NaCl, 1 mM TCEP, 10 μM ZnCl1H frequency. Data were processed using the NMRPipe/Draw suite of programs and analyzed using CCPN.All NMR experiments were carried out using Bruker Avance NMR spectrometers operating at 600, 700, 800 and 950 MHz, 2. For RBM10 RRM1–ZF protein backbone assignments for 1HN, 15N, 13Cα and 13Cβ and 13C’ chemical shifts were obtained from HNCACB, CBCA(CO)NH, HNCO and HN(CA)CO experiments. For RBM10 RRM2 protein backbone assignments for 1HN, 15N, 13Cα and 13Cβ chemical shifts were obtained from HNCA and CBCA(CO)NH experiments.Samples were at ∼400 μM protein concentration in 10 mM sodium phosphate (pH 6.9), 50 mM NaCl, 1 mM TCEP, 10 μM ZnCl2 with 10% D2O added for the lock. In order to determine longitudinal and transverse relaxation rates standard experiments were run , 50 mM NaCl, 1 mM TCEP, 10 μM ZnClwere run . For RRM15N-labeled samples of protein in 10 mM sodium phosphate (pH 6.9), 50 mM NaCl, 1 mM TCEP, 10 μM ZnCl2 were titrated with unlabeled RNA oligomers. 1H–15N SOFAST-HMQC spectra were recorded at each titration point at 298 or 310 K.All RNAs used in NMR titrations were purchased from Dharmacon, GE Healthcare. 40–100 μM 15N-labeled RBM10 RRM2 in 10 mM sodium phosphate (pH 6.9), 50 mM NaCl, 1 mM TCEP were prepared and individual RNA pools were added to the ratio of 1:2 as required. 1H–15N SOFAST-HMQC spectra were recorded for the free protein and for each of the protein–RNA samples at 298 K. Spectra were processed using NMRPipe and analyzed in CCPN. A subset of peaks, which shift in the fast exchange regime upon addition of RNA, was chosen. Shift perturbation for each of the peaks upon addition of each RNA pool was measured. For each position in the binding site, the shifts are normalized to the largest of the four then for each pool the shift values are averages over all the chosen peaks to give the final score.Protein samples of 40 μM 2 was added if protein constructs contained the ZnF domain). Typically, the sample cell contained RNA at 20 μM and the syringe RBM10 RRM1–ZF at 200 μM or RBM10 RRM2 at 190 μM. Titrations were carried out at 25°C using 19 injections of 2 μl with a delay of 180 s between injections. Areas under the peaks were integrated and fitted by least-square procedures assuming a 1:1 binding model using MicroCal Origin 7 software.All RNAs for ITC experiments were purchased from Dharmacon GE Healthcare. Binding of RBM10 constructs to the RNA oligomers was measured by ITC using an ITC200 instrument . Samples were dialyzed into in 10 mM sodium phosphate (pH 6.9), 50 mM NaCl, 1 mM TCEP  based on the PARalyzer tool  and cloned into the pEGFP-N1 plasmid at the XhoI and MfeI sites replacing the EGFP gene. Predicted RBM10 binding motif in the exon 7 was mutated by site-directed mutagenesis using Quikchange protocol and the following primers: 5΄-CCTTCTACTCCAGCCACAACAGCAGATAATGGTACTTCAGCATGG-3΄ and 5΄-CCATGCTGAAGTACCATTATCTGCTGTTGTGGCTGGAGTAGAAGG-3΄. pCMV6-Entry plasmid containing human RBM10 coding sequence with a C-terminal Myc-DDK tag was purchased from OriGene (RC200150). Human RBM10 siRNA mixture was from GE Dharmacon .A minigene containing TNRC6A exon 7 in its natural context – including partial sequences of exons 6 and 8 and the entire sequences of the two intervening introns – was generated by amplifying the corresponding genomic DNA fragment from human genomic DNA  with High-Fidelity DNA polymerase  and the following primers: 5΄-AATCHEK-293T cells were propagated in Dulbecco's modified Eagle's medium (DMEM) with GlutaMax  supplemented with 10% fetal bovine serum  and 1× penicillin/streptomycin . Cells were co-transfected with plasmids and siRNAs using Lipofectamine 2000  as recommended. RNA was extracted using Trizol reagent , reverse-transcribed with SuperScript III  and analyzed by semi-quantitative PCR using as described previously  and the corresponding CLIP-Seq motif . This conclusion is reinforced by the minimal effect on the NMR resonances of RRM1 of unfolding the ZnF, by chelating the Zn ion with EDTA  is significantly different from that of the ZnF (6.49 ± 0.81 ns) in the two-domain construct  affinity loss in ITC experiments Figure  and did Our data has shown that, RBM10 RRM1–ZnF recognition of the (C)UGUGGA sequence is a key element defining the RBM10-RNA interactome. However, RBM10–RNA interactome data indicate that additional RNA sequences are likely recognized by RBM10. Our RNAcompete assays show that the GGA signal is diluted when RRM2 is present in the construct and we reasoned that this domain might facilitate RBM10 interaction with sequences different from those recognized by the RRM1–ZnF module. Although the RRM2 domain of the RBM10 paralogue RBM5 has been shown to bind a diverse range of RNA sequences with similar affinity  the sequ15N T1 and T2 NMR relaxation data recorded on the RRM1–ZnF–RRM2 construct were used to calculate the rotational correlation times (τc) of the different domains. The τc of RRM2 is different from that of RRM1, and of the ZnF, and this indicates that the RRM2 re-orients quasi-independently in solution  intronic sequences immediately preceding (3΄ segments) and following (5΄ segments) exons  exons ,9,10 ands) exons . We thers) exons  even thos) exons .When we repeated this analysis for RBM10-interacting sequences, we noticed a general increase in the relative frequency of UGUGGACA-like sequences compared to CCCCCUGG-like sequences for all three regions Figure . This waRBM10 has been reported to control splicing of a large number of alternative exons. However, the only natural splicing target of this protein directly tested so far has been the C-rich intronic sequence preceding RBM10-repressed NUMB exon 9 , we analInspection of the list containing high-confidence RBM10 splicing targets  identifiin vitro unbiased analysis of the sequence specificity.RBM10 is a multi-domain RNA-binding protein with important functions in development and in cancer. RBM10 regulates alternative splicing in a large set of genes but how the protein selects its targets is unclear, and the requirements for its positioning in the proximity of the intron/exon junction are not understood. Here, we show that the RRM1 and ZnF domain of the protein, although not locked in a preformed structural interaction, come together to recognise CUGUGGA-like sequences, with the interaction between the ZnF binding and the GGA unit providing most of the recognition specificity. The specificity towards GGA dominates both the results of our analysis of RBM10–RNA interactome data and our In addition, we identify a second C-rich target sequence whose recognition is mediated by the RRM2 domain. This second signal, also found in the RBM10–RNA interactome and shown to be important for regulation, is masked by the RRM1–ZnF signal when evaluating the specificity of the three-domain protein in RNAcompete assays. The interference between RNA modules with different specificities explains, at least in part, the difficulties encountered in defining RBM10's principles of target selection. This study highlights what may be a common a problem in determining the rules of target selection in multi-domain splicing regulators.While we show that RBM10's multiple RNA-binding domains recognize different RNA sequences, we also question whether the two RNA recognition units described above play a different role in positioning the protein on the RNA. We analyzed the intron/exon partition of RBM10's binding sites and show that the GGA-based motif, but not the C-rich one, is enriched in exonic sites. This suggests the protein uses the high specificity of the ZnF domain to access GGA-rich exonic sequences and overall that the differential action of two RNA recognition units may facilitate efficient targeting of exonic versus exon-adjacent intronic regions. Of note, purine-rich sequences have been previously shown to function as exonic splicing enhancers interacting with cognate SR proteins ( and refeRegardless, testing the effect of the RRM1–ZnF interaction on the regulation of TNRC6A splicing using minigene assays, we showed that it results in exon exclusion, similar to the previously reported RBM10 intronic sites. These functional data support the idea of a common action for different RBM10 targets and suggest that the recognition of two diverse sequence motifs may be linked to the ability of this splicing regulator to access RNA regions on either side of the splice site.Microarray data were submitted to the GEO database under the accession number GSE96990.Supplementary DataClick here for additional data file.


Cellular life is based on interacting polymer networks that serve as catalysts, genetic information and structural molecules. The complexity of the DNA, RNA and protein biochemistry suggests that it must have been preceded by simpler systems. The RNA world hypothesis proposes RNA as the prime candidate for such a primal system. Even though this proposition has gained currency, its investigations have highlighted several challenges with respect to bulk aqueous media: (1) the synthesis of RNA monomers is difficult; (2) efficient pathways for monomer polymerization into functional RNAs and their subsequent, sequence-specific replication remain elusive; and (3) the evolution of the RNA function towards cellular metabolism in isolation is questionable in view of the chemical mixtures expected on the early Earth. This review will address the question of the possible roles of heterogeneous media and catalysis as drivers for the emergence of RNA-based polymer networks. We will show that this approach to non-enzymatic polymerizations of RNA from monomers and RNA evolution cannot only solve some issues encountered during reactions in bulk aqueous solutions, but may also explain the co-emergence of the various polymers indispensable for life in complex mixtures and their organization into primitive networks. Prebiotic chemistry is intimately linked with the emergence of life and should therefore have comprised most chemical processes that yielded biomolecules or at least their precursors relevant for the formation of precellular entities. It also determines at least in part the types of molecules that compose cells even today. Although the chemical definition of life is still debated, the ubiquitous presence of biopolymers at the core of the metabolism (peptides), information make-up  of cells and that of other molecule types (lipids for membranes) highlight the need for their early development, perhaps already during the time period in the Earth history that coincides with prebiotic chemistry. However, depending on the approach to that question, whether one decides to explore a top-down or a bottom-up perspective, one will be confronted with different questions that pertain to the core of prebiotic chemistry. The top-down approach is defined as a retrospective search that looks at common attributes of contemporary cells that likely were already present in the last common ancestor(s) (LCA) and might have been present at the origin of life. That is, the time interval between the emergence of the first “living” systems and the LCA, as well as the likely large difference in the organization of these systems leave the door open for intermediate systems, products and potentially victims of evolutionary processes, which might have left no traces in contemporary biochemistry.Considering the bottom-up approach, a prospective process, the main issue lies in the availability of the compounds on the prebiotic Earth, which is linked to the geochemical conditions on the early Earth. Unfortunately, the dearth of geological evidence on this period prevents us from having a clear picture. Indeed, this approach is clouded by uncertainties about the sources of prebiotic chemicals, which would depend on the flux and nature of extraterrestrial compounds delivered on the Earth and the nature of the primitive environment  and possibly landmass(es)). Even in the event that the chemical molecules might have existed, their reactivity  in this environment would have further defined whether they could accumulate, polymerize and later whether the level of chemical complexity required for the emergence of life could be attained.The RNA world hypothesis postulates that modern life, based on DNA, RNA and proteins, was preceded by a primitive form of life in which RNA replaced both DNA and proteins, as a genetic and catalytic polymer, respectively. Its prebiotic synthesis is therefore one of the most important steps in chemical evolution. ,2,3 ThisThe fundamental requirements of the RNA world hypothesis are (i) monomer (ribonucleotides) availability; (ii) efficient non-enzymatic spontaneous and template-directed polymerizations, that is, reactions whose RNA production must have at least outpaced the degradation of their products; (iii) the capacity of short non-enzymatically polymerized RNA oligomers to possess a catalytic/binding function and (iv) their capacity to self-assemble into long strands capable of evolved catalysis. These various stringent requirements have led some of the pioneers in the field to qualify the RNA world hypothesis as a “biochemist dream.” .The availability of the ribonucleotides has long been an unresolved issue. RNA monomers are conjugates of three different chemical parts, a phosphate, a ribose and a nucleobase A. One syIt should also be noted here that as for any prebiotically-relevant species, a multitude of related, but different compounds would have likely been “by-products” of prebiotic syntheses as exemplified by the formose reaction that only delivers a small amount of ribose  among a Assuming that RNA monomers were available, the next steps would be their incorporation into oligomers and polymers relying on their chemical reactivity and simple catalysts, as enzymes were absent on the early Earth. When considering this type of polymerization, one should differentiate between monomer self-condensation and template-directed (TD) polymerization . In the 2N) reaction 2+, or mixtures thereof in high-ionic strength media  and at low temperatures (0 °C to 4 °C) to enhance the intermolecular interactions between monomers and templates, as well as primers in the corresponding studies. Using a template/monomer system, the insertion of activated monomers on the homopolymeric templates with cognate residues only yielded good incorporations of activated G monomers on poly(C) templates . H. H81]. HTD polymerization on mineral surfaces was only superficially investigated on hydroxyapatite ,84 and fPorous minerals, formed by the precipitation of ions in ion-rich hot water in contact with colder water at hydrothermal vents , could hEutectic phases in water/ice  and highIn the salt/water eutectic phase system, the progressive evaporation of the solution containing monomers (NMPs) and inorganic salts at moderate temperatures (85 °C) leads to the crystallization of the salts concentrating the monomers within the remaining aqueous volume . The inoBoth approaches have delivered RNA analog products in relatively high monomer equivalent yields (over 60% monomer incorporation) even during self-condensation of monomer mixtures ,95. In tTD polymerization was only demonstrated in the water/ice system. Interestingly, the initial elongation rates of RNA hairpin  studied as a function of the monomer concentration were comparable for all canonical nucleotides with optimal concentrations lying between 100 s µM and less than 2 mM . The anaBesides the potential advantages of eutectic phases  and catalysts), several authors mention a possible pre-organization of monomers and interactions with the extended ice-surfaces, which could also be hypothesized for the salt/water eutectic. Such effects have yet to be demonstrated. However, it is clear at least for the water/ice eutectic that its 3D organization significantly affects the outcome of the polymerization. The nature of the solutes and their concentration, even those that are not reactants and do not act as catalysts, will influence reactions occurring in the eutectic phase and its characteristic . For exaThe eutectic phase in water/ice has also been shown to promote additional processes that are relevant to the RNA world hypothesis, such as the catalytic activity of ribozymes among other ligases ,101 and Contrary to previous heterogeneous media, self-assembled molecular systems require prebiotic synthesis of compounds, e.g., peptides , sugars The promotion of RNA monomers’ self-condensation and DNA monomer TD polymerization was investigated ,110 usinHowever, polymerization yields remained quite low (ca. 6% by weight). Interestingly, the length of the longest products depended on the number of different monomers present, as mixtures of two monomers (5′ UMP and AMP) yielded shorter polymers up to a length of 30 to 40 mers. The predominance of 2′-5′ linkages in the products can be surmised from the lack of product enzymatic digestion.DNA monomer TD polymerization has also been reported in amphiphile matrices  under coBased on the reaction conditions and on the observations made by X-ray , a polymThe temperature at which the dehydration proceeds, while being moderate compared to those at marine vents in acidic conditions, could still reduce the polymerization yields, as the report of product depurination  and hydrAs for the other heterogeneous media, the lipid matrices could also have played some significant, additional roles in the emergence of compartmentalized precellular systems or protocells (the nucleic acid products can be encapsulated in the reforming vesicles during rehydration) ,113 or eThe research undertaken to better understand the synthetic processes related to the emergence of the RNA world has significantly progressed in the last couple of years. In particular, several sets of “prebiotically-plausible” conditions have been found, which support the synthesis of RNA monomers, their efficient concentrating out of bulk aqueous solutions, their polymerization (self-condensation) and the replication of RNA sequences (TD polymerization). Interestingly, many of these processes require some form of “compartmentalization” that can be provided by several types of heterogeneous media. The relevance of any heterogeneous medium can be debated, as their basic properties  are derived from proposed and argued about scenarios for the early Earth environment. The similarities and complementarities between the various heterogeneous media cannot be overlooked, as most heterogeneous systems not only concentrate molecules out of the bulk aqueous solution, but additionally seem to promote their organization into molecular assemblies that are conducive to RNA polymerization. To some extent, they all protect both reactants and products against degradation. These various heterogeneous media are also complementary as far as they cover a large spectrum of environmental conditions that could have been present on the early Earth.In the exploration of this type of heterogeneous catalysis, the composition of the samples studied has gradually become more complex. Mixtures of monomers and catalysts , but also of various types of monomers/bystander molecules, from the point-of-view of their biochemical involvement in RNA polymerization, such polycyclic molecules , as wellThe long-standing problem of the RNA world hypothesis, i.e., the formation of polymeric RNA catalysts and their replication/amplification, has clearly not yet been resolved as the demonstration of the emergence of any catalytically-active RNA from monomers is still elusive. However, many potential environments that are plausible on the early Earth have been shown to promote some processes deemed essential for the emergence of an RNA world or its precursors. At this time, the evidence available about the early Earth conditions, as well as the fragmentary nature of the collected data preclude a definitive resolution of the question, but it is also conceivable that several heterogeneous media may have played a role either simultaneously (assuming an early Earth with various microenvironments) or at different stages of the RNA world emergence and evolution. Nonetheless, the most recent advancements in this research field have clearly highlighted the need for a systemic approach to the question that includes geochemistry, geophysics and more complex chemical systems (in opposition to the purified systems often used early on) to explore potential synergies between various biopolymers or their precursors, as well as other chemicals present in the prebiotic environment.


Translation of the pol gene is dependent upon readthrough of the gag UAG stop codon; whereas the env gene is translated from spliced mRNA transcripts. Here, we report the first high resolution analysis of retrovirus gene expression through tandem ribosome profiling (RiboSeq) and RNA sequencing (RNASeq) of MuLV-infected cells.The retrovirus murine leukemia virus (MuLV) has an 8.3 kb RNA genome with a simple 5′-gag stop codon readthrough to access the pol gene. Initiation of translation was found to occur at several additional sites within the 5′ leaders of the gag and env transcripts, upstream of their respective annotated start codons.Ribosome profiling of MuLV-infected cells was performed, using the translational inhibitors harringtonine and cycloheximide to distinguish initiating and elongating ribosomes, respectively. Meta-analyses of host cell gene expression demonstrated that the RiboSeq datasets specifically captured the footprints of translating ribosomes at high resolution. Direct measurement of ribosomal occupancy of the MuLV genomic RNA indicated that ~ 7% of ribosomes undergo These experiments reveal the existence of a number of previously uncharacterised, ribosomally occupied open reading frames within the MuLV genome, with possible regulatory consequences. In addition, we provide the first direct measurements of stop codon readthrough efficiency during cellular infection.The online version of this article (10.1186/s12977-018-0394-5) contains supplementary material, which is available to authorized users. Gammaretrovirus, has long served as a model for the study of retrovirus molecular biology. The viral genomic RNA (gRNA) is approximately 8 kb in length; encoding the gag, pol, and env genes in the 5′–3′ direction , the prototype ion Fig. a. Replicion Fig. a 1]. Th. ThGammaThe development of advanced RNA sequencing methodologies in recent years has made it possible to monitor viral gene expression at single nucleotide resolution –6. We caRattus norvegicus) monolayers at 10% confluence ).Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. These were blocked for 30–60 min with 5% powdered milk (Marvel) in PBST ] dripped onto the cells. Cells were scraped, collected and triturated with a 26-G needle ten times. Lysates were clarified by centrifugation for 20 min at 13,000g at 4 °C.At 96 h p.i. cells were treated with CHX , or HAR  then CHX . Cells were rinsed with 5 ml of ice-cold PBS, the dishes were submerged in a reservoir of liquid nitrogen for 10 s and then transferred to dry ice and 400 µl of lysis buffer [20 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM MgClCell lysates were subjected to RiboSeq and RNASeq as previously described . The metRattus norvegicus) and virus RNA using bowtie version 1 [Adaptor sequences were trimmed using the FASTX-Toolkit (hannonlab.cshl.edu/fastx_toolkit/) and reads shorter than 25 nt following adaptor trimming were discarded. Trimmed reads were then mapped to host  were counted. Histograms of the 5′ end positions (+ 12 nt offset) of host mRNA reads relative to initiation and termination codons were derived from reads mapping to RefSeq mRNAs with annotated coding regions ≥ 450 nt in length and with annotated 5′ and 3′ UTRs ≥ 60 nt in length. To estimate the gag stop codon readthrough efficiency, the expression levels  of the gag and pol ORFs were calculated by counting RPFs and RNASeq reads with 5′ end + 12 nt offset positions mapping within the respective coding regions, excluding 15 nt immediately upstream and downstream of the initiation and termination codons.To normalize for different library sizes, reads per million mapped reads (RPM) values were calculated using the sum of positive-sense virus RNA reads and host RefSeq mRNA reads as the denominator. For viral genome coverage plots, and for meta-analyses of host RefSeq mRNA coverage, mapping positions of RPFs were offset + 12 nt to approximate the location of the ribosomal P-site . To calcThe short read length used for RNASeq (~ 28–34 nt) prevented a robust de novo analysis of MuLV splice variants from these data.