Evolution of exon-intron structure of eukaryotic genes has been a matter of long-standing, intensive debate. The introns-early concept, later rebranded ‘introns first’ held that protein-coding genes were interrupted by numerous introns even at the earliest stages of life's evolution and that introns played a major role in the origin of proteins by facilitating recombination of sequences coding for small protein/peptide modules. The introns-late concept held that introns emerged only in eukaryotes and new introns have been accumulating continuously throughout eukaryotic evolution. Analysis of orthologous genes from completely sequenced eukaryotic genomes revealed numerous shared intron positions in orthologous genes from animals and plants and even between animals, plants and protists, suggesting that many ancestral introns have persisted since the last eukaryotic common ancestor (LECA). Reconstructions of intron gain and loss using the growing collection of genomes of diverse eukaryotes and increasingly advanced probabilistic models convincingly show that the LECA and the ancestors of each eukaryotic supergroup had intron-rich genes, with intron densities comparable to those in the most intron-rich modern genomes such as those of vertebrates. The subsequent evolution in most lineages of eukaryotes involved primarily loss of introns, with only a few episodes of substantial intron gain that might have accompanied major evolutionary innovations such as the origin of metazoa. The original invasion of self-splicing Group II introns, presumably originating from the mitochondrial endosymbiont, into the genome of the emerging eukaryote might have been a key factor of eukaryogenesis that in particular triggered the origin of endomembranes and the nucleus. Conversely, splicing errors gave rise to alternative splicing, a major contribution to the biological complexity of multicellular eukaryotes. There is no indication that any prokaryote has ever possessed a spliceosome or introns in protein-coding genes, other than relatively rare mobile self-splicing introns. Thus, the introns-first scenario is not supported by any evidence but exon-intron structure of protein-coding genes appears to have evolved concomitantly with the eukaryotic cell, and introns were a major factor of evolution throughout the history of eukaryotes. This article was reviewed by I. King Jordan, Manuel Irimia (nominated by Anthony Poole), Tobias Mourier (nominated by Anthony Poole), and Fyodor Kondrashov. For the complete reports, see the Reviewers’ Reports section. In a memorable phrase of Walter Gilbert, eukaryotes possess “genes in pieces” in which protein-coding sequences are interrupted by non-coding sequences denoted introns. The intGU(A/G)AGU sequence  at the donor splice signal is complementary to the 5’ end of the U1 snRNA, and this interaction appears to be the major requirement for splicing, h, h132], A. thaliana share ~25% intron positions. Th. Th53]. The reconstructions of the evolution of gene architecture in eukaryotes were performed on sets of orthologous genes with a single representative (or a single most conserved representative) in each of the analyzed genomes. Obviously, this type of reconstruction reflects only one facet of evolution of gene structure given that all eukaryotic genomes encompass numerous families of paralogous genes with broad distributions of the number of members. Reconstruction of parsimonious scenarios of gene structure evolution in paralogous gene families in animals, plants and malaria parasites revealed numerous apparent gains and losses of introns,142. In In contrast, comparison of the exon–intron structures of ancient eukaryotic paralogs reveals the absence of conserved intron positions in these genes Figure148]. T. T148]. The combined advances of comparative genomics and systems biology provide means to characterize genes by many features, for example expression level and connectivity in protein-protein interaction or regulatory networks. Various significant correlations have been demonstrated to exist between these variables; in particular, one of the most prominent, recurrent links is that the sequence of highly expressed genes tends, on average, to be more conserved-154. ConProbabilistic evolutionary reconstruction of gene structure yields gene-specific rates of intron gain and loss and thus provides for analysis of possible relationships between these rates and other characteristics of the respective genes. It has Drosophila have, on average, shorter introns than genes expressed at a lower level. St. St46]. It appears most likely that the Group II intron invasion was triggered by the establishment of the endosymbiosis between an α-proteobacterium and an archaeal host Figure. NotablyIndeed, it has to be emphasized that Group II introns are typical mobile elements that actively spread around the host genome when given a chance by weakness of purifying selection pressure.However, at the mechanistic level, the adaptation of the early eukaryotes to the swarms of genomic parasites , whereas the forms that never achieved high efficiency of purifying selection  followed the path to complexity.The elucidation of the general scenario of evolution of eukaryote gene architecture by no account implies that the main problems in the study of intron evolution and function have been solved. Quite the contrary, fundamental questions remains wide open.What are the mechanisms of intron loss and gain? There is very little direct evidence of any. A consensus seems to exist regarding the role of reverse transcription in intron loss although even this mechanism badly needs experimental corroboration. As for the mechanisms of intron gain, indications of the involvement of double-strand break repair notwithstanding, the study of this key problem has not even started in earnest.What are the sources of new introns? It is clear that duplication of pre-existing introns is a negligible route of intron evolution in many eukaryotic lineages although it seems to be important in some, whereas intronization of (parts of) exons appears to be a significant contribution throughout the evolution of eukaryotes. However, possible other routes of intron acquisition let alone their relative quantitative contributions remain unknown.What is the general role of introns in gene expression and function (if any) and why is it the case that new genes (such as those acquired from chloroplasts in plants) are saturated by introns at an apparent high rate? And a related question: why do a handful of introns  survive in the great majority of even the most streamlined eukaryotes? At best, only most general and largely speculative answers to these key questions are currently available. These are hard questions, and the only hope to obtain satisfactory answers is to combine comprehensive phylogenomic analysis with population genetic models and extensive experimentation.The authors declare that they have no competing interests.IBR, LC, MC and EVK wrote the manuscript which was read, edited, and approved by all authors.Reviewer #1: Dr. I. King Jordan, Georgia Institute of TechnologyIgor Rogozin and colleagues have written a comprehensive, synthetic and compelling review mainly covering the ‘evolution’ of spliceosomal introns. As in Darwin’s famous tome, the notion of ‘origin’ is actually given short shrift, but I will come to that point later. In any case, this ambitious work benefits from the broad perspective that the authors have gained over years of investigating the subject. Along with this perspective comes some inevitable bias, or perhaps it is more fair to say a favored world-view, as described below with respect to the authors position on ‘introns-kind-of-early’. But this does not represent a liability of the work in my opinion; the authors are clearly entitled to their views, and the conclusions they draw appear to be both nuanced and well-supported by the data. They cover a lot of ground herein and strike a nice balance between thoroughly reviewing the relevant literature and elucidating the most salient points from the large body of work on the subject. One of the major conclusions of this review relates to a resolution, or compromise really, of the ‘introns-early’ versus ‘introns-late’ debate that consumed the field for many years. The authors champion a merger of these two hypotheses into the ‘many introns early in eukaryotic evolution’ view, whereby the earliest eukaryotic lineages contained genomes that were already loaded with many introns and subsequent evolution was dominated by intron loss.The parts of the review that cover the origin of spliceosomal introns are the most speculative and least supported. This is not a critique per se; it may simply be the case that the study of origins must always be more speculative than the study of evolution. According to the ‘many introns early in eukaryotic evolution’ hypothesis, the earliest eukaryotic genomes were formed via massive intron invasion that resulted in genomes consisting of up to 80% intronic DNA. Crucially, the authors hold that this invasion was probably facilitated by low effective population sizes and the corollary weak purifying selection, following the influential Michael Lynch model for the non-adaptive evolution of eukaryotic genome complexity. This model accounts for population level dynamics but neglects the internal dynamics of the genome. If spliceosomal introns indeed evolved from Group II introns, as the authors maintain, then the initial intron invasion of eukaryotic genomes would have been driven, to some extent, by a kind of selfish genetic element with its own internal drive mechanism to replicate within the genome. In theory, such selfish replicators can efficiently increase in copy number even in the face of a selective cost to the host. Therefore, the early origin of introns may be attributed to an active internally driven process, rather than a solely passive drift related process, i.e. a mechanism akin to the molecular-drive concept of Gabriel Dover or the mutation bias emphasized by Arlin Stoltzfus. Such an active replicative process inside the genome could have actually outpaced selection’s ability to contain it. The authors actually touch on this notion, when they speculate as to whether introns are genomic parasites and how the host may have evolved the spliceosome as an adaptive response to intron invasion, but an explicit connection between their selfish drive to replicate and the origin of introns is not made.We agree on all accounts. Yes, it comes with the territory: discussion of origins is inevitably more speculative than the analysis of subsequent evolution. More importantly, the role of the active mobility of Group II introns certainly must not be under-appreciated, and we explicitly point this out in the revised manuscript: ‘Indeed, it has to be emphasized that Group II introns are typical mobile elements that actively spread around the host genome when given a chance by weakness of purifying selection pressure.’Authors’ response: One specific suggestion as to how the work can be improved relates to the abstract. Currently, the abstract is very short and concise, whereas the manuscript is rather long and presents a lot of material. I think it would be helpful to provide a more detailed abstract that specifically enumerates the authors’ most important points, something more like of a summary of the last two sections of the manuscript.Authors’ response: We fully agree, the original short abstract resulted from a misunderstanding regarding the limits on abstract length in review articles. In the revised article, the abstract was substantially expanded.Reviewer #1: Dr. I. King Jordan, Georgia Institute of Technology I have re-reviewed the manuscript of Rogozin et al. I am satisfied with the changes made, for the most part, and I recommend that the paper be accepted for publication in Biology Direct after the following point is addressed.I would like the authors to elaborate just a bit on their response the first comment that I made, in particular with respect to the connection between Group II intron dynamics and the evolution (emergence) of introns. I think I may have rambled a bit in my original comment and was not explicit enough. I would urge the authors to have a look at the manuscript of Donal Hickey from Genetics , which makes the point much better than I did in my comment. The population genetics models in the manuscript may be a bit simplistic by this time, but I think the ideas contained therein are highly relevant to their own work. In particular, Hickey makes an explicit connection between the genome dynamics of mobile elements, host selection pressure and the evolution of introns. The basic idea is that mobile genetic elements can spread in a population even in the face of a fitness cost to the host, and this kind of process could have resulted in the emergence and spread of introns. Below, I provide a comment in response to the authors' response to my first comment in an attempt to facilitate further discussion and consideration of this issue.Authors' response: We agree on all accounts. Yes, it comes with the territory: discussion of origins is inevitably more speculative that analysis of subsequent evolution. More importantly, the role of the active mobility of Group II introns certainly must not be under-appreciated, and we explicitly note in the revised manuscript: 'Indeed, it has to be emphasized that Group II introns are typical mobile elements that actively spread around the host genome when given a chance by weakness of purifying selection pressure.'Response: I would like the authors to further consider the possibility that mobile elements (such as Group II introns) can increase in frequency in a population, even when they impose a fitness cost on their host organisms, owing to the fact that a replicative transposition process results in a biased transmission rate relative to host genes. This idea was introduced by Donal Hickey 30 years ago, and he also connected this point to the evolution of introns (Hickey 1982 Genetics 101: 519). In other words, it is not simply a matter of weak purifying selection allowing active spread of the elements, but an effect of the element mutational dynamics introducing directional bias in the evolutionary process. This idea is very much analogous to the notion that mutation bias in the broader sense can be a cause of direction in evolution (e.g. see Yampolsky and Stoltzfus 2001 Evol Dev 3: 73).We agree that mutational dynamics of selifhs element could be an important driver of their spread. We think that once this additional exchange with the reviewer is published, the emphasis on this issue will be adequate.Authors' response: Reviewer #2. Dr. Tobias Mourier, University of Copenhagen (nominated by Dr Anthony Poole)This review provides a comprehensive overview of the current knowledge of intron evolution in eukaryotic genomes.The advent of numerous eukaryotic genomic sequences has consistently supported the 'many introns early in eukaryotic evolution' concept, as evident from the manuscript. But surely this hypothesis is not a merger from the introns early/late/first ideas . All recording of spliceosomal intron features comes from eukaryotic genomes, and regardless of how many eukaryotic genomes are sequenced, extant spliceosomal intron features will never allow one to synthesize past LECA.In the end of the manuscript, the authors present a scenario proclaiming that an intron-rich LECA is not inconsistent with the introns-late hypothesis. This is not a problem, but the structure of the manuscript may give the impression that this is a conclusion (or synthesis) directly from the current knowledge of eukaryotic gene architecture (that is nicely reviewed in the preceding text).Authors’ response: Actually, we do believe that the synthesis we present in the section of the review preceding the Conclusions follows from the comparative genomic results reviewed in the preceding sections. Certainly, not all parts of the article directly contribute to this synthesis: for instance, the discussion of the functional roles of introns is only tangentially relevant here albeit important in other respects. Nevertheless, we do maintain that in this section we present major implications of the comparative genomic study of eukaryotic gene structure.The review presents an overview of the comparative approaches taken to delineate intron-exon structures during evolution. The basis for such comparative analyses is well-aligned sequences around splice sites. If intron-exon structures to some extent evolve via mechanisms such as alternative splicing and intronization of exonic sequence, should this not result in sequences that are unlikely to meet the criteria for being included in the above analyses? I think it would be relevant to discuss the implications of this.Authors’ response: This issue is discussed in the section ’Evolutionary conservation of intron positions and routes of gene architecture evolution of eukaryotes’.Section "Functional elements and genes within introns"When discussing intronic RNA genes, I'm surprised there is no mentioning of the classical connection between vertebrate snoRNAs and introns .Authors’ response: Yes, this certainly is an important theme, and we added it to the section ‘Functional elements and genes within introns’.Very minor points:Page 11: "whereas the remaining 6 Xist" rather than "whereas remaining 6 Xist"Page 14: "and so does the strength" rather than "and so does and the strength"Page 18: should "introns are inserts or fixed" read "introns are inserted or fixed"?Authors’ response: All corrected, we appreciate the reviewer’s attention to these points.Reviewer #3. Dr. Manuel Irimia, University of Toronto (nominated by Dr Anthony Poole)Rogozin et al. have put together an impressively comprehensive review on the origin and evolution of splicesomal introns that will certainly become a major reference in the field. Overall, I found it easy and entertaining to read, as well as informative. I have only a few comments and suggestions, often regarding further literature, that I hope can help to improve the piece (listed according to their appearance in the main text):Authors’ response: We appreciate Dr. Irimia’s close attention to the details of this article. As detailed below, we found most of the suggestions fully pertinent and modified the manuscript accordingly.1) P3: The paragraph on splice site consensus sequences could provide a more detailed portrait of canonical intron signals across eukaryotes. For example, not all eukaryotes have polyT tracts between the branch point (BP) and the 3’ AG, and some fungal species even have polyT tracts upstream the BP . Also, some extremely intron-poor species intriguingly have strict GTATGT as consensus 5’sequence (including yeast), which may be worth pointing out. Finally, the 3’ consensus is closer to YAG than to CAG, at least in most species.Authors’ response: We added discussion of this issue to the revised text.2) P5: I found the (exciting) discussion on the ancestrality of U2 vs. U12 too short and a bit imbalanced. Personally, I think it is a good idea that the authors give their authoritative opinion/preference on this kind of discussions, but the opposite arguments should also be presented extensively. In this case, I think the arguments supporting an ancestral origin of U12  should be fully developed.Authors’ response: In our view, the questionable greater similarity of U12 introns to Group II introns does not immediately imply ancestral status of U12 introns. We added to the text ‘it might be tempting to speculate that the ancestral introns were of the U12 type  but have been subsequently converted to U2 introns.’3) P8: Pleiss et al. (PLoS Biol 2007) may be added supporting a global regulatory function of introns in yeast.Authors’ response: We added discussion of this important work to the section ‘Functions of introns associated with splicing’.4) P9: I missed a more comprehensive and complete review of the literature on the genome-wide dynamics of intron gain and loss in this section. For example, on the general slow paucity of intron gain, I missed references on vertebrates , flies , plants , apicomplexa , Entamoeba , Fungi ). On the opposite side: tunicates , diatoms , mitochondrial transfers . Given the overall level of comprehensiveness and detail of this review and that, as I said above, it is very likely to become a major reference in the field, I think it would be important to cite all relevant references in the main text, in particular from such an important and prolific subtopic.Authors’ response: There is indeed a lot of evidence on specific events in individual lineage. We appreciate their importance but it is hardly possible to discuss ‘everything’ in detail. That said, the revised version of the review cites all the references pointed out by the reviewer.5) P9: when commenting on ref 61, the use of the word “dispute” may give the impression that there is an ongoing controversy or a difference in opinions between the authors, which I guess is really not the case. Ref. 74 showed that most reported gains in ref. 61 were indeed losses by adding more species to the analysis that were not available by the time of the original study. This may not be clear to general readers that have not followed the specialized literature.Authors’ response: We added this explanation to the text.6) P11: the authors may wish to mention here the recent work by Cabili et al. (Genes Dev 2011), which describe >8,000 lincRNA genes, with an average of ~1.9 introns per Kbp and that are extensively alternatively spliced, with 2.3 isoforms per gene.Authors’ response: We added a brief description to the text.7) P14: I was quite surprised to read that the sequences at the 3’ of the intron behave completely different from those at the 5’. Many of the extremely intron-poor species  that show strict 5’ splice site consensus also have very strict BPs, and sometimes even very constrained branch-point-to-AG distances . I guess this apparent contradiction is due to the fact that these species are all missing from the analysis by Iwata and Gotoh , etc.).8) P14: also related to splicing signals, it would be interesting to include a comment on the effect of intron size on splicing signals  FigureAuthors’ response: We included an additional explanation in the legend: “An excess of protosplice sites in phase 0 is noticeable, however the ‘protosplice site’ hypothesis, which posits that introns are randomly inserted into protosplice sites, is unable to fully explain the observed over-representation of phase 0 introns.”10) P26: the authors may want to point out from the beginning that the “two competing hypothesis” they present are not necessarily mutually exclusive.Authors’ response: Added to the text as suggested.11) P28: ref. 158 also concludes that alternative splicing has emerged early in eukaryotic evolution, so it should be cited along with ref. 166 and not with 28.Authors’ response: Modified as suggested.12) P31: more references may be added supporting the low conservation of alternative splicing in mammals . Similarly, many other studies have dealt with the evolution of alternative splicing from the perspective of the splicing signals, not only regarding GC splicing donor sequences  and their polymorphism in human populations .Authors’ response: A brief discussion and references added as suggested.13) P32: perhaps the section “Functions of introns” would fit better before the section on alternative splicing (since the latter is one of those functions).Authors’ response: Alternative splicing is not exactly a function of introns, rather a mechanism of modulation of protein and RNA function. In the functional section we addressed specific functions of intron sequences. This might be debatable but we consider the original order of the sections acceptable.14) P33: the authors may want to add that some spectacular, functional exceptions are known to the general case that splicing occurs before mRNA is exported to the cytoplasm. For example, Buckley et al. (Neuron 2011) describe the case of some transcripts with retained introns, which drive subcellular location of the transcripts to the dendrites due to the presence of a particular transposable element within their sequence.Authors’ response: We appreciate the reviewer bringing our attention to this exiting work. Cited and briefly discussed.15) P34: the catalog of U12 introns by Alioto (Nucleic Acids Res 2007) could be referenced here.Authors’ response: Cited as suggested.16) P35: I think it could be useful to make a clearer distinction between Splice Leader (SL) trans-splicing and trans-splicing between two different genes from the beginning of the paragraph (I found it a bit confusing now). Also, the authors may wish to cite a very elegant analysis searching for trans-splicing in Drosophila using RNAseq on hybrids .Authors’ response: We agree and have included a brief discussion and references as suggested.17) P36: in this subsection I missed a paragraph on the (predictable and predictive) association between long introns and the presence of functional elements. For example, Denoeud et al. (Science 2011) found that the few genes with long introns in Oikopleura are enriched for key developmental regulators, and that those introns likely contain regulatory information. This has also been observed for many other developmental genes across metazoans  and for associated non-developmental genes (“bystander” genes) , with exciting implications for the evolution of genome architecture. Also, supporting the presence of regulatory elements, higher sequence conservation is often found in longer introns .Authors’ response: Brief discussion and references included as suggested.18) P38: the authors may add the report by Curtis and Archibald (Curr Biol 2010) to the list of different sources of spliceosomal introns.Authors’ response: Cited as suggested.Reviewer #4. Dr. Fyodor Kondrashov, Center for Genome Regulation, BarcelonaThis is a straightforward and extensive review of everything that is known about the evolution of introns and then some more. I do not have much to add in addition to what the authors have already said. The only thing that I am left wondering about after reading this review is whether or not the authors think that Group II introns in LECA were involved in the transport of mitochondrial precursor genes into what is now the cytoplasm across the novel intracellular membrane. In light of the previous reviews I would leave it up to the authors to space and moderate the level and format of speculation, even though I believe that the nice synthesis the authors have produced make the review more interesting and useful.Authors’ response: We appreciate this comment. We are not entirely clear about the exact meaning of the reviewer’s idea regarding mitochondrial genes. Is this about transfer of genes from the mitochondrial to the nuclear genome? If so, the possibility of involvement of the reverse transcriptase activity of Group II introns is intriguing but in the absence of specific evidence, one would think the main route was DNA recombination.


We report a systematic investigation of the temperature dependence of electrical resistance behaviours in tri- and four-layer graphene interconnects. Nonlinear current–voltage characteristics were observed at different temperatures, which are attributed to the heating effect. With the resistance curve derivative analysis method, our experimental results suggest that Coulomb interactions play an essential role in our devices. The room temperature measurements further indicate that the graphene layers exhibit the characteristics of semiconductors mainly due to the Coulomb scattering effects. By combining the Coulomb and short-range scattering theory, we derive an analytical model to explain the temperature dependence of the resistance, which agrees well with the experimental results. The e2 V−1 s−1-12. Most2 V−1 s−1,14 and t2 V−1 s−1. As more-1/2 dependence shows the evidence of the electron–electron Coulomb interaction in FLG. Our temperature-dependent resistance results reveal that the FLG interconnects display semiconductor properties and further confirm that Coulomb interaction can play a dominant role.In this letter, we report a systematic investigation of the temperature dependence behaviour of the four-terminal electrical resistance in FLG interconnects. The resistance of tri- and four-layer graphene, under direct current (DC) electric fields and in a temperature range from 5 to 340 K was measured. The T2 substrate. The number of graphene layers was confirmed by micro-Raman spectroscopy through the 2D-band deconvolution procedure [−7 mbar. Electronic transport measurements were performed on multiple samples, using the physical property measurement system  with a fixed excitation current of 0.01 mA; the temperature varied from 5 to 340 K.The graphene layers were produced by mechanical exfoliation techniques  from bulrocedure . The Ramrocedure ,17. ShowI-V) characteristics at different temperatures of (a) tri- and (b) four-layer graphene interconnects. Insets show the enlargement of the measurement results at low electric fields. For the tri- and four-layer graphene, the interconnect resistors display two distinct regions of ohmic characteristic: one at fields larger than 0.01 V/μm but less than 0.10 V/μm and the other at fields larger than 0.10 V/μm. The nonlinear behaviour of current–voltage characteristics at low threshold (<0.10 V/μm), and the second ohmic region in the strong DC electric field (>0.10 V/μm) can be explained by the heating effect [I-V over the whole temperature measurement range.Figure g effect . Within g effect ,19. At sGd = dI/dV of (a) tri- and (b) four-layer graphene as function of the temperature T−1/2 on a semi-logarithmic scale. As shown in the Figure In order to study the existence of electron–electron Coulomb interaction and how it plays an important role in our system, we adopted the resistance curve derivative analysis (RCDA) method to investigate the dominant scattering mechanism . Figure E. Shown in Figure RT/RT = 5K as a function of the electric field E at different temperatures, for (a) tri- and (b) four-layer graphene interconnects. Insets display the optical micrographs of the FLG interconnect. At a lower temperature range of 5 to 50 K, as the electric field increases from 0 to 0.6 V/μm, the resistance of the tri- and four-layer graphene interconnects show a reduction of about 30% and 70%, respectively. However, for the temperature range T ≥ 200 K, the corresponding resistance drop is smaller. The larger drops of the resistance at lower temperature range indicate that Coulomb scattering is the main transport mechanism in the FLG interconnects at this temperature range as it is proportional to the carrier density. Hence, Coulomb scattering is strongly dependent on temperature. We further note that with increasing temperature, the observed results indicate that the scattering induced by electric field from the substrate surface polar phonons is significantly screened by the additional graphene layers at room temperature [There are many experimental and theoretical reports on the mobility of one- to three-layer graphene from two- and four-terminal measurements. The main scattering mechanisms in one- to three-layer graphene are Coulomb scattering -23, shorperature ,22.RT/RT = 5K, whereby we investigate the temperature dependence change of the resistance. In Figure RT/RT = 5K is observed for decreasing temperature for both the tri- and four-layer graphene. This R-T behaviour indicates that the carriers transport in the graphene layers is non-metallic in nature. This implies that, the resistance does not originate from thermal activation but is attributed to ES VRH between localized states induced by the charge impurities [In order to further study the VRH and localized insulating behaviour, we investigate temperature dependence of electronic transport measurements on a tri- and four-layer graphene. Figure purities -23. ThisAt low temperature, the main scattering mechanisms in graphene are largely due to the Coulomb impurity and the short-range defect scatterings . Based o2-graphene interface, and the short-range scattering is constant. Then the energy average scattering time is deduced as [Based on a model proposed by Hwang et al. , we can duced as ,22:Ek is the wave vector energy and τ(Ek) is the transport scattering rate. For the low temperature limit, the scattering time averaged over energy can be written as 〈τ〉 ≈ τ(EF) [D(EF) in tri- and four-layer graphene is assumed to be a constant where  ≈ τ(EF) . The denructures -23.3-4 layers ∝ kBT) [In the high temperature regime, the Coulomb scattering is a strong function of temperature while the short-range scattering is independent of temperature. This is attributed to the density of states, the matrix element of graphene and the screening function being energy independent in FLG -23. Hencs ∝ kBT) . For triRsr−3–4 layers = C, and A, B and C are the fitting parameters. In our measurements, we have observed a linear approximation for the temperature-dependent normalized resistance of tri- and four-layer graphene:where we have defined These considerations explain qualitatively why the resistance of tri- and four-layer graphene decreases with the increasing temperature. We note that due to the complexity of the FLG band structure, these anomalous electrical properties are believed to originate in the unusual band structures near the Fermi level of graphene -29. MoreI-V curve shows unique combination of the low threshold of linearity and manifestation of the second ohmic region for the strong DC electric field in the FLG interconnects. With the RCDA method, our experimental results suggest that Coulomb interaction plays an essential role. The non-metallic temperature-dependent resistance is observed in the temperature range of 5 to 340 K. In this case, even though the FLG band structure as semimetal with zero-band gap, tri- and four-layer graphene resistors behave more like semiconductors. By combining the Coulomb and short-range scattering theories, an analytical model was developed, which well explains the experimental results.In conclusion, FLG interconnects were fabricated via a combination of mechanical exfoliation and photo lithography techniques. The temperature dependence of the electrical resistance of tri- and four-layer graphene was investigated. The observed The authors declare that they have no competing interests.YPL fabricated the device and performed the experiments. WQJ and WSL coordinated the project. ZWL and WSL provided key interpretation of the data. QJW and WSL drafted the paper. All authors read and approved the final manuscript.


Spliceosomal introns are important components of eukaryotic genes as their structure, sizes and contents reflect the architecture of gene and genomes. Intron size, determined by both neutral evolution, repetitive elements activities and potential functional constraints, varies significantly in eukaryotes, suggesting unique dynamics and evolution in different lineages of eukaryotic organisms. However, the evolution of intron size, is rarely studied. To investigate intron size dynamics in flowering plants, in particular domesticated grapevines, a survey of intron size and content in wine grape (Vitis vinifera Pinot Noir) genes was conducted by assembling and mapping the transcriptome of V. vinifera genes from ESTs to characterize and analyze spliceosomal introns.V. vinifera genome, otherwise inconsistent with overall genome size dynamics when comparing Arabidopsis, Populus and Vitis. In domesticated grapevine, intron size is generally not related to gene function. The composition of enlarged introns in grapevines indicated extensive transposable element (TE) activity within intronic regions. TEs comprise about 80% of the expanded intron space and in particular, recent LTR retrotransposon insertions are enriched in these intronic regions, suggesting an intron size expansion in the lineage leading to domesticated grapevine, instead of size contractions in Arabidopsis and Populus. Comparative analysis of selected intronic regions in V. vinifera cultivars and wild grapevine species revealed that accelerated TE activity was associated with grapevine domestication, and in some cases with the development of specific cultivars.Uncommonly large size of spliceosomal intron was observed in In this study, we showed intron size expansion driven by TE activities in domesticated grapevines, likely a result of long-term vegetative propagation and intensive human care, which simultaneously promote TE proliferation and repress TE removal mechanisms such as recombination. The intron size expansion observed in domesticated grapevines provided an example of rapid plant genome evolution in response to artificial selection and propagation, and may shed light on the important genomic changes during domestication. In addition, the transcriptome approach used to gather intron size data significantly improved annotations of the V. vinifera genome. Eukaryotic genes contain spliceosomal introns that are post-transcriptionally removed by the spliceosome, an RNA-protein complex . The lenPlant genomes usually have compact genes due to small introns . ArabidoTransposable elements (TEs) are known to be major components of plant genomes . The oriTo further investigate intron size evolution in plant genomes, we present a novel approach to identify and analyze introns of large size using genomic data for a domesticated grapevine cultivar (Vitis vinifera Pinot Noir). The specific objectives are to investigate the size distribution of introns; relationship between intron size and gene function, determine the contents of introns and examine the associations between intron size evolution and grapevine domestication. Generally, we show that introns of truly extraordinary size are widespread in cultivated grapevines, a phenomenon not observed in other plant genomes.Arabidopsis proteins and 15,683 proteins show homology to Populus proteins . BLAST searches against an EST database of other Vitis species showed that 15,089 clusters (60.5%) had high similarity hits in at least one other species of grapevine.In total, 353,748 publicly available EST sequences of V. vinifera were clustered into 26,111 clusters, 8,560 of which represented by only one EST sequence. The largest cluster is composed of 1,998 EST sequences. The distribution of cluster size suggested that one-third of V. vinifera genes are not expressed at very high levels (32.8% singletons) and a few (3 clusters contain more than 1000 ESTs) genes are highly expressed across the representative EST libraries. Among the 26,111 clusters, 5,678 clusters contain no intron, while 20,433 clusters were predicted to be spliced. Among all clusters, 17,059 clusters were predicted to encode proteins, in which 15,369 predicted proteins show homology to Vitis vinifera Pinot Noir, is slightly larger than other sequenced plants [The average intron size, predicted by the annotation of the first draft sequences of d plants . After mComparisons between the 2563 clusters (predicted genes from EST clusters) and existing gene annotations of the V. vinifera genome revealed that 1,812 clusters matched to known annotations with 100% identity, with 1,137 matches that showing identical transcription start/end sites and exon/intron structure between our predictions and known annotations. However, in 217 matches, multiple EST-predicted genes (487 clusters) matched to single annotated genes, while 188 clusters matched to multiple annotated genes. The later case represented one type of annotation error in V. vinifera genome, in which a single gene supported by expression data was incorrectly split into multiple annotated genes due to a large intron between coding regions. Among the 188 clusters, 99 clusters correspond to multiple, consecutively annotated genes, suggesting that additional introns are needed between annotated genes to correct the annotations  were identified , suggesting younger LTR retrotransposon insertions and recent LTR retrotransposition activity in these introns. Based on an estimate of substitution rate in LTR regions, age estimates of LTR retrotransposons in introns identified a burst of LTR retrotransposon activities: <10,000 ya (LTR similarity 100%)  Figure . For theIn the 39 genes with identical exon/intron structure/number in Arabidopsis and Populus, repetitive elements comprised roughly 90% of the 74 intronic regions, in which 10 complete LTR retrotransposons were identified. Among the 74 introns, 1 intron contains only a LINE; 1 intron contains only LTR elements; 15 introns contain LINEs and other repetitive elements; 15 introns contain LTR elements and other repetitive elements; 5 introns contain LTR, LINEs and other repetitive elements; 34 introns contain only other repetitive elements; and 3 introns do not contain repetitive elements. According to a BLAST search against the V. vinifera genome, 'other repetitive elements' were highly repetitive within V. vinifera and likely represented un-classified or unnamed grapevine-specific TEs. The three introns without any identified repetitive elements all contain LINE-like ORFs otherwise highly repetitive in the V. vinifera genome.Characterizations of four selected introns  in Vitis species and domesticated varieties revealed intron length variation across these species and varieties Table . None ofGenome-wide intron analysis has only been done in very few plants . In thisAnnotation errors such as splitting single gene into multiple ones as a result of unsually long introns, were revealed by comparing ESTs to current annotations Figure . This waLarge size introns are expected to require more energy to be transcribed and spliced, so large introns may decrease gene expression (energy cost hypothesis) . Such hyIn contrast, gene size and/or intron size seems to be not related to gene function in this study. Among identified large genes, there is no particularly overrepresented gene family. There are still about 20% of expanded intronic regions not accounted for by similarity searches or repeat masking, suggesting unique sequences in introns are also evolving. Most of these unique sequences may be evolving neutrally since there may not be any selective constraint on them. However, a few of them may contain very important regulatory elements controlling gene expression patterns thus under selective constraints.Populus specific intron contraction can explain the intron size difference between the two plants. The young LTR retrotransposons and unique presence of these TEs in domesticated grapevine both suggested that an intron size expansion is more likely to be the case. Wendel et al. [Comparisons of genome sizes and intron sizes between Populus and Vitis revealed that intron sizes has increased much more than genome size. Either grapevine specific intron expansion or l et al. ,24 propoA major force behind the intron size expansion in grapevine is the proliferation of TEs, consistent with previous observations . Among vV. vinifera expanded introns contain significantly more identical LTR pairs, suggesting that more young LTR retrotransposons have been preserved in introns of domesticated grapevine  were obtained from NCBI GenBank using the PartiGene package . Only V.artiGene ) and TGIartiGene . Both meartiGene  searchesartiGene . The proartiGene .EST consensus sequences were mapped to genomic sequences of V. vinifera Pinot Noir  to identV. vinifera proteins were subjected to HMM search of protein functional domains against Pfam database [Genes with putative large introns were submitted to the following analysis: 1, the coding regions were predicted and translated into protein sequences by ESTScan ; 2, preddatabase .Identified large introns (3 kb~100 kb-long) were subjected to the following analysis: 1, the intronic sequences were screened by RepeatMasker  to identTo characterize homologous introns in other Vitis species and varieties, intronic regions that met the following criteria were chosen: 1, there is only one repetitive element in the intron, suggesting a relatively recent expansion; 2, the total intron size without repetitive elements does not exceed 3 kb (to facilitate PCR amplifications). Primers were designed for the flanking exon sequence as well as conserved regions within the repetitive elements to allow scoring either the presence or absence of the elements in a given intron (primer sequences available upon request). PCR amplifications of selected intronic regions were performed for one individual each of six wild grapevine species: V. rotundifolia, V. californica, V. girdiana, V. aestivalis and V. labrusca, V. jacquemontii, and seven V. vinifera cultivars: Cabernert Sauvignon, Chardonnay, Dolcetto, Pinot Noir, Riesling, Sangiovese and Zinfandel. Alleles with or without TE insertions were determined by the size of the PCR product.The authors declare that they have no competing interests.KJ and LRG conceived the study, designed and carried out the experiments, analyzed data, and wrote the manuscript. All authors read and approved the final manuscript.


However, it is currently unclear whether—and to which degree—the liberated 3′-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of reverse transcription. Here, we employed a direct assay for the initiation of L1 reverse transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3′ end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3′ overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3′ end to allow L1 reverse transcription. Based on these data we propose that the specificity of L1 reverse transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate reverse transcription within the L1 RNA poly(A) tail, a process known as target-primed reverse transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate  Jumping genes are DNA sequences present in the genome of most living organisms. They contribute to genome dynamics and occasionally result in hereditary genetic diseases or cancer. L1 elements are the only autonomously active jumping genes in the human genome. They replicate through an RNA–mediated copy-and-paste mechanism by cleaving the host genome and then using this new DNA end as a primer to reverse transcribe its own RNA, generating a new L1 DNA copy. The molecular determinants that influence L1 target site choice are not fully understood. Here we present a quantitative assay to measure the influence of DNA target site sequence and structure on the reverse transcription step. By testing more than 65 potential DNA primers, we observe that not all sites are equally extended by the L1 machinery, and we define the rules guiding this process. In particular, we highlight the importance of partial sequence complementarity between the target site and the L1 RNA extremity, but also the high level of flexibility of this process, since detrimental terminal mismatches can be compensated by an increasing number of interacting nucleotides. We propose that this mechanism contributes to the distribution of new L1 insertions within the human genome. Retrotransposons are highly repetitive and dispersed sequences, accounting for almost half of our DNA cis with their encoding mRNA L1 retrotransposition starts with the transcription of a 6 kb L1 RNA driven by an internal Pol-II promoter The current model for non-LTR retrotransposon integration, named target-primed reverse transcription (TPRT), was originally deduced from biochemical studies on the insect R2Bm element 5′-TTTT/A-3′) and are often preceded by T-tracts of variable length in vitro, without apparent sequence or structure requirement, or any need for primer-template complementarity As regards L1, conclusive evidence on whether primer-template complementarities are required for efficient reverse transcription initiation is lacking. Most L1 pre-integration sites contain an EN recognition sequence  tail of the L1 RNA for efficient priming of reverse transcription. To achieve this goal, we validated a direct L1 extension assay (DLEA) to quantitatively measure the ability of native L1 RNPs to initiate reverse transcription. Then we systematically assayed more than 65 DNA substrates varying in sequence and structure, allowing us to define the preferential rules of L1 reverse transcription priming. Our results clarify the importance of base-pairing between the L1 RNA template and the target site DNA for this process and demonstrate its exceptional flexibility.To test the DNA primer requirements for initiating L1 reverse transcription, we set up a direct L1 extension assay (DLEA), which would avoid PCR and therefore would allow us to quantitate L1 priming efficiencies. The L1 retrotransposition machinery is notoriously difficult to express and to detect in most experimental systems. To obtain sufficient amounts of L1 RNPs for direct detection, we modified the protocol developed by Kulpa and Moran 18 primer in the presence of radiolabelled 32P-dTTP. This reaction would mimic the initiation step of L1 reverse transcription, which starts at the poly(A) tail of the L1 RNA. After a 4 min incubation at 37°C, we purified the reaction products and resolved them on sequencing gels. A short end-labeled oligonucleotide was added after the reaction as a recovery control (RC). No or minimal extension was detected in vector or RT-defective controls consistent with the presence of only minimal amounts of endogenous hL1 activity in RNP preparations . We chose to use 2 µg of RNPs, at the upper end of the linear range, for all following experiments and to set to 100% the level of extension obtained with an oligo(dT)18 primer under these conditions. Based on the dynamic range of the initial RNP titration, primer extension efficiencies as low as 5% should therefore be reliably quantified. The products are heterogeneous in length, consistent with the expected products of poly(A) reverse transcription and range from 19 nucleotides (nt) to approximately 150 nt (We reasoned that if L1 RNPs were active enough we should detect the extension of an oligo(dT)arations . In contarations , showingin vivoTo further confirm that the ladder observed results directly from the reverse transcriptase activity of the transfected L1 element, we performed additional controls. RNase treatment reduced primer extension to undetectable levels , the reabona fide initiation of reverse transcription by native mammalian L1 RNPs through the direct incorporation of radiolabeled dTMP in a primer extension reaction. Importantly, DLEA is quantitative since it demonstrates a linear relationship between the signal and RNP quantities under the reaction conditions employed.Altogether these results show that DLEA detects 18 primer to a non-T nucleotide in order to prevent base-pairing of the primer 3′ end to the L1 poly(A) tail , in agreement with previous reports 3 to V6), DNA polymerization becomes undetectable under the employed reaction conditions  tail . Althougnditions  as a prinditions , lane 8.Next, we measured the influence of each individual terminal base on primer extension. Although all terminal mismatches reduced the efficiency of reverse transcription initiation to some extent, a terminal G was the most detrimental, whereas a C or an A was better tolerated . Thus th1 to T6). Initiation of reverse transcription is robustly detected only when the single-stranded primer ends with at least 4 Ts and trace activity can already be detected with 3 terminal Ts  RT to prime reverse transcription under identical experimental conditions. In these experiments, exogenous poly(rA) was added as a template together with quantities of the AMV RT that lead to similar levels of extension as the L1 RNP using the (dT)et al. observed processing in 33/81 (39%) of the analyzed clones obtained with HeLa cells, while Kopera et al. found 5/45 (11%) of processed primers in CHO-derived cell lines. In comparison, we obtained 2/70 (3%) clones showing a processed primer with RNPs prepared from HEK293T cells.It was previously reported that a nuclease activity in the RNP preparations could process primers before their extension Altogether these observations show that native L1 RNPs efficiently prime reverse transcription at DNA ending with 4–6 terminal matching nucleotides, although it can accommodate terminal mismatches with lower priming efficiencies.bona fide insertion sites. We designed 35 primers corresponding to previously published insertion sites recovered from new hL1 retrotransposition events obtained in cultured cells 20 oligonucleotide.L1 EN-mediated nicking at a consensus target site produces a 3′-OH DNA ending with four Ts in vivo without processing We observed that all sites are not equally extended see . The lev5′-TTTC-3′ and their respective levels of extension are LOU527<LOU538<LOU525, which roughly follows the number of Ts close to the 3′ end. This suggests a compensation mechanism allowing the extension of primers ending with suboptimal sequences.We categorized primers based on their potential of extension . To evaluate the number of terminal nucleotides that contribute to priming efficiency, we further correlated the priming efficiency with position-weighted T-density, taking into account a variable number of terminal nucleotides. The goodness-of-fit (R2) increases steadily up to 10 considered nucleotides and then reaches a plateau (2 = 0.8276).To address the significance of this phenomenon more quantitatively, we calculated for each oligonucleotide two parameters: (i) the density of Ts (number of Ts/length of the oligonucleotide), which simply reflects the abundance of Ts in the primer, and (ii) the position-weighted T-density, which is similar but the weight of each T is inversely proportional to the distance from the 3′ end  tail and the last 10 nucleotides of the target DNA plays a role in extension at the target site, the last 4 nucleotides being the most critical. Suboptimal primers with a mismatch in their last 4 nucleotides are extended with a lower efficiency, which can be partially compensated by increasing the number of Ts in the upstream sequence.To illustrate these findings, we propose that the four terminal bases of the primer, which overlap with the EN nuclease recognition sequence, act as a specific snap and the upstream six bases act as a weaker velcro strap . When th4. The velcro strap was considered as tightly fastened if the position-weighted T-density score of this region was at least half of its maximum value alue see . In agrein vivo, at the genomic level, L1 elements would more frequently insert at putative EN recognition sites with a closed snap and a tightly fastened velcro strap; and that a tightly fastened velcro would favor insertions as compared to similar sites with an open velcro. To test this model, we searched in the human reference genome (hg19) for the position of all potential EN targets: R/TTTT, which corresponds to a closed snap; or R/VTTT, R/TVTT, R/TTVT and R/TTTV, which correspond to open snaps . For each of them, we extracted the 10 nucleotides upstream of the nick position and categorized each on the basis of its snap/velcro status to obtain the exact frequency of each category in hg19. Then we extracted the exact insertion sites for all the L1HS polymorphic insertions present in dbRIP in vitro data, given a snap status, insertions are more frequent at sites with a tightly fastened velcro than with a loosely fastened velcro. Other studies have previously reported that T-richness extends beyond four nucleotides upstream of the cleavage site in vivo is consistent with the snap-velcro model at the genomic level, but it should also be stressed that, in vivo, other determinants are likely to influence L1 insertion profiles.A testable prediction of this model is that, An alternative pathway of L1 integration uses preformed double-stranded DNA lesions instead of EN-mediated cleavage. To determine whether the L1 RNP is able to directly initiate reverse transcription at blunt DNA ends, we designed model hairpins ending with four or six Ts at their 3′ terminus .18 primer , which depends on primer length see , is indi8 primer , lane 2,0 to H6). In contrast to reactions performed with blunt or 3′-recessed hairpin substrates, initiation of mL1 reverse transcription is easily detected as soon as the 3′ overhang reaches a length of 6 nt, as shown by the mL1-specific ladder which appears above 50 bp (Since L1 elements are believed to integrate into double-stranded genomic DNA and L1 RNPs can efficiently extend single-stranded oligonucleotides (see above), we reasoned that L1 RNPs might be able to prime DNA synthesis on double-stranded primers ending with a 3′ overhang. To test this hypothesis we designed model hairpins extended by a 3′ overhang of increasing size  partial sequence complementarity between the 10 terminal nucleotides of the target site and the L1 RNA poly(A) tail impact reverse transcription initiation  foitiation ; (iii) titiation ; (iv) thitiation . Based oitiation . This moin vitro provides detailed molecular insights into specific steps of the retrotransposition process. This is a useful complement to retrotransposition cellular assays, which offer a more global view of this mechanism. Nevertheless, a number of differences between the in vitro and in vivo situations, and between endogenously and ectopically expressed L1, should be emphasized. First, reverse transcription initiation is uncoupled from the cleavage of the target DNA, in primer extension assays such as LEAP or DLEA. Thus, we cannot completely exclude that L1 RNPs would utilize a different priming mechanism in the context of a L1 TPRT reaction. Likewise, it is possible that the detected activity results from a minor fraction of the RNPs, which can only extend exogenous primers. This situation is reminiscent of L1 reverse transcription initiation at existing DNA lesions as hypothesized for EN-independent integration events in vitro has been described In vivo, L1 elements carrying this endonuclease variant still integrate in extended T-rich sequences, which shows that additional factors other than the EN specificity contribute to L1 insertion profile in vivo. Our data suggest that primer-template complementarity might be one of these factors, by promoting the initiation of reverse transcription, but it is also very likely that additional partners or inhibitors influence L1 targeting in vivo, modulating or relaxing EN or RT specificity. Indeed, L1 insertions occasionally take place at sites that do not strictly follow the rules described here  versions of the human L1.3 element in a pCEP4 backbone (a kind gift of N. Gilbert) CNQYKNGNNALEKTRR-C) or human (N-CERNNRYQPLQNHAKM-C) ORF1p were synthesized and coupled to the KLH protein as a carrier. The first cysteine (underlined) is not present in the ORF1p sequence but was added for the coupling reaction with the carrier protein. KLH-coupled peptides were used to immunize rabbits (Eurogentec). For immunoblotting the mORF1p antiserum (SE-0560), the hORF1p antiserum (SE-6798), and the S6 protein antibody  were used at a dilution of 1∶2000.Peptides corresponding to the C-termini of mouse (N-2. Eluted oligonucleotides were precipitated with ice-cold ethanol (3v). After centrifugation for 30 min at 4°C at 16'000 g, the pellets were washed with 70% ethanol, air-dried and dissolved in 10 mM Tris-HCl pH 8.0, 1 mM EDTA.One hundred micrograms of each lyophilized oligonucleotide was dissolved in 10 µl of 98% deionized formamide, 1 mM EDTA, 0.01% (w/v) xylene cyanol and 0.01% (w/v) bromophenol blue and resolved in 10% polyacrylamide-urea denaturing gels. Full length oligonucleotides were visualized by UV shadowing, excised from the gel and eluted overnight at 37°C in 0.3 M sodium acetate, 0.1% SDS and 10 mM MgCl6 cells per 10 cm Petri dish. Twenty-four hours after plating, the cells were transfected with 24 µg of plasmid DNA (see plasmids above) per dish using the calcium phosphate method. Growth medium was changed 5 hours later. One day post-transfection, cells were split into two plates in growth medium supplemented with 1.5 µg/mL puromycin  or 100 µg/mL hygromycin . Cells were collected 4 days post-transfection by trypsinization, pooled and washed in PBS. Cell pellets were lysed in 500 µL of CHAPS lysis buffer  CHAPS, 10% (v/v) Glycerol, supplemented before use with Complete EDTA-free protease inhibitors cocktail (Roche) and 1 mM DTT). After incubation at 4°C for 15 min, cell debris was removed by spinning down extracts at 4°C for 10 min at 16'000 g. Supernatants were transferred to clean tubes and 500 µL of lysis buffer were added to each of them.L1 RNPs were produced in HEK293T cells grown in Dulbecco's Modified Eagle Medium  containing 2 mM L-Glutamine, 4500 mg/L D-Glucose, 1 mM Sodium Pyruvate, 10% (v/v) fetal bovine serum (Life Technologies) and 100 units/mL penicillin/streptomycin (Life Technologies). Cells were plated at 3×102, 1 mM DTT and Complete EDTA-free protease inhibitors cocktail (Roche). For each sucrose cushion, 1 mL of cell lysates, prepared as described above, was used. Samples were centrifuged for 2 h at 178'000 g at 4°C and the pelleted material was resuspended in 100 µL H2O. Total protein concentration was determined by Bradford assay (Biorad). The samples were diluted in 50% (v/v) glycerol, quick frozen in liquid nitrogen and stored at −80°C until use.L1 RNPs were prepared as previously described Protein A-Sepharose beads (Sigma) were blocked overnight at 4°C in PBS containing 0.5 mg/mL of bovine serum albumin (BSA) and washed twice in 1 mL of IP buffer . Eight microliters of preimmune or anti-mORF1p serum were bound to 70 µl of blocked beads for 3 h at 4°C. For each immunoprecipitation, 200 µL of L1 RNPs (2 µg/µL) were diluted 1∶1 (v/v) in IP buffer. The RNPs were precleared with blocked beads for 1 h at 4°C and incubated for 3 h at 4°C with antibody-bound beads on a rotating wheel. After 4 washes in IP buffer, the bead slurry was split equally into 7 tubes (6 for RT reactions and 1 for immunoblotting). Beads were pelleted for 5 min at 4°C at 750 g, supernatants were removed and the RT reaction mixture was directly added to the beads (see below).2, 10 mM DTT, 0.05% (v/v) Tween-20 and 10 µCi of α-32P-dTTP . In reactions using the Avian Myeloblastosis Virus RT , the RNPs were replaced by 0.04 U of AMV RT and 250 ng of poly(rA) template (Roche). Reactions were stopped by the addition of 8.3 mM EDTA and 0.83% SDS final. Trace amounts of a 32P-labelled 14- or 30-mer DNA oligonucleotide were added as recovery control (noted RC (14) or RC (30) in the figures). Products were purified by phenol-chloroform extraction and ethanol precipitation with 10 µg of glycogen as a carrier and 0.1 mM sodium acetate [pH 5.2]. DNA pellets were resuspended in 98% deionized formamide containing 10 mM EDTA, 0.02% (w/v) xylene cyanol and 0.02% (w/v) bromophenol blue, heated to 95°C for 5 min, and analyzed on 13% polyacrylamide-urea sequencing gels. After drying, gels were exposed to a PhosphorImager screen.Reverse transcriptase assays were carried out for 4 min at 37°C in 25 µL reactions containing 2 µg of RNPs, 400 nM of primer, 50 mM Tris-HCl [pH 7.5], 50 mM KCl, 5 mM MgClFor primers used in For gel or spot quantification, the reaction without primer obtained with a given RNP preparation was used as background and was subtracted from the reaction with primers. Only the signal above the primer size was quantified for the hairpin oligonucleotides.32P incorporation was RNase sensitive  as triphosphate derivatives were obtained from Biocentric. They were added to reactions at a final concentration of 10 µM  Tween-20, 20 U RNasin (Promega), 200 µM dNTP, and 0.4 µM LEAP primer. Eventually, unextended primers were eliminated through an S-400HR size-exclusion spin column . Reverse transcription products (1 µL of the LEAP reaction) were PCR-amplified in 50 µL reactions containing 1 U of Platinum Taq DNA Polymerase (Life technologies), 0.2 µM of primers LOU851 and LOU312, 200 µM dNTP, 3 mM MgCl2 in the Platinum Taq buffer. A first step at 94°C for 2 min was followed by 35 cycles of . The final extension was at 72°C for 5 min. PCR products were analyzed by 2% agarose gel electrophoresis in 1x TBE. Gels were stained by SYBR Safe (Life technologies) or ethidium bromide. LEAP products were gel-purified with a gel extraction kit (Macherey Nagel) and cloned into the pGEM-T-easy vector (Promega), according to manufacturer's protocol. Clones from isolated colonies were sequenced by GATC. Regions with low quality (Phred<Q20) were trimmed or filtered out using Geneious 5.LEAP was performed as previously described 2 and 5 mM DTT. Primer pairs used for PCR were LOU851/LOU312 (mOrfeus or L1.3) or LOU852/LOU312 (GAPDH). PCR products were resolved by 2% agarose gel electrophoresis in 1x TBE.Total RNA was extracted from 30 µg of L1 RNP using TRIzol extraction (Molecular Research Center Inc) following the manufacturer's instruction. RNA was resuspended in 20 µL of milliQ water and quantified by Nanodrop. One microgram of RNA was digested by 1 U of RNase-free RQ1 DNase (Promega) in 10 µL reaction in the manufacturer's buffer at 37°C for 30 min. DNase was heat-inactivated for 10 min at 65°C. Then, cDNA synthesis was performed at 50°C for 1 h in 20 µL reactions containing 6 µL of the DNase reaction, 200 U of SuperScript III Reverse Transcriptase (Life technologies), 500 µM dNTP, 50 pmol of RACE primer, 40 U RNAseOUT (Life technologies), 50 mM Tris-HCl [pH 8.0], 75 mM KCl, 3 mM MgClT-density is calculated by dividing the number of Ts in the oligonucleotide by the length of the oligonucleotide. The position-weighted T-density gives more weight to Ts which are close the 3′ extremity of the primer. The weight is inversely proportional to the distance from the 3′ end.The For example:position-weighted T-count equal to:Primer LOU519 has a position-weighted T-count equal to:Primer LOU541 has a position-weighted T-density of a given primer is calculated by dividing the position-weighted T-count of this primer to the maximum position-weighted T-count. Thus the position-weighted T-density of LOU519 is equal to 2.23/3.60 = 0.62 and the position-weighted T-density of LOU541 is equal to 3.60/3.60 = 1The position-weighted T-count for the upstream 6 nucleotides (velcro region) and we divided it by the maximum value (1/5)+(1/6)+…+(1/10) = 0.84563492 to obtain the velcro position-weighted T-density. We consider a velcro as fastened if its position-weighted T-density is ≥0.5  and opened otherwise.The snap is considered open if the 4 terminal nucleotides contain a non-T nucleotides and closed if the last four nucleotides are 4 Ts. We calculated a All putative integration sites with a perfect or degenerate EN recognition sequence  were recovered from both strands of the reference human genome (hg19) or from its repeatmasked version (hg19 RM). For each putative EN site, snap and velcro status were defined as described above. The C++ program used to achieve this task is available in Protocol S1. Polymorphic L1 insertions were extracted from dbRIP Figure S118 primer extension by hL1 RNP. (D) Formation of long cDNA species upon addition of all four dNTPs. Reactions were performed with hL1 RNPs in presence of α-32P-dTTP and a (dT)18 primer, with (lanes 3 & 6) or without (lanes 1–2 & 4–5) cold dATP, dCTP and dGTP .Additional characterization of the L1 RNP RT activity by DLEA. (A) RNA-dependent DNA polymerase activity of L1 RNPs. Murine L1 RNPs were incubated for 1 h at 37°C in the presence (lane 3) or in the absence (lane 4) of RNases before the start of the reaction. (B) RT inhibitors prevent primer extension by L1 RNPs. Reactions were performed with mL1 RNPs in the presence of thymidine analogs , or in the presence of water as a negative control (lane 2). (C) Time-course of (dT)(TIF)Click here for additional data file.Figure S232P-dTTP. RC denotes a 14 nt recovery control added after the reaction but before DNA purification. The black dots on the left side of each lane indicate the expected start of reverse transcription. Their position varies since primer length varies. Quantification of primer extension (% Activity) was relative to levels of extension obtained with oligo(dT)18. Primers are identical to The murine L1 RNP preferentially extends primers ending with at least 4 Ts. DLEA showing the extension of single-stranded primers by mL1 RNPs in the presence of α-(TIF)Click here for additional data file.Figure S3LEAP with hL1 RNPs and mismatched primers. (A) Primers with terminal mismatches. LEAP was performed with RNPs prepared from hL1-transfected cells (top panel), from vector-transfected cells (middle panel), or without RNPs (bottom panel). Primers are identical to those used in (TIF)Click here for additional data file.Figure S432P-dTTP. Note that the products observed with hairpin primers (lanes 3–7) result from contaminating cellular activities  Absence of extension by hL1 RNPs of double-stranded primers with blunt or 3′-recessed end in the presence of α-(TIF)Click here for additional data file.Protocol S1Source code of the software used to find putative endonuclease sites in the human genome and to calculate their associated snap/velcro scores.(GZ)Click here for additional data file.Table S1List of oligonucleotides used in this study.(XLSX)Click here for additional data file.Table S2Data used to calculate genomic enrichment of L1 insertions depending on the snap-velcro status of the target. The table sheets are the following: (hg19) For each potential L1 EN target site present in hg19, the snap status was defined and the position-weighted A density was calculated. Sites with position-weighted A density equal to or above 0.5 were considered as having a closed velcro strap. (hg19 RM) Same as above but with a repeatmasked hg19 reference genome. (dbRIP sequences) L1HS dbRIP entries used in (XLSX)Click here for additional data file.


Amphimedon queenslandica, Suberites domuncula, Suberites ficus and Suberites pagurorum and showed that RPG introns from A. queenslandica share position conservancy and some structural similarity with “higher” metazoans. Moreover, our study indicates that mobile element insertions play an important role in the evolution of their size. In four sponges 51 snoRNAs were identified. The analysis showed discrepancies between the snoRNA pools of orthologous RPG introns between S. domuncula and A. queenslandica. Furthermore, these two sponges show as much conservancy of RPG intron positions between each other as between themselves and human. Sponges from the Suberites genus show consistency in RPG intron position conservation. However, significant differences in some of the orthologous RPG introns of closely related sponges were observed. This indicates that RPG introns are dynamic even on these shorter evolutionary time scales.Ribosomal protein genes (RPGs) are a powerful tool for studying intron evolution. They exist in all three domains of life and are much conserved. Accumulating genomic data suggest that RPG introns in many organisms abound with non-protein-coding-RNAs (ncRNAs). These ancient ncRNAs are small nucleolar RNAs (snoRNAs) essential for ribosome assembly. They are also mobile genetic elements and therefore probably important in diversification and enrichment of transcriptomes through various mechanisms such as intron/exon gain/loss. snoRNAs in basal metazoans are poorly characterized. We examined 449 RPG introns, in total, from four demosponges:  Amphimedon queenslandica revealed that intron position and phase are conserved relative to other metazoans Trichoplax adhaerens are not conserved in human A. queenslandica, which may indicate that metazoan complexity correlates with an increasing number of miRNAs Nme6 gene, but not in the sponge ortholog, contain miRNAs also supports that thesis cis-regulatory architecture in promoters of RPGs appears not to be drastically changed from sponge to human A. queenslandica RPG introns are similar in many structural characteristic to “higher” metazoans. Furthermore, the sponges A. queenslandica and S. domunucula show as much conservancy of RPG intron positions between each other as between themselves and human. However, when we compared RPG introns with three species from the genus Suberites of sponges, there was a discrepancy in snoRNA pools between the genus Suberites and A. queenslandica. Even though the intron positions in the Suberites genus are conserved, there are significant differences in certain RPG introns which indicate that they are dynamic even on these shorter evolutionary time scales.The ribosome is a protein-RNA complex, fundamentally the same in all three domains of life, and is the crucial cell component for protein synthesis. X-ray crystal structures of ribosomal subunits were used to determine ribosome structure and function in detail A. queenslandica expressed sequence tags (ESTs) encoding homologs of human RPs TBLASTN  was used. The set of sponge RPGs was obtained using the NCBI WGS (whole genome shotgun) Trace Archive as well as the assembled draft genome available at http://spongezome.metazome.net/cgi-bin/gbrowse/amphimedon/http://codonw.sourceforge.net/), while the GC content of the whole genome was calculated using geecee from the EMBOSS suite Homo sapiens, Strongylocentrotus purpuratus, Drosophila melanogaster, Caenorhabditis elegans, Nematostella vectensis, Trichoplax adhaerens, Monosiga brevicollis, Saccharomyces cerevisiae and Arabidopsis thaliana). One RPG from T. adhaerens, four from N. vectensis and ten from M. brevicolis were assembled manually from data available on the NCBI Trace Archive to give a total set of 55 orthologs from nine organisms. Due to the lack of a fully annotated set of RPGs from the nine organisms mentioned, it was not possible to analyze the entire set. RPGs analyzed are listed in http://www.psc.edu/biomed/genedoc). MEME was used for searching for over-represented motifs in RPG introns http://snoopy.med.miyazaki-u.ac.jp/) and the snoRNA-LBME database Suberites domuncula RPGs, the corresponding and neighboring introns were sequenced. All primer sequences used in this study are given in Suberites ficus and Suberites pagurorum from genomic DNA isolated as previously described S. domuncula were cut into pieces, frozen in liquid nitrogen and ground to a fine powder. Approximately 3 µg of small RNAs were obtained from 260 mg of tissue powder by the mirPremier microRNA Isolation Kit (Sigma), according to the manufacturer's protocol for plant tissue. Polyadenilation of 1 µg of small RNAs was achieved by incubation with 5 U of the E. coli Poly(A) Polymerase (BioLabs) for 15 minutes at 37°C. All 12 µl of the poly(A) tailing reaction mixture was then reverse transcribed using the SuperScript II Reverse Transcriptase (Invitrogen) and a modified poly-d(T) primer (modpolydT). The resulting cDNA was diluted to 100 µl and each PCR was performed on 3 µl of cDNA using the HotStarTaq DNA Polymerase (Qiagen), a universal reverse primer (uni) and forward primers specific for predicted snoRNAs. The products were cloned into the pGEM-T vector (Promega). Positive clones were sequenced using the ABI PRISM BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit and T7/pUC primers.To identify A. queenslandica (AQ) genome, and the gene structure for 78 of them was completely ascertained. The RPS14 gene was not completely determined due to the absence of WGS sequences indispensable for assembling one long intron. This gene was not considered in calculating the average values shown in N. vectensis and 73 from placozoan T. adhaerens were used as control. Sponge AQ RPGs contained an average of 4.01 introns. The RPP0 gene had the largest number of introns - 10, while the only gene without introns was RPL35 (RPS27 gene (2263 bp), and the shortest one was the first intron of the RPS21 gene (37 bp). Human RPGs have significantly longer introns of 760 bp on average RPS9 gene. Most of the introns found between translational start and stop codons, were phase 0 (52%), 27% were phase 1 and 21% were phase 2. These results support the so-called “50/30/20 rule” of intron phase distributions. It has been found that across many studied organisms, approximately 50% of introns are phase 0, 30% are phase 1 and 20% are phase 2 S. domunucula (SD) RPGs have a preference for C- and G- ending codons Full-length cDNA sequences coding for 79 RPs were identified in the marine sponge as RPL35 . In 78 cas RPL35 , which iH. sapiens (HS), S. purpuratus (SP), D. melanogaster (DM), C. elegans (CE), N. vectensis (NV), T. adhaerens (TA), M. brevicollis (MB), S. cerevisiae (SC) and A. thaliana (AT). The total number of analyzed introns within the coding regions of the RPGs in ten organisms was 1491  and S. pagurorum (SP) which produced a candidate set of 9 C/D box snoRNAs and 2 H/ACA snoRNAs in each of the sponge  and invertebrates  and checked for the presence and the position of the SNORD100 ortholog. There is an obvious tendency of this snoRNA to colonize the RPS12 gene from basal metazoans to “higher” vertebrates , found in the second intron of the RPL7A gene. It is predicted to guide the 2′-O-ribose methylation of 28S rRNA cytosines at position 2338 and 2352 in human RPP0 gene and is most similar to human SNORD83A/SNORD83B (U83A/U83B) found in the fifth and seventh intron of the human RPL3 gene, respectively  of this snoRNA are still unknown RPL13A gene. While AQ possesses only one copy, in SD, SF and SP this snoRNA was found duplicated in the neighboring intron. The SD copies are 94% identical to each other, while one shares only 48% and the other 49% identical nucleotides with those from AQ. Although overall not well conserved, all essential snoRNA elements and target sites are maintained  and S. pagurorum (SP). The total of 126 RPG introns examined  of these rRNA's target sequences are well conserved in sponge, three targets of three snoRNAs are unknown, two targets are not determined due to incomplete sponge 28S rRNA sequences, and seven are poorly conserved. Presence of the majority of target rRNA sequences lead us to seek out whether these snoRNA orthologs are nested somewhere else in the genome of AQ. We checked for orthologs of 19 C/D box snoRNAs with conserved target rRNA. Six conserved orthologs were found see . This inFrom all these results we can conclude that sponge RPG introns share many structural characteristics with “higher” metazoans. These similarities are probably important because RPG introns function as carriers of snoRNAs. Intensive mobility of snoRNAs is probably the reason why sponges from different genera show discrepancy in snoRNA pools in orthologous RPG introns. Their mobile nature is also a plausible reason for almost equal RPG intron positions' conservation between sponges from different genus as between sponges and human, which indicates their more important role in diversification and enrichment of transcriptomes through mechanisms such as intron/exon gain/loss. Within the same genus large insertions in orthologous RPG introns were detected. Mechanisms of intron dynamics on this shorter evolutionary time scale is influenced by factors not correlated with mobility of snoRNAs.Figure S1Percentage change from the consensus sequence of RPG introns from three species of the Suberites genus. Introns (4L28 through 2L14 on the y axis) from S. domuncula (SD), S. ficus (SF) and S. pagurorum (SP), were aligned and the percent change from the resulting consensus sequence is shown per species. The average column shows the average percent change per intron for all three species. The last set of bars is the percent change from the consensus for the concatenated sequences of all introns from a species (CONCAT on the y axis).(TIF)Click here for additional data file.Figure S2Human (HS) RPG-sited snoRNAs identified in sponge (AQ) introns of non-RP genes. All essential snoRNA elements and methylation (Me) guide sites are designated.(TIF)Click here for additional data file.Table S1Characteristics of 55 RPGs from seven model organisms.(DOC)Click here for additional data file.Table S2Sequences of primers used in this study.(DOC)Click here for additional data file.Table S3Characteristics of RPG introns in three basal metazoans.(DOC)Click here for additional data file.Table S4Comparison of 55 RPGs in ten organisms.(DOC)Click here for additional data file.Table S5Percentages of amino acid identity  and overall similarity  extracted from GeneDoc.(DOC)Click here for additional data file.Table S6Percentages of intron positions shared among organisms.(DOC)Click here for additional data file.Table S7The sequences of the C/D and H/ACA box snoRNAs identified in RPG introns of sponges A. queenslandica (Aq), S. domuncula (Sd), S. ficus (Sf) and S. pagurorum (Sp). Conserved snoRNA elements are boxed and predicted methylation guide sites are shaded. Experimentally verified snoRNAs are marked with asterisk (*). Lowercase letters indicate nucleotides that were not sequenced due to the snoRNA cloning strategy. Letters in brackets indicate nucleotides which do not belong to snoRNA, although they were predicted by snoSeeker.(DOC)Click here for additional data file.Text S1126 introns sequences from three Suberites species: S. domuncula (SD), S. ficus (SF) and S. pagurorum (SP).(TXT)Click here for additional data file.