Wild mammalian species, including bats, constitute the natural reservoir of betacoronavirus . Different hosts or host tissues provide different cellular environments, especially different antiviral and RNA modification activities that can alter RNA modification signatures observed in the viral RNA genome. The zinc finger antiviral protein (ZAP) binds specifically to CpG dinucleotides and recruits other proteins to degrade a variety of viral RNA genomes. Many mammalian RNA viruses have evolved CpG deficiency. Increasing CpG dinucleotides in these low-CpG viral genomes in the presence of ZAP consistently leads to decreased viral replication and virulence. Because ZAP exhibits tissue-specific expression, viruses infecting different tissues are expected to have different CpG signatures, suggesting a means to identify viral tissue-switching events. The author shows that SARS-CoV-2 has the most extreme CpG deficiency in all known betacoronavirus genomes. This suggests that SARS-CoV-2 may have evolved in a new host (or new host tissue) with high ZAP expression. A survey of CpG deficiency in viral genomes identified a virulent canine coronavirus  as possessing the most extreme CpG deficiency, comparable with that observed in SARS-CoV-2. This suggests that the canine tissue infected by the canine coronavirus may provide a cellular environment strongly selecting against CpG. Thus, viral surveys focused on decreasing CpG in viral RNA genomes may provide important clues about the selective environments and viral defenses in the original hosts. Coronaviruses (CoVs) evolve in mammalian hosts and carry genomic signatures of their host-specific environment, especially the host-specific antiviral and RNA modification activities. Many pathogenic single-stranded RNA viruses, including CoVs, exhibit strong CpG deficiency . Two mamExperimental evidence is consistent with the interpretation that CpG deficiency in RNA viruses has evolved in response to these cytoplasmic CpG-specific antiviral activities. During natural evolution of HIV-1 within individual patients, viral fitness decreased with increasing CpG dinucleotides . ExperimAnother antiviral enzyme is APOBEC3G, found in innate immune cells. APOBEC3G was originally thought specific to single-stranded DNA, such as reverse-transcribed HIV-1, but is now known to modify a variety of RNA viruses, deaminating C to U , 2019. TBoth ZAP and APOBEC3G exhibit tissue-specific expression patterns in human . Both arThe differential expression of ZAP and APOBEC3G in different host or host tissues is expected to leave different genomic signatures on viral RNA genomes. We may use the conventional index of CpG deficiency  implemenICpG, which is significantly (P < 0.0001) smaller than their null expectation of 1.The index is expected to be 1, with no deficiency or excess; <1, if deficient; and >1, if excess. The 1252 betacoronavirus (BetaCoV) full-length genomes deposited in GenBank  have mean±SE value of 0.516 ± 0.0017 for ICpG during viral genomic evolution. In contrast, a CoV infecting a specific host tissue for a long time would experience the same cellular antiviral and RNA modification environment and is consequently expected to have similar and stable ICpG.If a CoV infects a different host tissue with different ZAP abundance, then its RNA genome will experience different selection pressure against its CpG. This difference in cellular antiviral activity would result in differences in ICpG among its close relatives, both being outliers in a plot of viral genomic ICpG vs. GC%  have the lowest  vs. GC% . Three g vs. GC%  and circ vs. GC% . Group 3ted CoVs  and the ted CoVs , 2) civeted CoVs , and 3) Rhinolophus affinis) in Yunnan Province in 2013 but only sequenced by Wuhan Institute of Virology after the outbreak of SARS-CoV-2 infection in late 2019. This bat CoV genome is the closest phylogenetic relative of SARS-CoV-2 (ICpG) in viral RNA genomes and increased virulence, not only in HIV evolving within individual patients . Nature had essentially inoculated BetaCoVs into various Rhinolophus lineages and allowed genomic evolution to happen  genomes . There are two genomes sequenced for CRCoV (accession nos. JX860640 and KX432213). Their genomic e system A. Thus, ICpG and GC% values similar to those observed in SARS-CoV-2 and BatCoV RaTG13 . Viruses are propagated in cells that expresses the right cellular receptor for viral entry, but do not mount an immune response to kill the virus or get killed by the virus  regaining CpG faster than a slow-evolving lineage . Second, many experimental studies , was isolated from a fecal swab. These observations are consistent with the hypothesis that SARS-CoV-2 has evolved in mammalian intestine or tissues associated with intestine.Humans are the only other host species observed to produce CoV genomes with low-genomic  genomes . This vi genomes . In a co genomes , one pat genomes . Stool s genomes , corrobo genomes . In part genomes . In thisICpG values. One example is viruses isolated from pangolins. Nine SARS-CoV-2-like genomes have recently been isolated and sequenced from pangolin and deposited in GISAID database (gisaid.org). The one with the highest sequence coverage (GISAID ID: EPI_ISL_410721) has an ICpG value of 0.3929, close to the extreme low end of ICpG values observed among available SARS-CoV-2 genomes. Thus, SARS-CoV-2, BatCoV RaTG13, and those from pangolin may either have a common ancestor with a low ICpG or have convergently evolved low ICpG values.ICpG in large CoV RNA genomes, although it has been observed to relocate from the nucleus to cytoplasmic stress granules . Second, the presumably strong selection against CpG in the viral RNA genome in canid intestine resulted in rapid evolution of the virus, with many CpG → UpG mutations leading to reduced genomic Although the specific origins of SARS-CoV-2 are of vital interest in the current world health environment, this study more broadly suggests that important evidence of viral evolution can be revealed by consideration of the interaction of host defense with viral genomes, including selective pressure exerted by host tissues on viral genome composition.Molecular Biology and Evolution online.msaa094_Supplementary_DataClick here for additional data file.


Tombusviridae and Amalgaviridae viral families commonly associated with land plants. This suggests that the evolutionary history of these viruses extends to divergence events between algae and land plants. Seven Ostreobium sp-associated viruses exhibited sequence similarity to the mitoviruses most commonly found in fungi, compatible with horizontal virus transfer between algae and fungi. We also document, for the first time, RNA viruses associated with chlorarachniophytes, including the first negative-sense (bunya-like) RNA virus in microalgae, as well as a distant homolog of the plant virus Virgaviridae, potentially signifying viral inheritance from the secondary chloroplast endosymbiosis that marked the origin of the chlorarachniophytes. More broadly, these data suggest that the scarcity of RNA viruses in algae results from limited investigation rather than their absence.Our knowledge of the diversity and evolution of the virosphere will likely increase dramatically with the study of microbial eukaryotes, including the microalgae within which few RNA viruses have been documented. By combining total RNA sequencing with sequence and structural-based homology detection, we identified 18 novel RNA viruses in cultured samples from two major groups of microbial algae: the chlorophytes and the chlorarachniophytes. Most of the RNA viruses identified in the green algae class Ulvophyceae were related to the  Viruses are likely to infect every cellular organism and play fundamental roles in biosphere diversity, evolution, and ecology. Those studies of the global virosphere performed to date have revealed marked heterogeneities in virus composition. For example, while RNA viruses are commonplace in eukaryotes, they are less often found in bacteria and are yet to be conclusively identified in archaea. Rather, both the bacteria and archaea are dominated by DNA viruses ,2. It isThe advent of “omics” technologies has fueled more intensive efforts to assess global viral diversity, especially in marine environments ,4,5,6,7.Riboviria”; VirusHostdb, April 2020 release). Most (85%) of the 27 RNA viruses described from algae to date have been identified in diatoms [Totiviridae, Reoviridae, Marnaviridae, Endornaviridae, Flaviviridae, Narnaviridae and Alvernaviridae. It is currently unclear, however, whether the seemingly differing distributions of DNA and RNA viruses reflect a major switch in virus composition that occurred during the expansion of land plants or is indicative of the inherent limitations in sampling and cultivation of both algae and their viruses, or the difficulties in identifying highly-divergent RNA viruses [Since the first cultivation in 1979 ,18, the  diatoms . Althoug viruses . Indeed, viruses , phyloge viruses . As a co viruses ,26, inclGiven the phylogenetic diversity and array of genomic features characterized to date in algal RNA viruses , as well as their wide range of habitats and ubiquity, it is reasonable to expect that microalgae will harbour an abundance of RNA viruses. In addition, 72,500 species of microalgae have been identified to date distributed across diverse branches of the eukaryotic phylogeny , Archaeplastida, Haptista, Cryptista and “excavates” supergroups ,28,29), We aimed to reveal more of the RNA virosphere in cultured samples of two clades of microalgae: (i) the green algae (Chlorophyta), that are part of the Archaeplastida eukaryotic supergroup, and (ii) the chlorarachniophytes, a lineage of Rhizaria that obtained a chloroplast through secondary endosymbiosis of a green alga . To thisMicrorhizoidea pickettheapsiorum [Kraftionema allantoideum [Chlorarachnion reptans and Lotharella sp. from the Wye River, Victoria, Australia) or coral skeletons , or obtained from the NIVA Culture Collection of Algae . Cultures were maintained in K-enriched seawater medium (transferring every other week) at either 26 °C (Ostreobium) or 16 °C  under cool white LED lights at 1 Photosynthetic active radiation (PAR) (Ostreobium) or 15 PAR . Cultures were pelleted by centrifugation in falcon tubes and stored in RNAlater at −80 °C until RNA extraction.Algal strains were isolated from marine sand , and total RNA was extracted using the Qiagen Dolichomastix tenuilepis and Microrhizoidea pickettheapsiorum (Mamiellophyceae) and Kraftionema allantoideum (Ulvophyceae) were pooled into meta-transcriptome ‘ALG_1′; (ii) the ulvophyte Ostreobium sp. comprised ‘ALG_2′; and (iii) the two chlorarachniophytes Chlorarachnion reptans and Lotharella sp. were pooled into ‘ALG_3′  the chlorophyta  ‘ALG_3′ .RNA quality was assessed and TruSeq stranded libraries were synthetized by the Australian Genome Research Facility (AGRF), using either (i) TruSeq stranded with a eukaryotic rRNA depletion step  for ALG_2, or (ii) the SMARTer Stranded Total RNA-Seq Kit v2—Pico Input Mammalian libraries  for ALG_1 and ALG_3 , due to The RNA-seq data were first subjected to low-quality read and Illumina adapter filtering using the Trimmomatic v0.36 program . Ribosom−10 and 10−05 as e-value cut-offs and the more sensitive option in BLASTx. RNA virus-like sequences were also identified using BLASTx against all RdRp protein sequences available on NCBI/GenBank. False-positive signals for RNA viruses were removed by BLASTing RdRp-like sequences against the nr database and discarding sequences displaying a nonviral sequence as the best hit, based on BLASTx scores.The similarity of contigs to the current NCBI nucleotide (nt) and protein (nr) databases was determined using the BLASTn v2.2.30 and Diamond BLASTx v0.9.32 programs , respecthttp://vogdb.org) [To detect especially diverse RdRp-based sequences, orphan contigs  were compared to the Pfam RdRp-protein profiles ‘MitoVir_RdRp’ (PF05919), ‘Birna_RdRp’ (PF04197), ‘Viral_RdRp_C’ (PF17501), ‘RdRP_1′ (PF00680), ‘RdRP_2′ (PF00978), ‘RdRP_3′ (PF00998) and ‘RdRP_4′ (PF02123), as well as to the entire VOG profile database (gdb.org)  using thgdb.org) . To checTo infer a structural model for the distant RdRp signals detected using profiles, sequences displaying a RdRp-like signal were subjected to the normal mode search of the Protein Homology/analogY Recognition Engine v 2.0 (Phyre2) web portal . Brieflyhttps://github.com/ebi-pf-team/interproscan).Total non-rRNA reads were mapped onto RNA virus-like contigs using Bowtie2 v2.3.3.1 and heterogeneous coverage and potential mis-assemblies were manually resolved using Geneious v11.1.4 . Open reA challenge faced by all metagenomic studies is confidently assigning each viral sequence to a particular host in a given sample. We used algal cultures to minimize the number of potential additional cellular hosts. These cultures were, however, nonaxenic , with mainly bacteria present. To evaluate the possibility of additional microeukaryotic cells in the sample, we obtained taxonomic identification for contigs in the meta-transcriptome by aligning them to the NCBI nt database using the KMA aligner v1.2.11a and the CCMetagen program ,51 v1.1.https://www.ncbi.nlm.nih.gov/genome/viruses/). To depict the evolutionary relationships of the newly discovered viruses as meaningfully as possible, the closest unclassified BLASTx homologs were used in the phylogenetic analysis. This resulted in alignments of 237, 76, 198, 558 and 325 RdRp protein sequences for Amalga-like, Mito-like, Tombus-like, Virga-like and Bunya-like virus groups, respectively.RdRp amino acid sequences were aligned using the L-INS-I algorithm and default parameters in the MAFFT program v7.402  and trimrbcL and tufA marker genes were used as PCR positive controls using sets of primers designed in [Viral contigs were validated experimentally and associated to individual algal sample using Reverse Transcription  followed by PCR (Platinum ™ SuperFi ™ DNA polymerase – Invitrogen ™), with specific primer sets for each contig. The igned in . All priThe libraries sequenced here are available at the Sequence Read Archive (SRA) under BioProject PRJNA668187. The consensus sequences of the all novel viruses identified here have been submitted to GenBank and assigned accession numbers MW086576-MW086593.Our aim was to determine the RNA viromes of six microalgae cultures from six different algal species classified into two highly phylogenetically distinct algal clades: the chlorarachniophytes (Rhizaria) and the green algae  A,B. To tWhile our limited understanding of chlorarachniophyte viruses can be explained by the small number of species characterized to date (only 15 in Algaebase database), the Ulvophyceae is an abundant and diverse algal lineage in existence since the late Proterozoic and comprises at least 1933 species . It contBecause of variable RNA quality and quantity, fewer nonrRNA reads were obtained for the ALG_1 and ALG_3 libraries. This may in large part explain the limited length of contigs, the reduced number of estimated full-length transcripts, and ultimately the lower number of viral sequences, compared to ALG_2 .Chlorarachnion reptans and may constitute the first negative-sense (ssRNA(−−−)) virus identified in microalgae. Importantly, the presence of these viruses was validated by RT-PCR on each total extracted RNA  RNA or single-stranded positive-sense (ssRNA(+)) genomes . Howevercted RNA . In eachOstreobium sp. fell within the Narnaviridae or Tombusviridae. In contrast, five viral sequences from Ostreobium sp. and K. allantoideum do not fit into defined taxonomic groups and were instead related to the broad set of ‘partiti-like’ viruses that comprise the Partitiviridae, Totiviridae and Amalgaviridae. Finally, more divergent but detectable sequence similarities to the Virgaviridae (+ssRNA) were obtained for samples from the chlorarachniophyte library.Eight of the viruses identified in An additional attempt to detect even more divergent RNA viruses was conducted was using protein structure. In particular, it is possible that highly divergent viruses are part of the unknown orphan sequences  that comprise between 50–60% of total contigs obtained in this study A.Accordingly, we attempted to detect evolutionary-conserved features of protein structural and functional motifs in orphan sequences that encode unknown ORFs, using a cut-off of 200 amino acids (600 nucleotides): we chose this size because it is shorter than most RdRps  yet shouBryopsis cinicola [Bryopsis-replicons in fact represent viral RdRp sequences [To manually assess the level of confidence of the RdRp signal detected in the HMM comparisons, the protein sequences of the three RdRp candidates were aligned to amino acid sequences retrieved from the RdRp_C, RdRp_4 and RdRp_1 PFAM profiles. The ALG_2_DN594 contig displayed similarities with the RdRp_C profile that represents the C-terminal of the RdRp (Protein A) found in alphanodaviruses. Unfortunately, this C-terminal region lacks the key functional motifs usually associated with the RdRp, preventing us from definitively establishing the ALG_2_DN594 contig as a true RNA virus. Similarly, the ALG_3_DN34624 alignment with the viral RdRp sequences that comprise the PFAM RdRp_4 profile (PF02123) does not show conservation of the crucial functional residues at the A, B and C-motifs within the RdRp, particularly the canonical motif C that is normally GDD, yet GFD in ALG_3 contig : strikincinicola  .Ostreobium sp. culture, with viral-like sequences constituting between 0.01 (considered as average abundance) and 1.2% (considered as very high abundance) of the total non-rRNA reads . A nonannotated ORF from a K. allantoideum contig, ALG_1_DN2506, aligned with the N-terminal ORF of Ostreobium sp. amalga-like virus contigs that potentially encode the virus coat protein, but the high level of divergence prevented us from performing any phylogenetic analysis of proteins other than the RdRp. Because of their co-occurrence in K. allantoideum . Although sequences assigned to Lingulodinium polyedrum potentially result from GenBank mis-annotation and were likely of bacterial origin, the Coolia malayensis (Dinophyceae), Amphidinium sp. (Dinophyceae) and Cyanophora paradoxa (Glaucophyta) associated sequences likely constitute true assignments. We therefore suspect that these additional micro-eukaryotic transcripts may have arisen from cross-contamination with additional algae samples that were extracted and prepared at the same time and sequenced in the same run. Importantly, however, none of the viruses identified in the three libraries studied here could be detected in the transcriptomes of co-processed Dinophyceae and Glaucophyta cultures, suggesting that these low-abundance contaminants are not the hosts of the viruses reported here. In addition, a minor portion of the contigs in ALG_2 and ALG_9 was assigned to bacterial species  and found at various levels of abundance , although they share only 28–32% sequence identity  and foridentity . BDRM wacroalgae  and lateequences  form an Amalgaviridae full-length genomes available. The closest matches to existing Amalgaviridae sequences were retrieved for each newly discovered virus, and the resulting pairwise identity distributions compared with those observed within and between Amalgaviridae genera  belonging to the order Cladocera [Narnaviridae.Seven viral sequences from itovirus . With thaviridae , right, aviridae , these vladocera . These “Narnaviridae (ICTV report 2009), we identified seven new mito-like viral species  viruses (Nodularia douglasiae) [Ostreobium sp. sample (File S2), and the average abundance level associated to this virus  . AccordiTombusviridae and their relatives  viruses traditionally associated with plants and display diverse genomic organizations. The short length of the Virga-like bellevillovirus associated with chlorarachniophytes indicates that this sequence likely comprises a partial genome sequence only. Moreover, the multi-segment structure of the closest relatives suggests that the partial genome recovered in ALG_3 could also contain additional segments not yet identified. Although further host confirmation is required, this newly described RNA virus-like sequence would constitute the first algae virus from the Hepe-Virga group.One sequence identified in the pergroup  and is pChlorarachnion reptans sample at an abundance of 0.01% of total non rRNA reads. However, this sequence is highly divergent and cannot be formally assigned to any previously described viral family. Despite this, it is striking that the sequence clusters with a Bunya-Arena-like virus, Shahe bunya-like virus 1  [Bunyavirales   that typavirales . In addiviticola  that areOstreobium sp.) representing 99% of all assigned contigs, makes it likely that most if not all of these 18 viruses infect algae hosts. In addition, we identified a number of narnaviruses, a group previously observed in algae [Bunyavirales-like sequence is similarly in accord with a study that presented evidence for the presence of bunya/phlebo-like viruses in brown algae [We aimed to better characterize the RNA virus diversity in two major algal lineages, the chlorarachniophytes and the ulvophytes, for which no RNA viruses had previously been reported. Our investigation of the RNA virus diversity in samples from six microalgae species led to the identification of 18 new and divergent RNA viruses, although with clear homology to five established viral families. While an unequivocal host assignment cannot be formally established on these metagenomic data alone, that the algae studied were the dominant host species in the metagenomic sequencing data, in one case  or if tiviridae . More bres hosts ,87,88,89C. reptans) associated viral sequence and land-plant infecting viruses was striking. The Rhizaria and Archaeplastida are assumed to have diverged before the cyanobacteria primary endosymbiosis event, ca. 1.5 billion years ago [C. reptans, the presence of such land plant-like viruses in Chlorarachniophyte would reflect their more recent acquisition in chlorarachniophytes through either horizontal transfer by common vector/symbiont/parasite ancestors or secondary endosymbiosis (eukaryote-to-eukaryote) events. Indeed, a secondary endosymbiosis event of a green alga in the core Chlorophyta, possibly related to Bryopsidales, led to the origin of the plastid of chlorarachniophytes between 578–318  Ma [Lepidodinium where distinct secondary endosymbiosis with green algae have also occurred, as well as to cryptophytes that also contain the remnant nucleus of its red algae endosymbiont [The apparent similarity between a Rhizarian , their association with chlorarachniophyte hosts is plausible. However, given its very low abundance in C. reptans, additional work is clearly needed to retrieve the complete virus genome and to confirm the association of such bunya-like viruses with the chlorarachniophytes.Our detection of a One of the greatest challenges in viral genomics is the ability to detect distant homologies, especially in rapidly evolving RNA viruses. As a first attempt to retrieve such ephemeral evolutionary signals, we scanned orphan contigs using RdRp protein structural information in addition to the standard primary amino acid sequence. Notably, both the RdRp profile and 3D protein structure comparison led to the identification of highly divergent RNA virus candidates, although these remain difficult to annotate. Efforts to better describe the repertoire of sequence and structure of viral RdRps are therefore central to unveiling the RNA virosphere in overlooked eukaryotic organisms.


However, we still lack approaches to directly study how the set of marks for a given RNA molecule shape its fate. In this perspective, we first review current leading approaches in the field. Then, we propose an experimental and computational setup, based on direct RNA sequencing and mathematical modeling, to decipher the functional consequences of RNA modifications on the fate of individual RNA molecules and isoforms.It has been known for a few decades that transcripts can be marked by dozens of different modifications. Yet, we are just at the beginning of charting these marks and understanding their functional impact. High-quality methods were developed for the profiling of some of these marks, and approaches to finely study their impact on specific phases of the RNA life-cycle are available, including RNA metabolic labeling. Thanks to these improvements, the most abundant marks, including N More than a 100 RNA modifications have been identified since the 1950s . They we6-methyladenosine (m6A) emerged as one of the most abundant modifications of coding transcripts  , revealed the preferential, while not exclusive, association of the mark with the central adenosine in the RRACH sequence context around the stop codon of messenger RNAs   , recogniMETTL14) , and remMETTL14) , suggestC, or U) . Notablyhis mark . Howeverhis mark . AltogetNA decay  and tranNA decay , while dNA decay .6A, or other RNA modifications, with nascent transcripts, but also allows to quantify the impact of these marks on the dynamics of all key steps of the RNA life cycle, and specifically on the kinetic rates of RNA synthesis, processing, and degradation. The application of this technique confirmed the role of m6A on the regulation of RNA stability, and suggested its influence on the dynamics of RNA synthesis and processing , and the exogenous modification used for the labeling . As we discuss in the following sections, this is a complex task that can be only indirectly implemented through current approaches.The joint analysis of the information gained from RNA metabolic labeling experiments, together with the profiling of specific RNA modifications, would be extremely powerful for the study of the functional consequences of these marks on specific RNA life cycle steps. However, while the application of metabolic labeling for the profiling of nascent RNA  and for 6A) or exogenous  RNA modifications. A first class of methods is based on the enrichment of modified RNAs before the sequencing. This relies either on the use of specific antibodies [e.g., MeRIP-seq for m6A detection . DART-seq , leading to single transcript m6A detection. The key downside of this method is the required cells transfection with APOBEC1-YTH fusion protein. Finally, the ability to quantify the abundance of m6A marks remains to be established.Numerous protocols based on srRNA-seq were developed for the identification of either endogenous  is a major obstacle for the analysis of individual RNA molecules, despite the development of methods to infer isoforms expression from these data  RNA modifications simultaneously.Besides the specific limitations of each technique, all available protocols for the profiling of RNA modifications through srRNA-seq share some key limitations. ications . Second,sitivity . Third, ese data . As a co1-methyladenosine, 5-methylcytosine, and pseudouridine . Hence, reduced base calling accuracy is not considered a major issue in the field of RNA modifications but, on the contrary, represents an opportunity for aiding the identification of modified bases  modified nucleotides was tested . Rather, the performance decreased when the tool was applied on in vivo yeast data and benchmarked with MeRIP-seq m6A calls for the same conditions (accuracy: 87% and recovery: 32%). In terms of downsides, EpiNano requires prior knowledge on the sequence motif for the mark of interest, and it cannot achieve single molecule resolution, since it aggregates the information derived from multiple reads alignments.EpiNano relies on a support vector machine, and exploits the increased frequency of alignment errors and the low base quality caused by the presence of the modification of interest . The tooin vitro fully modified transcripts, rRNAs from various species, and a human lymphoblastoid cell line. Like Tombo, the main downside of ELIGOS is in terms of false positive rates.ELIGOS aims at the unbiased identification of any RNA modification that would impact bases errors frequencies. It relies on the comparison between dRNA-seq of native and cDNA-converted transcripts, the latter used as a reference that is devoid of any mark due to the retro-transcription to cDNA . ELIGOS 6A identification that was recently released is called MINES , the tool returns the corresponding changes in the signal to identify all marks occurrence, while mark-specific calls are not possible. Advantages and disadvantages of the tools discussed above are reported in Nanocompore is a novel tool recently released, which is based on the comparison of a condition of interest with a condition where the writer for a specific mark was depleted or removed . The ide6A, with the quantification of the corresponding RNA dynamics, via metabolic labeling and profiling of exogenous modifications such as 4sU or 5eU . Notably, the frequency and the specific position of occurrence of the marks is increasingly recognized as an important factor. For example, the fate of RNAs carrying multiple m6A marks was shown to be influenced by a liquid–liquid phase separation processes driven by the binding of readers of the YTH family. Eventually, those transcripts were shown to be targeted to specific cellular compartments, including stress-granules and P-bodies, with important consequences for their translation and stability  is the most direct data type for the identification of the marks, compared to more indirect measurements, such as the error rate. While tools, such as EpiNano, showed a good performance by only using the latter, we would recommend trying to incorporate information from the former. Indeed, indirect measurements could be completely or partially originated by unexpected causes, which could lead to high false positive rates with in vivo datasets.Fourth, the quantification of RNA dynamics should include the step of premature RNA processing. This is often neglected, by assuming the corresponding rate being constant. However, RNA synthesis and processing are tightly coupled, then when the former is modulated, which often occurs, the latter is also expected to be altered  and exogenous  marks at single molecule resolution are in place, they could be used, in series, for the identification of the four possible classes defined by the presence or absence of each modification. The performance of such an approach should be tested on a dataset generated ad hoc. The genesis of reads with both the RNA modifications, or missing only the exogenous mark, is feasible by using or avoiding long-time metabolic labeling, respectively. Instead, reads devoid of both the base analogs can be produced sequencing the corresponding cDNA. It is more difficult to generate transcripts that lack only the endogenous modification, which could be obtained by knocking-out the corresponding writer (for those marks for which this is known). However, genetic compensation (Once proficient algorithms for the detection of the endogenous (e.g., mensation  or writeThe study of the impact of RNA modifications on the RNA life cycle dynamics would largely benefit from the development of a unified computational framework. This, starting from long reads dRNA-seq data, should manage the RNA kinetic rates inference, according to their modification status, at the level of individual transcriptional units or specific isoforms.A convenient starting point could be INSPEcT , a tool INSPEcT has been recently extended by implementing a novel approach that allows the inference of synthesis, processing and degradation kinetic rates without nascent RNA profiling . This apIn conclusion, a number of recent and on-going technology advancements are significantly facilitating the study of the functional consequences of RNA modifications on the fate of marked transcripts. In particular, the combined application of RNA metabolic labeling, for the profiling of nascent transcripts and the quantification of the kinetic rates governing the RNA life cycle dynamics, and of long-reads direct RNA sequencing, is particularly promising. Indeed, they promise to deliver data of unprecedented quality and resolution, and should allow studying the impact of RNA modifications at the level of individual molecules and isoforms.MF and MP conceived the study. MF led the writing and produced the figures. MP supervised the study and the writing of the manuscript. All authors contributed discussing and writing 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.


With recent advances in nanotechnology, graphene nanomaterials are being translated to applications in the fields of biosensing, medicine, and diagnostics, with unprecedented power. Graphene is a carbon allotrope derived from graphite exfoliation made of an extremely thin honeycomb of sp2 hybridized carbons. In comparison with the bulk materials, graphene and its water-soluble derivative graphene oxide have a smaller size suitable for diagnostic platform miniaturization as well as high surface area and consequently loading of a large number of biological probes. In this work, we propose a nanotechnological method for concentrating total RNA solution and/or enriching small RNA molecules. To this aim, we exploited the unique trapping effects of GO nanoflakes in the presence of divalent cations  that make it flocculate and precipitate, forming complex meshes that are positively charged. Here, we demonstrated that GO traps can concentrate nucleic acids in the presence of divalent cations and that small RNAs can be selectively released from GO-magnesium traps. GO nano-concentrators will allow better analytical performance with samples available in small amounts and will increase the sensitivity of sequencing platforms by short RNA selection. With advances in next-generation sequencing, the human transcriptome has been deciphered and the regulatory roles of a large fraction of small RNAs suggest that they might represent poorly invasive disease biomarkers in diagnostics . Liquid In recent years, the carbon allotrope graphene has become extremely popular in the biomedical field, due to its unique interactions with cells and organic compounds . GraphenBoth GO and its reduced by-product rGO have a high affinity for single-stranded nucleic acids (ssNAs) via hydrogen bonds and/or π-π interactions, especially for short ssNAs , given the slower diffusivity of longer ssNAs . This obGraphene oxide binding to nucleic acids is based on the interaction with backbone phosphate, and this could lead to indiscriminate reactions with either double-stranded nucleic acids (dsNAs) or ssNAs, and to reduced sensitivity in distinguishing the dsNAs to the ssNAs. For this reason, rGO has been preferred, given its direct interaction with nucleotide residues exposed in ssNAs like miRNAs . On the 2+, which is known to form RNA clamps in cells  in solution. To this aim, we developed a protocol based on the unique trapping effects of GO nanoflakes in the presence of divalent cations. Indeed, cations like calcium and magnesium quickly adsorb on GO surface. After adsorption, GO flocculates and precipitates forming complex structures with size and stability largely dependent on the type and concentration of the ion involved . This hain cells . This mein cells  and tearin cells .2 or MgCl2. After the interaction, samples were diluted to avoid multiple scattering previous DLS measurements.Graphene oxide (4 mg/ml) was purchased from GrapheneA (Spain) and diluted in MilliQ RNA and DNA free water for further experiments. GO size and thickness were evaluated by atomic force microscopy (AFM)  as previously reported . GO hydr® SQ Blood DNA Kit (Omega BioTek), according to the manufacturer’s instructions. Total RNA samples were collected by TRIzol reagent method (Invitrogen), using the standard protocol (Genomic DNA (gDNA) and total RNA samples were alternatively extracted from U87 human glioblastoma cells (purchased from ATTC). Cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum , 2% penicillin–streptomycin (Sigma-Aldrich), and 2% L-glutamine (Sigma-Aldrich). gDNA was extracted using E.Z.N.A.protocol . gDNA an2O, CaCl2, or MgCl2. After 10 min, 9 μl of gDNA or total RNA (60 ng/μl) have been added to the solution. The final concentration of cations in solution was 5, 1, or 0.5 mM. A synthetic miRNA  was added to RNA samples, to a final concentration of 30 pM, to be assessed as an exogenous control for small RNAs for qPCR analysis. In particular, the use of this exogenous miRNA allowed overcoming the possible issue deriving from the different expression levels of any endogenous cellular miRNAs among biological samples. Samples were incubated for 15 min and then centrifuged for 10 min at 14,000 × g. Nucleic acid concentration was measured in the supernatant of samples by measuring absorbance at 260 nm with Cytation 3 Take 3 reader  or fluorescence peak at 520 nm when 5′6-FAM-labeled ssNAs were used in the experiment. In particular, a 22-nucleotide-long DNA sequence (ACTGCGGCTTAGGTTAGCATTG) was randomly designed, considering a sequence with the same length and CG and AT content of a typical miRNA, and purchased by IDT Integrated DNA technologies with a 5′-56 FAM modification. The presence and integrity of total RNA in the supernatant of samples have been additionally evaluated by agarose gel electrophoresis in all the experimental conditions.Ten microliters of GO  have been added to 1 μl of ddHGAPDH  transcripts and of cel-miR-39-3p in samples were evaluated using reverse transcription quantitative real-time PCR (qPCR). More in detail, the same quantity of each sample was reverse transcribed using GoScript Reverse Kit (Promega) and GAPDH transcript levels were subsequently amplified with StepOne system (Applied Biosystems), using Syber Green master mix  and specific primer pairs  (TM Advanced miRNA cDNA Synthesis Kit (ThermoFisher), according to the manufacturer’s instructions. cel-miR-39-3p levels were amplified using TaqManTM Fast Advanced Master Mix (ThermoFisher) and a specific TaqManTM Advanced miRNA Assay .The levels of CATGGAC) . In para2+ and Mg2+ ions among cations known to change GO properties in solution mainly because heavy metal cations  destabilize too strongly GO . The addition of divalent cations caused an increase of RH up to ∼1400 nm from ∼500 nm as reported in 2+ and Mg2+, confirming a positive charging of GO surface in the presence of cations. GO meshes created with cations can be separated from the rest of the solution by precipitation or more quickly with centrifugation; this avoids functionalization with magnetic beads or other nanoparticles for further analysis. Representative images of GO aggregates in the presence of Ca2+  without significant differences between the cations.Our results showed that GO (1 mg/ml) does not adsorb gDNA , only if2+ or Mg2+ concentration is added to GO prior to the incubation with RNA. Twenty micrograms of GO adsorbed 0.4 and 0.55 μg of total RNA with 5 mM Ca2+ and 5 mM Mg2+, respectively. This result might seem in contrast with reports from literature that have shown, even if in low amounts, adsorption of RNA even from bare GO . First, we incubated GO (2 mg/ml) with total RNA  added with the synthetic cel-miR-39-3p as an exogenous control for small RNA  . As obse bare GO . We want bare GO . This le2+-RNA and GO-Mg2+-RNA had surface charge substantially different from GO-Ca2+ and GO-Mg2+ pellets, respectively.Analysis of surface zeta potential of GO pellets after RNA adsorption confirmed that RNA molecules were deposited on GO surface  only in Electrophoresis analysis of supernatants confirmed the UV spectroscopy results  and in pGAPDH transcript amplification  values correspond to a lower amount of transcript levels in the sample, we have compared the different samples amplifying the same volume of each supernatant. Our data showed that longer RNAs are significantly adsorbed more at high concentration of cations, as demonstrated by the increased Ct for  present . Very inAltogether, these data demonstrate efficient trapping of total RNA in solution by GO-divalent cations with faster recognition of small RNAs.2O and with incubation of 5 min with 1.5 mM Tris buffer (pH 8.8) at room temperature. These conditions were chosen based on literature data that indicate that the adsorption is favored at lower pH and low temperature (It is important to release small RNAs bound to GO surface for further PCR/sequencing analysis. Indeed, at the critical concentration of 4 μg/ml, PCR reactions are inhibited by the GO in solution . The desperature . Furtherperature .2+samples (both small and long RNAs; 2+ with our buffer affected only smaller RNAs and was not concentration-dependent, whereas the longer RNAs remained “trapped” and there is a small release of large RNA only at high Mg2+ concentration (see line profiles in In tris buffer, the RNA was successfully released from GO-Ca2+ ions. Conversely, with calcium, all RNA species are released (2+ ability to make bidentate RNA clamps with large RNAs markedly influenced released studies. Indeed, it has been demonstrated that magnesium, due to its size and charge density, binds more intimately than calcium to the oxyanions of RNA, so that magnesium clamps are made stable by electrostatic interactions, charge transfer, polarization, and exchange interactions (2+ based selection represents a fast, reliable method for rRNA depletion. Our results also suggested that GO-Ca2+ complexes can instead represent a suitable method to isolate and concentrate total RNA, hence providing a tool for the simultaneous isolation and analysis of both small and large RNA species. This study can pave the way toward the development of novel methods to improve liquid biopsy performance and may contribute to developing innovative approaches for miRNA and/or total RNA detection (With recent advances in nanotechnology, nanomaterials are being translated to applications in the fields of biosensing, medicine, and diagnostics, with unprecedented power . In thisreleased . Indeed,ractions . This “practions . rRNA inractions . Our GO-etection .The datasets generated for this study are available on request to the corresponding author.VP, LD, and MB performed experiments and contributed to manuscript writing. VP and MP designed the experiments and methodology and revised the manuscript. GP characterized the graphene samples. WL, OP, and MD contributed to manuscript writing and revision. All authors have read and agreed to the published version 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.


C. elegans. The fungus Drechmeria coniospora produces spores that attach to the cuticle, before hyphae invade the body. Mutants with an altered surface coat, the outermost layer of the cuticle, including bus-2, bus-4, bus-12 and bus-17 show increased adhesion of fungal spores . We unexpectedly found that D. coniospora spores attach unusually densely around the mouth of unc-119 mutants. Interestingly, this phenotype is not rescued by the C. briggsaeunc-119 construct that is conventionally used to rescue neuronal unc-119 phenotypes.If the cuticle acts as a protective barrier against environmental insults, several pathogens have developed strategies that use it as a way to infect  C. elegans and D. coniospora, we made use of MosSCI strains, constructed in an unc-119(ed3) background . We noticed that a variety of these strains exhibited a greatly increased susceptibility to infection. Upon further examination, we determined that this was due to an increase in the adhesion of fungal spores, most prominently at the tip of the head (C. elegansunc-119 rescuing construct in an unc-119(e2498) background. But the increased spore adhesion was visible in both the unc-119(ed3) and the unc-119(tm4063) background even in the presence of the standard C. briggsae unc-119 rescuing construct. The phenotype was absent from these same transgenic strains in which unc-119(ed3) was eliminated by out-crossing (Table 1 below). unc-119 function has been extensively analysed in the nervous system. Notably, some expression in the epidermis was reported recently . While we have not determined the precise cause, since spore adhesion is a major determinant of infection progression , such effects need to be taken into account when interpreting experiments involving any strain that has an unc-119 allele in it, which has been often employed as selectable marker for transgenesis.As part of our investigations of the interaction between the head . The pheE. coli OP50 and cultured at 25°C until the L4 stage (40 h) before being exposed to fungal spores as previously described . Images were taken of worms mounted on a 2% agarose pad on a glass slide anesthetized with 0.25 mM levamisole, using a Zeiss AxioCam HR digital colour camera and AxioVision Rel. 4.6 software (Carl Zeiss AG).Eggs, prepared by the standard bleach method, were allowed to hatch in 50 mM NaCl in the absence of food at 25°C overnight. Synchronized L1 larvae were transferred to NGM agar plates spread with unc-119(ed3); ttTi5605 II, JR667 unc-119(e2498::Tc1) III; wIs51 V and OP533 unc-119(tm4063) III; wgIs533 strains were provided by the CGC (Caenorhabditis Genetics Center), which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). In addition, the following strains were tested for spore adhesion: IG1604 unc-119(ed3); frSi6 II , IG1622 unc-119(ed3); frSi9II, IG1623 frSi9 II , AX6672 unc-119(ed3); npr-1(ad609); ilcr-1(tm5866);  II , IG1633 frSi6 II and IG1629 unc-119(ed3); frSi10 II, this study. Both pNP145 and pNP150 were derived from pCFJ151 – ttTi5605_MCS, that was a gift from Erik Jorgensen  .N2, EG6699


The telomere repeats containing RNA (TERRA) is transcribed from the C-rich strand of telomere DNA and comprises of UUAGGG nucleotides repeats in humans. The TERRA RNA repeats can exist in single stranded, RNA-DNA hybrid and G-quadruplex forms in the cell. Interaction of TERRA RNA with hnRNPA1 has been proposed to play critical roles in maintenance of telomere DNA. hnRNPA1 contains an N-terminal UP1 domain followed by an RGG-box containing C-terminal region. RGG-motifs are emerging as key protein motifs that recognize the higher order nucleic acid structures as well as are known to promote liquid-liquid phase separation of proteins. In this study, we have shown that the RGG-box of hnRNPA1 specifically recognizes the TERRA RNA G-quadruplexes that have loops in their topology, whereas it does not interact with the single-stranded RNA. Our results show that the N-terminal UP1 domain in the presence of the RGG-box destabilizes the loop containing TERRA RNA G-quadruplex efficiently compared to the RNA G-quadruplex that lacks loops, suggesting that unfolding of G-quadruplex structures by UP1 is structure dependent. Furthermore, we have compared the telomere DNA and TERRA RNA G-quadruplex binding by the RGG-box of hnRNPA1 and discussed its implications in telomere DNA maintenance. The C-rich strand of telomere DNA in eukaryotes is transcribed into a long non-coding RNA called the telomeric repeats containing RNA (TERRA) . Due to 4-RGG-X4-RGG, where X is any amino acid) and a single RGG repeat that is present nine amino acids away towards the N terminal region of the tri-RGG motif  and UP1+RGG (residues 1–249) of hnRNPA1 (Uniprot identifier P09651-2) were sub cloned into pET28a E. coli expression vector. Both the constructs contain a C-terminal hexa-histidine (6x His) affinity purification tag. The proteins were expressed in BL21 Rosetta (DE3) cells in LB media. The cells were induced with 0.5 mM IPTG and incubated for overnight at 30°C after growing them at 37°C till the culture reached an optical density (OD) at 600 nm of 0.6. First step of purification involved 6x His-tag–Ni-NTA affinity chromatography followed by cation exchange and size exclusion chromatography (SEC) using a Superdex 75 column. The isolated RGG-box was sub cloned into pGEX-6P-1 vector and a TEV cleavage site was introduced between the GST tag and the protein. Glutathione-sepharose column was used for the purification of RGG-box, followed by the GST tag removal using TEV protease. SEC using Superdex 75 column was performed at the final step of purification of the RGG-box. The final purity of all the proteins was ascertained using SDS-PAGE and MALDI-MS analyses and the proteins were adjudged to be >95% purified.1H NMR spectra.The RNA sequences were ordered from Eurofins . For ITCIsothermal titration calorimetry (ITC) experiments were performed using iTC200 instrument (GE) at 25°C. The protein and the RNA samples were thoroughly de-gassed before the experiment. For the titrations, 100–200 μM of the protein was titrated into 5 μM of the RNA filled in the sample cell. 20 injections containing 2 μl of the titrant each were made keeping an interval of 180 s between each injection. The integrated heat data for all the runs was corrected for the heat of dilution, the protein being injected at each step. The data was then fitted for one site binding model using ORIGIN software provided by the vendor (GE). All the parameters were kept floating during the data fitting. All the experiments were repeated at least two times for data consistency.The structural changes in RNA G-quadruplexes  by UP1 and UP1+RGG were monitored using CD spectroscopy. CD titrations were carried out using JASCO J-715 Spectrophotometer. The wavelength scans for the RNAs were done from 325 to 225 nm. The average of three spectral scans was taken for each titration step. Baseline correction was done for each run and the data was normalized to molar ellipticity per residue. The titration experiments were performed in 1 cm path length cuvette from Hellma Analytics at 25°C at a scanning speed of 100 nm/min with a response time of 4 s. 5 μM of the RNA sample was titrated with increasing protein concentration .For the melting studies of the RNA G-quadruplexes, the change in ellipticity was measured at 262 nm as a function of increasing temperature. The ellipticity was monitored from 10°C to 100°C with an increase of temperature of 1°C every minute. Data points were recorded after every 0.5°C.1H NMR and 2D 1H–15N HSQC spectra of protein and 1D 1H NMR spectra of RNA were recorded on Bruker 700 MHz, equipped with a cryoprobe, at 298 K. Water suppression for the 1D 1H experiments was done using excitation-sculpting. 550 μl of 50 μM of RNA sample was prepared in 10 mM potassium phosphate (pH 6.5) containing 100 mM KCl. The RNA was then titrated with increasing concentration of UP1 and UP1+RGG (in K+ buffer).1D 1H–15N HSQC titration experiments, 500 μl of 138 μM of the 15N labeled RGG-box sample was prepared in 10 mM potassium phosphate (pH 6.5) and 100 mM KCl. 10% D2O was added to the sample for the deuterium lock. The protein was titrated with increasing concentration of RNA  followed by 10 min of incubation. In case of abasicloopsTERRA-24 titration with the RGG-box, 120 μM of 15N labeled RGG-box sample was titrated with increasing concentration of the RNA G-quadruplex. 2D 1H–15N HSQC spectrum of the protein was recorded at each step of the titration. The chemical shift changes were monitored in each step of the titration. The NMR data was processed using Bruker TOPSPIN 3.1 and analyzed using NMRFAM-SPARKY ,The observed chemical shift changes for the RGG-box—TERRA24 titrations were calculated to quantify the binding affinity. The following formula was used, 4, 24mer] were used as single stranded RNA control 4, 24mer] RNA sequences are designed to form tetrameric, dimeric, and intramolecular monomeric RNA G-quadruplexes of parallel topologies respectively , enthalpy change (ΔH), entropy change (ΔS), and stoichiometry (n) of the complex formation under the experimental conditions. For the ITC titrations, 5–10 μM of RNA solutions were titrated with 100–200 μM of UP1 (residues 1–196) and UP1 along with RGG-box . All the interactions were exothermic at 25°C. Figures Kd of 2.37±0.49 μM and a stoichiometry of ∼1  we have used isothermal titration calorimetry (ITC) method. ITC directly measures the binding enthalpy upon complex formation and provides the information on equilibrium constant  was observed for UP1 and UP1+RGG binding to all three G-quadruplexes compared to the ssRNA-18  interaction between UP1+RGG and TERRA-24 in the presence of 20% PEG 200 with a Kd of 3.0±0.55 μM   and 2D 1H–15N HSQC spectrum was recorded at each step of the titration.Recently, we had shown that the isolated RGG-box of hnRNPA1 is an intrinsically disordered region of the protein . Poor ch1H–15N HSQC spectrum at each step of the titration. We observed no significant chemical shift perturbations in the 2D 1H–15N HSQC spectrum of the RGG-box  and recorded a 2D x Figure . In the ractions . Thereforactions  and B. T15N-uniformly labeled RGG-box was titrated with increasing concentration of physiologically relevant intramolecular TERRA-24 RNA G-quadruplex . Therefore, both ITC and NMR titration results unambiguously showed that the RGG-box of hnRNPA1 specifically interact with the intramolecular TERRA RNA G-quadruplex structures.The interaction between TERRA-24 and the RGG-box was also probed using ITC experiments at 25°C Figure . We obse15N labeled RGG-box and a 2D 1H–15N HSQC spectrum of the protein was recorded at each step. Interestingly, we observed very small chemical shift perturbations in few residues at an oversaturated 1:2 protein–TERRA-6 ratio . AbasicloopsTERRA-24 contains only sugar phosphate backbone in the loop residues. The formation of G-quadruplex of parallel topology by abasicloopsTERRA-24 was validated through NMR and CD spectroscopy . The RGGn Figure  and B. Tn Figure  and B. TUP1 is a well-established DNA G-quadruplex destabilizing protein ,30,47. HIn the next set of experiments, we used UP1+RGG and monitored the unfolding of TERRA-6, TERRA-12 and TERRA-24 G-quadruplexes by recording CD spectrum at each step of the titration. Same trend in the unfolding of the RNA G-quadruplexes was observed: TERRA-24 was unfolded to the maximum extent followed by TERRA-12 and finally TERRA-6 at 1:5 molar ratio of RNA to protein Figure –F and H.1H spectra of different G-quadruplexes were followed at different ratio of RNA to proteins (UP1 or UP1+RGG). Disappearance of H-bonded imino peaks, therefore suggests unfolding of G-quadruplexes. In case of tetrameric TERRA-6 G-quadruplex, both UP1 and UP1+RGG destabilized the G-quadruplex structure, however inefficiently. The observation of imino proton peaks at 1:1.5 RNA to protein molar ratio suggest that both UP1 and UP1+RGG could not unfold TERRA-6 completely  in the 1D y Figure . Howevery Figure . Similary Figure .1H NMR spectra here emanate from the hydrogen bonded guanine residues in the G-quartets (1H NMR spectra.We observed that the efficient unfolding of the TERRA RNA G-quadruplexes was achieved at higher RNA to protein ratio (lower protein concentrations) in NMR experiments compared to the CD titrations. The imino proton peaks monitored in the 1D quartets . During Overall, the CD and NMR results showed that hnRNPA1 destabilizes the TERRA RNA G-quadruplexes, wherein the loop containing TERRA RNA G-quadruplexes  than its equivalent intramolecular Tel22 DNA G-quadruplex (Tm = 68°C) under similar conditions ..Tm = 80.In case of protein and RNA G-quadruplex interactions, the 2′OH of the ribose sugar contributes towards additional hydrogen bond interactions . FurtherKd = 2.37±0.49 μM) with about two-fold lower affinity compared to the single-stranded DNA (22mer Tel22ss with Kd = 1.23±0.14 μM) under the same experimental conditions reported earlier (Kd = 1.71±0.17 μM) with ∼3.3-fold higher affinity compared to the intramolecular Tel22 DNA G-quadruplex (Kd = 5.62±1.09 μM) reported earlier (Kd = 0.92±0.17 μM) compared to intramolecular Tel22 DNA G-quadruplex (Kd = 4.85±0.60 μM)  . A stoic0.60 μM) . While, 0.60 μM) ,55. Base0.60 μM) ,54. Howe0.60 μM) .In a recent study, using electrophoretic gel mobility shift assays (EMSA) and photo cross-linking experiments the interaction of hnRNPA1 with the single stranded RNA and TERRA G-quadruplexes was probed ,29. Thes1H–15N HSQC spectra of the RGG-box upon binding to TERRA-24 RNA G-quadruplex compared to Tel22 DNA G-quadruplex binding  than the isolated RGG-box interaction with Tel22 DNA G-quadruplex (Kd = 349 ± 35 μM) reported earlier (The RGG-box is the second most frequently occurring RBDs in human genome after the RNA Recognition Motif (RRM domain) . Often, g Figure  and B. B earlier . TherefoRecently, it was proposed that the amide groups present in the backbone of glycine and the side chain of arginine residues of the RGG-box in different proteins are important for mediating protein and nucleic acid interaction . In addiIn vivo, the arginine residues in the RGG-box are known to undergo methylation (In summary, in this study we have reported the structure dependent recognition of TERRA RNA G-quadruplex by the RGG-box of hnRNPA1. We showed that the RGG-box of hnRNPA1 binds to the loops containing TERRA RNA G-quadruplexes but not with the single-stranded RNA. This provides the specificity to the UP1 domain to bind and enhances unfolding of the intramolecular TERRA RNA G-quadruplex. hylation ,61–63. Tgkaa134_Supplemental_FileClick here for additional data file.


The modern version of the RNA World Hypothesis begins with activated ribonucleotides condensing  to make RNA molecules, some of which possess (perhaps slight) catalytic activity. We propose that noncanonical ribonucleotides, which would have been inevitable under prebiotic conditions, might decrease the RNA length required to have useful catalytic function by allowing short RNAs to possess a more versatile collection of folded motifs. We argue that modified versions of the standard bases, some with features that resemble cofactors, could have facilitated that first moment in which early RNA molecules with catalytic capability began their evolutionary path toward self-replication. There remains a robust debate about the transition from a primordial soup of molecules to organisms with Darwinian descent, a moment that Carl Woese , 2 calleEach of the stages of evolution—going from simple molecules, like cyanide, to more complex molecules, like nucleic acids, to polymers capable of complex functions and ultimately, to systems capable of catalysis and templated replication—represents distinct and poorly understood problems. Although these steps involve vastly different processes and undoubtedly involve progressively increasing molecular complexity, each also required some form of partitioning to allow molecules the chance to not lose each other in the vastness of the early Earth. For later steps, such as energy utilization and genetics, formation of membranes and eventually, cells could have created the needed molecular localization. For early chemical condensations, the partitions could have been small primordial pools, where proximity was facilitated by evaporation or the presence of rocks composed of useful minerals . It has In this paper, we have tried to focus only on one issue within this long chain of events: how is it possible to evolve catalytic RNAs that function as short molecules with enough catalytic power to bootstrap the development of those short, chemically derived RNAs into replicators/genetics? In other words, what were the earliest ribozymes in an RNA World, and how did they get their start? These huge questions are addressed, along with many others, in wonderful (but slightly dated) reviews of the RNA World Hypothesis , 8. Bernglobularity) as well as the availability of nucleobases with catalytic function could facilitate the emergence of a first set of ribozymes from shorter RNAs than one might have predicted, making the start of life more likely.Scientists have imagined that RNAs randomly assembled at sufficient lengths are all one might need to achieve a transition to a stable RNA World. We argue that length is less important than catalytically competent shape, which we refer to as “globularity.” Globularity in RNAs  experiments to address the problem inherent in short RNA molecules, and in doing so, perhaps, we have increased the plausibility of the RNA World Hypothesis.That is, in the chemisphere that might have existed, the two rate-limiting problems could have been solved simultaneously. A single early chemistry that provides globularity and catalytic power to short RNAs would have led to the “enzymatic” replication of template RNAs  that had been made initially without replication either by spontaneous condensation of monomers or in reactions catalyzed by other RNAs. We have used our own data about oligonucleotide globularity coupled with Harold B. White’s ideas about catalytic RNA –11 and aAptamers are single-stranded nucleic acid-based affinity reagents evolved from random libraries using SELEX  , 13. Enh1417SI Appendix, Fig. S1). For DNA hairpins containing a stem region of four Watson–Crick base pairs and a loop of seven thymidine nucleotides (the T7 variant), the Tm increases steadily as up to five of these thymidines are replaced with 5-(N-benzylcarboxamide)-deoxyuridines (Bn-dU), with a total change of 14 °C (N-benzylcarboxamide) base substitutions and 2′-O-methyl ribose substitutions  suggests that an ordered loop structure can exist if simple hydrophobic adducts are substituted into hairpin loop regions. The data suggest that base modifications can create additional stable structures, which may facilitate the formation of pockets for substrate binding in single-stranded oligonucleotides.lac repressor protein searched Escherichia coli DNA for the single chromosomal lac operator. In the latter case, the needed three-dimensional collision between a protein and its binding site on a large genome was reduced to one-dimensional diffusion on the double-helical DNA genome.We believe that we have reduced two questions to one, where the presence of the hydrophobic modifications facilitates solving both the length and catalysis problems simultaneously. This reduction in the number of independent variables can be thought of as an example of a reduction of dimensionality. This concept, while more commonly applied to machine learning and statistics, has been used in a biological context to describe solute/membrane receptor reaction rates mediated by two-dimensional diffusion along membrane surfaces –28 and bOften, a reduction in dimensionality makes searches more efficient through the removal of an independent spatial dimension , such asWe are aware that the benefits of this reduction of dimensionality are intimately tied to a complication associated with the need to distinguish one specific type of a modification  over other similar modifications (or no modifications) in order to preserve sequence information through multiple cycles of replication. The simplest solution to this problem is to propose that there was only one or at least a predominant modification in relevant prebiotic conditions so as to ensure uniform substitution throughout the RNA sequence that can be faithfully replicated . There is unsurprisingly an analogy with in vitro evolution. We use uniform modification at defined positions with our SELEX libraries precisely because multiple modifications at any one position, while having the desired effect of increasing chemical complexity in starting libraries, would dilute the number of exact copies of the original sequence with a precise pattern of modifications with each cycle of replication, thereby reducing the likelihood of copying the desired sequence for the next round of selection. The notion that only one or few modifications were present in prebiotic conditions seems unlikely. If there was a rich array of modifications, a mechanism would need to exist to selectively favor incorporation of certain modifications over others, which is also challenging. In the context of the RNA World, short functionally active sequence length enabled by modifications, as we are proposing here, would help with this problem but only to a limited extent. Another solution to this problem would be to propose that the introduction of specific modifications can be done by specific catalysts dedicated to such reactions , but this of course assumes that such entities already existed in prebiotic conditions. We do not provide a solution to this difficult problem here.In our work in the development of nucleic acid-based affinity reagents, we have transitioned from RNA-based aptamers to DNA-based SOMAmer reagents –16. To dThe positions of some hydrophobic modifications in SOMAmer–protein cocrystals are shown in We have also observed some modification-driven intermolecular contacts for 5-position–modified sequences. This suggests that the hydrophobic modifications may provide yet another evolutionary component—solving what can be deemed “the proximity problem.” Formation of oligomers can be facilitated entropically by aligning to individual building blocks in space. Interaction of hydrophobic moieties in a hydrophilic environment could potentially provide the scaffolding for this alignment for intermolecular as well as intramolecular interactions, which is another mechanism for reducing the number of independent variables.As pointed out by Cermakian and Cedergren , early mThe advantages of expanding the repertoire of functional groups to develop better catalysts have been duly recognized in the context of in vitro evolution from random libraries, and indeed, modified side chains that can serve as general acid or general base catalysts mimicking those observed in proteins have been used for this purpose –40. FinaN-benzylcarboxamide) groups to each of the seven uridines using PyMOL Builder  and at the same time, stabilize the stem-loop structure C suggestic acids  and Tm dge bonds . The preHarold B. White  suggesteAbsent chemical fossils, we cannot know what the primordial cofactors were except that they could have enhanced both the catalytic power of early RNAs and their ability to function at shorter lengths. Two examples seem relevant to the discussion.tRNAs have the highest densities of modification of any class of RNAs , 47. OneModified tRNAs have different stability of overall three-dimensional structure than unmodified tRNAs. Sampson and Uhlenbeck  showed tFurthermore, stable loops can be constructed from present day nucleotides. For example, CUUCGG and GAAA hairpin loops are known stabilizers of short stems. Tuerk and coworkers  showed tClearly, only a few instances of unmodified sequences survived the test of time for hairpin loop stabilization. This is in striking contrast to our observation that most additions of a modified nucleotide to the loop resulted in an incremental increase in stability, strengthening the notion that modifications could have facilitated the early evolutionary development of catalytically capable nucleic acid structures.The White paper on modified bases adds to the discussion of the function/catalysis problem, while the stabilization of a hairpin through modified loop bases potentially adds to the discussion of the length problem. The presence of prebiotic nucleobases modified at the 5-position has been proposed by Robertson and Miller . Uracil The RNA World Hypothesis faces the problem first declared by Leslie Orgel . How do Joyce and Orgel  discussem data and inspiration from reading insightful papers, that cofactor-like primordial nucleobases simultaneously provide catalytic activity and diminish the oligonucleotide lengths needed to create intramolecular frameworks/active sites in RNAs. Our addition to the RNA World Hypothesis, dependent on Harold B. White’s brilliant idea of primordial cofactor-like RNA nucleobases, may provide a step toward the evolution and replication of early genomes and structured catalytic centers.To that desire for a magic catalyst, we add, based only on our T2+ and other divalent cations, spermidine, proflavine, etc.) on the stability of these hairpin structures and also on the outcome of these proposed selections.David Perrin and coworkers , 55, 56 However, the deeper question is not would our experiment be successful but how close to the chemical building blocks available in the chemical world would that experiment be. This problem seems to be at the heart of all experiments about the moment that chemistry became biology. As pointed out by Cairns-Smith  and YaruWe hope that people will tackle the length question systematically so that shorter modified RNAs could also be tested for catalytic breadth. The early Earth might have broken into biology from both short RNAs and broad catalytic opportunities.Our proposal for the usefulness of hydrophobic modifications in early evolution creates additional questions with regard to specialization of function. For example, how is it possible that modifications were maintained throughout evolution along functional/structural paths, such as the folding of modern tRNAs, but did not persist with regard to storage of genetic information? In addition, what sequence of events is plausible for the loss of modifications along the heritability pathway? Considering the White hypothesis and the modern presence of modified nucleotides as cofactors, this may have correlated with the transition to the protein-based world. While these questions certainly cannot be intelligently addressed from the limited data presented here, we hope that these questions will be tackled as well.2, 1.0-K molecular weight cutoff polyethersulfone filter and a Cole Parmer Masterflex peristatic pump at a flow rate that kept the pressure below 30 psi. The purity of each oligonucleotide was determined by ion-pairing reverse-phase ultraperformance liquid chromatography as described previously  protecting groups (t-butyl amine:methanol:water (1:1:2) overnight at 37 °C, deprotection of the 2′-O-TBDMS ethers was accomplished using an ammonium fluoride/dimethylsulfoxide scheme  and 2) determining the temperature at which the fraction of molecules in the double-stranded state (θ) was equal to 0.5. Although Tm values calculated for each hairpin via the slope method were a few degrees higher than those obtained by determining the temperature at which θ = 0.5, the magnitude of the changes and the trends in the datasets were comparable. Only the Tm values calculated via θ = 0.5 are presented in SI Appendix, Fig. S1. For comparison and presentation, all data were normalized to an absorbance of 1.0 at 95 °C.Optical melting experiments were done via absorbance at 260 nm in 1 M NaCl, 50 mM sodium phosphate, pH 7, as described previously ( methods : 1) deteThe genome sequence of Enterobacteria phage T4 was obtained from GenBank (NC_000866.4). We searched the genome for all occurrences of the sequence CTTCGG using Blat . We thenThere are no data associated with this paper.Supplementary File


Cortical theta oscillations of neuronal activity are a fundamental mechanism driving goal-directed behavior. We previously identified in the rat orbitofrontal cortex (OFC) a neuronal correlate of individual preferences between cocaine use and an alternative nondrug reward (i.e. saccharin). Whether theta oscillations are also associated with choice behavior between a drug and a nondrug reward remains unknown. Here we investigated the temporal structure between single unit activity and theta band oscillations (4–12 Hz) in the OFC of rats choosing between cocaine and saccharin. First, we found that the relative amplitude of theta oscillations is associated with subjective value and preference between two rewards. Second, OFC phase-locked neurons fired on opposite phase of the theta oscillation during saccharin and cocaine rewards, suggesting the existence of two separable neuronal assemblies. Finally, the pharmacological influence of cocaine at the moment of choice altered both theta band power and theta phase-locking in the OFC. That is, this drug influence shifted spike-phase relative to theta cycle and decreased the synchronization of OFC neurons relative to the theta oscillation. Overall, this study indicates that the reorganization of theta phase-locking under the influence of cocaine biases OFC neuronal assemblies in favor of cocaine choice and at the expense of a normally preferred alternative, a neuronal change that may contribute to drug preference in cocaine addiction. OFC neurons encode incentive value and the relative preference when choosing between two rewards of the same kind 10. We recently extended these findings to choice between drugs of abuse  and nondrug alternative rewards 12. Specifically, we found that drug and nondrug rewards are selectively encoded by two non-overlapping populations of OFC neurons, and that OFC spiking activity reflect the relative preference between the two types of options12.The orbitofrontal cortex (OFC) plays a crucial role in processing reward information about competing options to influence choice and preference in humans, monkeys, and rats13, also plays a fundamental role in cognitive functions and behavior16. In particular, theta oscillations (4–12 Hz) have been observed across several cortical brain regions and implicated in cognitive functions such as reward processing and goal-directed behavior19. Moreover, within individual brain areas, oscillations can synchronize neurons thereby creating coherent cell assemblies20. Consistent with this, it has been shown that reward information encoded by OFC neurons in rats becomes highly synchronized to OFC theta oscillation in anticipation of the reward18. Importantly, spike-field phase locking synchronization was stronger when anticipating a positive rather than a negative outcome, suggesting that OFC theta oscillations encode subjective reward value.In addition to single cell coding, oscillatory activity of the local field potential (LFP), which results from the sum of incoming currents across all excitable membranes within a local volume22. To address these questions, we analyzed the LFP data from a previous set of experiments11. Briefly, LFP and neuronal spiking activities were recorded in the OFC of rats trained to choose between sweet water and cocaine. Specifically, we first examined the temporal modulation of theta oscillations in the OFC and then the relationships between OFC single unit activity and theta oscillations during both sampling and choice trials. Finally, we compared OFC theta oscillatory activity and spike field phase-looking when choices were made drug free versus under the influence of cocaine.Whether OFC theta oscillations are modulated and synchronize OFC firing neuronal activity during choice between cocaine and a nondrug reward remains to be investigated. Moreover, we also sought to determine whether and to what extent the drug influence at the moment of choice could alter these OFC oscillatory activity dynamics. We recently demonstrated that while rats prefer the nondrug option when not under the drug influence, they shift their choice toward cocaine nearly exclusively when making their choice under the drug influence11, we reported OFC neuronal spiking activity in rats choosing between a drug  and a nondrug reward (sweet water). Here we looked more specifically at LFP activity in the OFC. Briefly, two groups of animals were trained to sample and choose between two levers associated with different rewards  = 6.84; p < 0.001) but this effect was not significantly different between the two samplings and choice trials  = 1.14; NS). In contrast, theta power increased within 1 s before lever press  = 19.36; p < 0.001). Notably, this increase in theta power before the press differ depending on the trials type  = 3.21, p < 0.001). To further quantify and compare theta modulation across trials type, theta power (1 s after lever insertion and 2 s before lever press) was normalized as the percentage of power during baseline  = 5.40, p < 0.05), but not by lever insertion  = 2.13), indicating a predominant role of the theta oscillation during lever press. Importantly, the increase in theta power was greater for the preferred  than for the non-preferred alternative option  . Thus, as with OFC single activity neurons, the amplitude of theta oscillations seems to represent individual preferences. However, the theta modulation during sampling for the preferred reward was similar to that during the choice trials , suggesting a similar implication of theta oscillations in the OFC during sampling and choice trials.We first examined the temporal modulation of theta oscillations in the S/S group by calculating mean averaged peri-event spectrograms around lever insertion (1 s before and 1 s after lever insertion) and lever press (2 s before and 1 s after lever press) during both sampling and choice trials Fig. . Theta p18. However, in this report, we based our phase-locking analysis only on the pre-press period (2 s before lever press) mainly for two reasons: 1) it was not possible to define a similar post-press consumption period in the S/C group and thus to compare the different groups; 2) OFC neurons increased their firing during the pre-press period11. The proportion of theta phase-locked neurons was slightly higher during sampling for the preferred reward and during choice than during sampling for the non-preferred reward . Importantly, the distribution of theta phases became even more concentrated during choice trials (Rayleigh’s test: p < 0.01), suggesting an increase in theta-mediated synchrony among OFC neurons during choice. Consistent with this, the strength of phase locking was higher during choice than during each of the two samplings  = 4.05, p < 0.001; time x trial type interaction: F = 1.01, NS), but did not change significantly before lever press  = 0.78; NS), suggesting that the presence of the drug option abolished theta power increase before lever press. Alternatively, it is also possible that different electrode placements between the S/S and the S/C groups could explain this lack of increase in theta power. However, normalized theta power 1 s before the press was still greater during choice and during sampling for the preferred than for the non-preferred alternative option   and lever press (2 s before and 1 s after lever press) was also observed in the S/C group Fig. . Theta p05) Fig. . Spike-L05) Fig. , phase-l05) Fig. . Specifi05) Fig. . The mea22. As expected, in all initially saccharin preferring animals pre-choice cocaine shifted preference from saccharin to cocaine almost exclusively  = 23.8; p < 0.01). We hypothesized that passive, pre-choice administration of cocaine will alter the theta power and the timing of neuronal firing with respect to local theta activity. Compared with the control condition where no pre-choice cocaine was administered, passive, pre-choice administration of cocaine blocked theta power increase before lever press  = 5.2; p < 0.01; Fig. p < 0.01 and NS for no pre-choice and pre-choice cocaine, respectively; Fig. Finally, we assessed LFP activity and synchrony in a separate group of rats (n = 5) that made choices under the influence of cocaine versus not under this influence . The influence of cocaine was induced by passively administering cocaine before each choice trial  before each choice trial, a pharmacological intervention known to shift choice to cocaine45. Indeed, interneurons can modulate theta oscillations and control dynamically the firing phase of pyramidal neurons47. It is therefore possible that the shift in theta phase after pre-choice cocaine was due to a change in interneurons’ firing activity. Consistent with this hypothesis, a preliminary analysis revealed that the activity of OFC interneurons, as identified by their electrophysiological profile48, increased dramatically and rapidly after passive pre-choice administration of cocaine  = 1.8; p < 0.001, K. Guillem et al., unpublished data) and remained significantly elevated during several minutes. Such an increase in inhibitory efficacy after cocaine pre-trial administration could constrain the pyramidal cells to fire primarily at an earlier phase  of the theta cycle, hence decreasing synchrony. Future research will need to confirm this hypothesis.GABAergic inhibitory interneurons have been shown to play a crucial role in the generation of theta cycle in several brain areas including the cortexRegardless of the mechanism involved, this shift in phase-locking could cause a profound reorganization of the dynamics of OFC network leading to the formation of different neuronal assemblies associated with different behavior. Consistent with this, distinct ensembles of phase-locked neurons were recruited under control condition and after cocaine priming. Under cocaine priming, only a minority (31%) of phase-locked neurons were also phase-locked under control condition while the vast majority (69%) was not. Importantly, these two phase-locking ensembles are associated with two different choice behaviors: one phase-locking ensemble fired close to the peak of the theta oscillation and is associated with saccharin choices, while the other phase-locking ensemble fired closer to the trough and is associated with cocaine choices. Thus, our results suggest that pre-choice cocaine decreased the activity of the neuronal assembly associated with saccharin choices, while it increased the activity of another neuronal assembly associated with cocaine choices. Though the mechanisms underlying this shift in phase-locking remain to be fully elucidated, this aberrant synchronization within the OFC and consequent of the neurocircuitry underlying choice may contribute to drug preference in cocaine addiction.48. Rats were initially housed two per cage. After surgery, they were housed individually in a temperature- and ventilation-controlled environment under a reversed 12 h light/dark cycle . All experiments were carried out in accordance with standard ethical guidelines (European Communities Council Directive 86/609/EEC) and approved by the committee on Animal Health and Care of Institut National de la Santé et de la Recherche (Agreement A5012052).Male Wistar rats (n = 16) were surgically prepared with a catheter in the right jugular vein and rectangular arrays of 16 teflon-coated stainless steel microwires  were implanted unilaterally in the OFC  as previously described in detail elsewhere46. Briefly, each chamber was equipped with 2 automatically retractable levers (Imetronic), a commercially-available lickometer circuit (Imetronic), two syringe pumps, a single-channel liquid swivel , an electrical commutator , and two pairs of infrared beams to measure horizontal cage crossings.Operant chambers (30 × 40 × 36 cm) used for all behavioral training and testing  have been described in detail elsewhere11. Briefly, each session consisted of 44 trials spaced 10 min apart and divided into two successive phases: sampling  and choice   schedule to obtain different rewards depending on the group: between two concentrations of sweet water , or between sweet water and cocaine  Fig. . In the ls) Fig. . During 21. A group of rats (n = 5) was first trained to choose between cocaine and saccharin until stabilization of their choice performance before the non-contingent pre-trial administration of cocaine test, as described elsewhere11. This was done in one session using a choice procedure identical to that described above during a total of 24 choice trials, except that the last 12 choice trials were each preceded 5 minutes before by a pre-choice administration of cocaine .We recently found that a non-contingent administration of cocaine  before each choice trial caused rats to immediately shift their preference from sweet water to cocaine12. The quality of individual-neuron recordings will be ensured with the following criteria: <3% of all interspike intervals exhibited by the unit are <2000 µs, and the average amplitude of the unit waveform is at least three times larger than that of the noise band . These units were classified into putative interneurons and pyramidal neurons according to the waveform spike width and average firing rate, as previously described48. Electrophysiological data were analyzed using NeuroExplorer (Plexon) and Matlab .During recording sessions, electrodes will be connected to headstages and voltage signals from each electrode will be recorded, amplified, bandpass filtered (from 250 Hz to 8 kHz for unit activity and from 0.7 Hz to 170 Hz for LFPs) and digitally captured . Neuronal activity was digitized at a rate of 40 kHz and LFPs were down sampled to 1 kHz. Behavioral events in the operant task will be streamed to the Plexon system via TTL pulses to allow the neuronal data to be accurately synchronized to these behavioral events. Single units spike sorting was performed off-line using Offline Sorter software (Plexon) and analyzed using Neuroexplorer (Nex Technologies) as previously describedThe power spectral density (PSD) of each LFP was calculated with the NeuroExplorer PSD function using a multitaper fast Fourier transform (FFT), with a Hamming window of 2048 points (2 s), 50% overlap and 1 Hz resolution. PSD values were expressed as the percent of the total power spectrum within the frequency range considered (1–30 Hz). For each trial of a given condition , the power density analysis was performed using a 2-s time window period centered around the behavioral events of interest  and averaged across each trial of a given condition  to obtain a mean power spectrum. To measure the temporal modulation of LFP theta oscillations during specific behavioral events of interest  peri-event spectogramms (PESPs) of power density were calculated (NeuroExplorer) and averaged across animals (Matlab), using the following time windows: 1 s before and 1 s after lever insertion, and 2 s before and 1 s after lever press. The LFP theta frequency band was taken as the frequency at maximum power within the 4–12 Hz frequency band of the time-averaged spectrogram. Averaged theta power (% of baseline ± s.e.m.) was normalized to the average spectral power during the baseline period (a 1 s window beginning 2 s before lever insertion).11. LFP data were first Hann filtered to the theta (4–12 Hz) (NeuroExplorer). The phase vector was then calculated using the Hilbert transform of the filtered signal yielding values ranging from 0–360° (180° corresponds to the trough), thus providing an indication of the current phase position of the oscillation over time. Only neurons with >50 spikes during the period analyzed were used. Phase-locking was quantified as the circular concentration of the resulting phase vector distribution which is defined as the mean resultant vector length of the phase angles. The resultant vector length is a real number ranging from 0 (low phase-locking) to 1 (perfect phase-locking). Measures based on mean vector length can be biased by the number of spikes entered into the computation. Thus, we tried to prevent this bias by keeping constant the number of spikes (n = 50) used when comparing between different rewards and trials types, as was done previously49. The statistical significance of spike-LFP phase locking was addressed using the circular statistics . A neurons was significantly phase-locked if the distribution of spike phase angles deviated from a uniform circular distribution .To investigate the relationship between single unit activity and LFPs we performed spike-LFP phase locking for each trial of a given condition . This phase-locking analysis was conducted only on the pre-press period (2 s before lever press) because this period was preserved and identical between the two groups, and OFC neurons increased their firing during that pre-press period


RNA-binding proteins (RBPs) play a pivotal role in the lifespan of RNAs. The disfunction of RBPs is frequently the cause of cell disorders which are incompatible with life. Furthermore, the ordered assembly of RBPs and RNAs in ribonucleoprotein (RNP) particles determines the function of biological complexes, as illustrated by the survival of the motor neuron (SMN) complex. Defects in the SMN complex assembly causes spinal muscular atrophy (SMA), an infant invalidating disease. This multi-subunit chaperone controls the assembly of small nuclear ribonucleoproteins (snRNPs), which are the critical components of the splicing machinery. However, the functional and structural characterization of individual members of the SMN complex, such as SMN, Gemin3, and Gemin5, have accumulated evidence for the additional roles of these proteins, unveiling their participation in other RNA-mediated events. In particular, Gemin5 is a multidomain protein that comprises tryptophan-aspartic acid (WD) repeat motifs at the N-terminal region, a dimerization domain at the middle region, and a non-canonical RNA-binding domain at the C-terminal end of the protein. Beyond small nuclear RNA (snRNA) recognition, Gemin5 interacts with a selective group of mRNA targets in the cell environment and plays a key role in reprogramming translation depending on the RNA partner and the cellular conditions. Here, we review recent studies on the SMN complex, with emphasis on the individual components regarding their involvement in cellular processes critical for cell survival. Post-transcriptional mechanisms governing gene expression depend on the concerted action of RNA-binding proteins (RBPs) and RNAs. Soon after transcription commences, the mRNA associates with a distinct type of RBPs, giving rise to dynamic ribonucleoprotein (RNP) entities, which control gene expression as a function of the components present in the complex . In this7GTP residue (or cap) located at the 5′end of most mRNAs. During cap-dependent initiation, eukaryotic initiation factors (eIFs) recruit the small ribosomal subunit to the 5′end of the mRNA . Th. Th74]. Beyond self-dimerization, the identification of p85-Gemin5-associated partners has disclosed relevant unknown functions of this versatile protein. The top gene ontology (GO) terms retrieved by p85 are splicing-related components, translation control members, and snRNP assembly factors, fully consistent with the functions reported for this protein . InteresGiven that the dimerization mutant disrupts the p85-Gemin5 interaction, hampers the association with proteins regulating splicing and translation processes, and abolishes translation control, it is conceivable that defects in Gemin5 dimerization will occur in severe diseases, as shown in mutations affecting the TPR domains of other proteins . MoreoveThe diverse composition of the cellular Gemin5 interactome reflects its functional versatility. A comprehensive view of the Gemin5 proteome reveals that most partners copurify with the long N-terminal domain , includi1-1287 polypeptide as well are detected on heavy polysome fractions, reinforcing their ribosome-binding capacity  pull-down assays, suggesting that Gemin5 can form a binary complex with these proteins. L3 and L4 are conserved proteins with terminal extensions located on the solvent side of the ribosome ,86,87. LIn recent years, Gemin5 has emerged as a critical factor during translational reprogramming events, although the mechanisms underlying this phenomenon are still poorly known . In thishttps://www.encodeproject.org) shows a 30% overlap with RBS1 hits, further supporting the RNA-binding capacity of RBS1. These studies identified an internal region of the Gemin5 mRNA, designated H12 for hits 1-2, among the RNA targets of both Gemin5 and RBS1. The biochemical and functional characterization of this target has demonstrated that the RBS1 polypeptide physically interacts with the H12 RNA region upregulating mRNA translation, thereby providing a regulatory feedback loop that counteracts the negative effect of Gemin5 on translation control. Further supporting this feedback loop, the expression of RBS1 alone stimulates the translation of the endogenous Gemin5 mRNA [The marked differences in RNA recognition reported by different studies are compatible with the capacity of the individual domains of Gemin5 to recognize still-unknown RNAs. This possibility prompted the search for cellular RNA targets of the RBS1 domain using a modified crosslinking and immunoprecipitation (CLIP)-based approach in HEK293 cells. A prominent feature of the RNA hits specifically recognized by the RBS1 domain is the ability to fold as thermodynamically stable hairpins  in full in5 mRNA . In addiIn summary, Gemin5 could contribute to translation control in different ways using distinct functional domains and their respective RNA partners . The negBesides the different functions of Gemin5 in normal conditions, the possibility that this protein can have an important impact on translation control under cellular stress is still an open question. As mentioned before, Gemin5 was identified as a factor bound to two viral IRES elements—hepatitis C (HCV) and FMDV . MoreoveRegarding the role of Gemin5 in the regulation of gene expression under severe stress as it occurs during viral infections, it has been shown that the protein is redistributed to the viral factories during Sindbis virus (SINV) infection co-localizing with SINV RNA . Indeed,RBPs participate in many steps of the mRNA lifespan and are frequently associated in large macromolecular complexes. However, the role of individual RBPs in the expression of a given RNA in living organisms and how RBPs cooperate to form networks are still poorly understood. The works reviewed here recapitulate the involvement of the SMN members in various cellular processes, including snRNP assembly and translation control. Numerous studies have shown that RBPs with functions related to neuromuscular diseases, such as the components of the SMN complex, display a modular structure, allowing their interaction with different partners presumably in response to specific cellular signals. Beyond its RNA-binding activity in the SMN complex, Gemin5 cooperates in translation control and ribosome binding through separate structural and functional domains of the protein . Collect